HK40004013A - Efficacy of anti-trop-2-sn-38 antibody drug conjugates for therapy of tumors relapsed/refractory to checkpoint inhibitors - Google Patents

Description

Efficacy of anti-TROP-2-SN-38 antibody drug conjugates for therapy of checkpoint inhibitors of relapsed/refractory tumors

RELATED APPLICATIONS

This application is entitled to provisional patent application 62/328,289, filed 2016, 4, 27, which is hereby incorporated by reference in its entirety, in accordance with 35U.S. c.119 (e).

Sequence listing

This application contains a sequence listing that has been filed in ASCII format through EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on day 5/4/2017 was named IMM366WO2_ sl.

Technical Field

The present invention relates to therapeutic uses of antibody-drug conjugates (ADCs) comprising an anti-Trop-2 antibody or antigen-binding fragment thereof and a camptothecin, such as SN-38, with improved ability to target various cancer cells in a human subject. In preferred embodiments, the antibody and therapeutic moiety are linked by an intracellularly cleavable linkage, which increases the therapeutic efficacy. In a more preferred embodiment, the ADC is administered at a specific dose and/or specific administration regimen to optimize the therapeutic effect. Surprisingly, optimized doses and administration regimens of SN-38-conjugated antibodies such as saxilizumab gavitic (sacituzumab) show unexpectedly superior efficacy, which is unpredictable from animal model studies, allowing for effective treatment of tumors resistant to checkpoint inhibitor antibodies such as atelizumab (atezolizumab), pembrolizumab (pembrolizumab), nivolumab (nivolumab), pidilizumab (pidilizumab), dulamuzumab (durvalumab), alezumab, ipilimumab (ipilimumab), or tremelimumab (tremelimumab). In particular embodiments, the ADC may be administered to a human subject having a Trop-2-positive cancer at a dose of 3mg/kg to 18mg/kg, more preferably 4mg/kg to 12mg/kg, most preferably 8mg/kg to 10 mg/kg. Surprisingly, the anti-Trop-2-SN 38 Antibody Drug Conjugates (ADCs) are effective in treating Trop-2 positive cancers, such as pancreatic cancer, triple negative breast cancer, small cell lung cancer, endometrial cancer, urothelial cancer, and non-small cell lung cancer, in patients who relapse from or show resistance to checkpoint inhibitor therapy.

Background

Scientists in the field of specifically targeted drug therapy have been working for many years on the specific delivery of toxic agents to human cancers using monoclonal antibodies (mabs). Conjugates of tumor-associated mabs and suitable toxic agents have been developed, but have been half-defeated in the therapy of human cancer, and have had little application in other diseases such as infectious and autoimmune diseases. The most common toxic agents are chemotherapeutic drugs, although particle emitting radionuclides or bacterial or phytotoxins are also conjugated to MAbs, particularly for Cancer therapy (Sharkey and Goldenberg, CA Cancer J Clin.2006, 7-8 months; 56(4): 226-.

The advantage of using MAb-chemotherapeutic drug conjugates is that (a) the chemotherapeutic drug itself is well-defined in structure; (b) chemotherapeutic drugs are typically linked to MAb proteins at specific sites remote from the antigen binding region of the MAb using well-defined conjugation chemistry; (c) MAb-chemotherapeutic drug conjugates can be made more reproducibly and are generally less immunogenic than chemical conjugates involving mabs and bacterial or phytotoxins, and are therefore easier to commercially develop and to regulatory approve; (d) MAb-chemotherapeutic drug conjugates are systematically several orders of magnitude less toxic than radionuclide MAb conjugates, especially for radiation-sensitive bone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumor agents. Irinotecan (Irinotecan) (also known as CPT-11) and topotecan (topotecan) are CPT analogs that have been approved for use as Cancer therapeutics (Iyer and Ratain, Cancer Chemother. Phamacol.42: S31-S43 (1998)). CPT inhibits topoisomerase I enzyme by stabilizing The topoisomerase I-DNA complex (Liu et al, The Camptothecins: Unfolding therir anticerancer Potential, Liehr J.G., Giovanella, B.C., and Verschraegen (eds.), NYAcad Sci.e., NY922:1-10 (2000)). CPT presents particular problems in the preparation of conjugates. One problem is that most CPT derivatives are not soluble in aqueous buffers. Second, CPT provides specific challenges for structural modification of conjugation to macromolecules. For example, CPT itself contains only tertiary hydroxyl groups in ring-E. The hydroxyl functionality in the case of CPT must be coupled to a linker suitable for subsequent protein conjugation; and in potent CPT derivatives such as SN-38, the active metabolites of chemotherapeutic CPT-11, and other C-10-hydroxy containing derivatives such as topotecan and 10-hydroxy-CPT, the presence of a phenolic hydroxy group at the C-10 position complicates the necessary derivatization of the C-20-hydroxy group. Third, the instability of the delta-lactone moiety of the E-ring of camptothecin under physiological conditions leads to a greatly reduced antitumor efficacy. Thus, the conjugation scheme is performed such that it is performed at a pH of 7 or lower to avoid lactone ring opening. However, conjugation of bifunctional CPTs having amine reactive groups, such as active esters, will typically require a pH of 8 or higher. Fourth, the intracellular cleavable moiety is preferably incorporated into a linker/spacer that connects the CPT and the antibody or other binding moiety.

There is a need for more efficient methods of preparing and administering antibody-CPT conjugates, such as anti-Trop-2-SN-38 conjugates (e.g., saxizumab govitegam). Preferably, the methods include optimized doses and administration regimens that maximize efficacy and minimize toxicity for use in patients with Trop-2-positive cancers that relapse from or are resistant to checkpoint inhibitor antibodies.

Disclosure of Invention

As used herein, unless otherwise specifically indicated, the abbreviation "CPT" may refer to camptothecin or any derivative thereof, such as SN-38. The present invention addresses an unmet need in the art by providing improved methods and compositions for making and administering CPT-antibody ADCs. Preferably, the camptothecin is SN-38, and the antibody is an anti-Trop-2 antibody (e.g., saxilizumab gaulthikang). The disclosed methods and compositions may be used to treat diseases that are refractory or poorly responsive to other forms of therapy, such as Trop-2+ cancers that are resistant to treatment with or relapse from checkpoint inhibitor antibodies.

Preferably, the antibody portion of the ADC is a monoclonal antibody, antibody fragment, bispecific or other multivalent antibody or other antibody-based molecule or compound. The antibody may be of various isotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferably including human IgG1 hinge and constant region sequences. The antibody or fragment thereof may be a chimeric human-mouse, chimeric human-primate, humanized (human framework and murine hypervariable (CDR) regions) or fully human antibody, and variants thereof, such as a half IgG4 antibody (referred to as "monoclonal antibody"), as described by van der Neut Kolfschoeten et al (Science 2007; 317: 1554) -1557. More preferably, the antibody or fragment thereof may be designed or selected to comprise a human constant region sequence belonging to a specific allotype, which may result in reduced immunogenicity when the ADC is administered to a human subject. Preferred allotypes for administration include non-G1 m1 allotypes (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1, 2. More preferably, the allotype is selected from the group consisting of: nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

An exemplary anti-Trop-2 antibody is a humanized RS7(hRS7) antibody comprising the light chain C DR sequence CDR1(KASQDVSIAVA, SEQ ID NO:1), CDR2(SASYR YT, SEQ ID NO:2) and CDR3(QQHYITPLT, SEQ ID NO:3), and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2(WINTYTGE PTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ I D NO: 6).

In a preferred embodiment, the chemotherapeutic moiety is selected from Camptothecin (CPT) and analogues and derivatives thereof, and more preferably SN-38. However, other chemotherapeutic moieties that may be used include taxanes (e.g., baccatin III, paclitaxel), epothilones (epothilones), anthracyclines (anthracyclines) (e.g., doxorubicin (doxorubicin) (DOX), epirubicin (epirubicin), morpholino doxorubicin (morpholino-DOX), cyanomorpholino doxorubicin (cyanomorpholino-DOX), 2-pyrroline doxorubicin (2-PDOX) or prodrug forms of 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (eds.), Systemphosium series 574, exemplified by American Chemical Society, Washington DC, 1995 (p. 332) and Nagy et al, Proc. Natl. Acad. Sci. USA 93:2464-2469,1996), exemplified by geldanamycin (geldanamycin) (J. ansamycin: 23; Nebiles et al.),442, Invest.New Drugs 17:361-373,1999), and so on. Preferably, the antibody or fragment thereof is linked to at least one chemotherapeutic moiety, preferably 1 to about 5 chemotherapeutic moieties, more preferably 6 or more chemotherapeutic moieties, most preferably about 6 to about 12 chemotherapeutic moieties.

Various embodiments may relate to the use of the methods and compositions of the invention to treat human Trop-2 expressing cancers, including but not limited to, carcinomas such as those of the esophagus, pancreas, lung, stomach, colon and rectum, bladder, breast, ovary, uterus, kidney, urothelium and prostate.

In certain embodiments directed to the treatment of cancer, the drug conjugates can be used in combination with surgery, radiation therapy, chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy, immunomodulators, vaccines and the like. These combination therapies may allow lower doses of each therapeutic agent to be administered in such combinations, thereby reducing certain serious side effects and possibly shortening the required course of treatment. The full dose of each agent may also be administered when there is no or minimal overlapping toxicity. In alternative embodiments, the ADC may be administered in combination with an interferon, checkpoint inhibitor antibody, bruton's tyrosine kinase inhibitor, PI3K inhibitor, PARP inhibitor, or microtubule inhibitor as discussed below.

Preferred optimal doses of ADC may include intravenous doses of 3mg/kg to 18mg/kg, preferably administered once a week, twice a week or once every other week. An optimal dosing regimen may include two weeks of continuous treatment followed by one, two, three or four weeks of rest; or alternating between treatment weeks and rest weeks; or treatment for one week followed by rest for two, three or four weeks; or for three weeks followed by a rest of one, two, three or four weeks; or for four weeks, followed by a rest of one, two, three or four weeks; or for five weeks followed by a rest of one, two, three or four weeks; or once every two weeks, once every three weeks, or once a month. The treatment may be extended for any number of cycles, preferably at least 2 cycles, at least 4 cycles, at least 6 cycles, at least 8 cycles, at least 10 cycles, at least 12 cycles, at least 14 cycles, or at least 16 cycles. Exemplary dosages for use may include 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg and 18 mg/kg. Preferred doses are 4mg/kg, 6mg/kg, 8mg/kg, 9mg/kg, 10mg/kg or 12 mg/kg.

In certain alternative embodiments, where the ADC is administered subcutaneously, the dosage of an ADC such as a saxizumab govitegam (IMMU-132) may be limited by the ability to concentrate the ADC without precipitation or aggregation and the amount of administration that can be given subcutaneously (preferably 1ml, 2ml or 3ml or less). Thus, for subcutaneous administration, the ADC may be administered at 1.5mg/kg to 4mg/kg daily for 1 week, or 3 times per week for 2 weeks, or twice per week for two weeks, followed by rest. In the case of multiple site subcutaneous injections, doses up to 8mg/kg may be provided. Maintenance doses of ADC may be administered intravenously or subcutaneously every two to three weeks or once a month after induction. Alternatively, 2 to 4 cycles (each cycle being the administration of ADC on days 1 and 8 of two 21-day cycles with a one week rest period) may be administered intravenously at 8mg/kg to 10mg/kg, followed by one or more subcutaneous injections of the active dose per week or induction as maintenance therapy. The dose may be adjusted based on the provisional tumor scan and/or by analyzing Trop-2 positive circulating tumor cells.

One of ordinary skill will recognize that in selecting the optimal dose of ADC, a variety of factors may be considered, such as age, general health, specific organ function or weight, and the effect of previous therapy on a specific organ system (e.g., bone marrow), and that the dose and/or frequency of administration may be increased or decreased during the course of treatment. Where evidence of tumor shrinkage is observed after as little as 4 to 8 doses, the dose can be repeated as needed. The optimized dosages and administration regimens disclosed herein exhibit unexpectedly superior efficacy and reduced toxicity in human subjects, which is unpredictable in animal model studies. Surprisingly, the superior efficacy allows for the treatment of tumors previously found to be resistant to one or more standard anti-cancer therapies, including checkpoint inhibitor therapies.

The method of the invention may comprise the use of CT and/or PET/CT or MRI to periodically measure the tumor response. Blood levels of tumor markers such as CEA (carcinoembryonic antigen), CA19-9, AFP, CA15.3, or PSA can also be monitored. Depending on the results of the imaging and/or blood levels of the markers, the dosage and/or administration regimen may be adjusted as needed.

A surprising result of the presently claimed compositions and methods is that high doses of antibody-drug conjugates are unexpectedly tolerated even under repeated infusions, with only relatively low toxicity observed to be severe and vomiting or with controllable neutropenia. Another surprising result is the lack of accumulation of antibody-drug conjugates, unlike other products with SN-38 conjugated to albumin, PEG, or other carriers. The lack of accumulation is associated with improved tolerability and lack of severe toxicity even after repeated or increased dosing. These surprising results allow optimization of dosage and delivery regimens with unexpectedly high efficacy and low toxicity. The claimed methods provide 15% or more, preferably 20% or more, preferably 30% or more, more preferably 40% or more, shrinkage in solid tumor size (as measured by longest diameter) in individuals with previously resistant cancer. One of ordinary skill will recognize that tumor size may be measured by a variety of different techniques, such as total tumor volume, maximum tumor size in any dimension, or a combination of size measurements in several dimensions. This may be done using standard radiological procedures such as computed tomography, ultrasonography, and/or positron emission tomography. The method of sizing is less important than observing the tendency of ADC treatment to cause a reduction in tumor size, which preferably results in tumor elimination.

While the ADC may be administered in periodic bolus injections, in an alternative embodiment, the ADC may be administered by continuous infusion of the antibody-drug conjugate. To increase Cmax and extend PK of ADC in blood, continuous infusion can be administered, for example, through an indwelling catheter. Such devices are known in the art, such asOr PORT-A-Catheters (see, e.g., Skolnik et al, the Drug Monit32:741-48,2010) and any such known indwelling catheter may be used. Various continuous infusion pumps are also known in the art and any such known infusion pump may be used. The dose range for continuous infusion may be between 0.1 mg/kg/day and 3.0 mg/kg/day. More preferably, the ADCs may be administered by intravenous infusion over a relatively short period of 2 to 5 hours, more preferably 2-3 hours.

In particularly preferred embodiments, the ADC and dosing regimen are effective in patients resistant to standard therapy. For example, anti-Trop-2 saxizumab govietin can be administered to patients who do not respond to prior therapy with irinotecan (the parent agent of SN-38). Surprisingly, irinotecan-resistant patients could show a partial or even complete response to saxizumab govitegam. The ability of ADCs to specifically target tumor tissue can overcome tumor resistance by improving targeting and enhancing delivery of therapeutic agents. ADCs may exhibit similar improved efficacy and/or reduced toxicity compared to alternative standard therapeutic treatments, such as checkpoint inhibitor antibodies.

A particularly preferred subject may be a metastatic colorectal cancer patient; patients with metastatic pancreatic cancer; patients with triple negative breast cancer; HER +, ER +, progesterone + breast cancer patients; patients with metastatic non-small cell lung cancer (NSCLC); metastatic small cell lung cancer patients; patients with metastatic gastric cancer; patients with metastatic renal cancer; patients with metastatic urothelial cancer; patients with metastatic bladder cancer; patients with metastatic ovarian cancer or patients with metastatic uterine cancer.

Drawings

Figure 1 summary of CT scan results of patients treated with 10mg/kg of saxizumab gavatinib with checkpoint inhibitor resistant metastatic TNBC.

Fig. 2. baseline CT images of patients with checkpoint inhibitor resistant metastatic TNBC showing axial images (top row) and sagittal images (bottom row). Tumors are indicated by arrows.

FIG. 3 comparison of CT scans performed on target lesions 1 and 2 before and after treatment with 10mg/kg of Saxizumab govitegam (IMMU-132). Baseline images are shown on the top and secondary response assessments after the treatment of saxizumab govitikang are shown on the bottom. Tumor shrinkage induced by IMMU-132 was clearly observed.

Figure 4 comparison of CT scans performed on target lesion 3 before and after treatment with 10mg/kg of saxizumab govitegam (IMMU-132). Baseline images are shown on the top and secondary response assessments after the treatment of saxizumab govitikang are shown on the bottom. Tumor shrinkage induced by IMMU-132 was clearly observed.

Figure 5 summary of the results of patients with checkpoint inhibitor resistant tumors treated with IMMU-132.

Detailed Description

Definition of

In the following description, a number of terms are used, and the following definitions are provided to facilitate understanding of the claimed subject matter. Terms not explicitly defined herein are used according to their ordinary and customary meaning.

Unless otherwise indicated, all references to FIGSA/aMeans "one or more".

Term(s) forAboutAs used herein, is intended to mean a value plus or minus ten percent (10%). For example, "about 100" refers to any number between 90 and 110.

As used hereinAntibodiesRefers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., an IgG antibody) or an antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. Antibodies or antibody fragments may be conjugated or otherwise derivatized within the scope of the claimed subject matter. Such antibodies include, but are not limited to, IgG1, IgG2, IgG3, IgG4 (and IgG4 subtypes), and IgA isotypes. As used below, the abbreviation "MAb" is used interchangeably to refer to an antibody, antibody fragment, monoclonal antibody, or multispecific antibody.

“Antibody fragments"is part of an antibody, such as F (ab')₂、F(ab)₂Fab', Fab, Fv, scFv (single chain Fv), single domain antibodies (DAB or VHH), etc., including the above cited half molecule of IgG4 (van der Neut Kolfschoeten et al, (Science 2007; 317 (9.14 th): 1554 1557.) regardless of structure, the antibody fragment used binds to the same antigen recognized by the intact antibodyForming a complex and acting like an antibody. For example, antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which the light and heavy variable regions are joined by a peptide linker ("scFv proteins"). Fragments can be constructed in different ways to produce multivalent and/or multispecific binding forms.

Naked antibodyTypically intact antibodies that are not conjugated to a therapeutic agent. Naked antibodies can exhibit therapeutic and/or cytotoxic effects, e.g., through Fc-dependent functions, such as complement fixation (CDC) and ADCC (antibody-dependent cellular cytotoxicity). However, other mechanisms such as apoptosis, anti-angiogenesis, anti-metastatic activity, anti-adhesion activity, inhibition of heterotypic or homotypic adhesion, and interference in signaling pathways may also provide therapeutic effects. Naked antibodies include polyclonal and monoclonal antibodies, naturally occurring or recombinant antibodies, such as chimeric, humanized or human antibodies and fragments thereof. In some cases, "naked antibody" may also refer to "naked" antibody fragments. As defined herein, "naked" is synonymous with "unconjugated," and means not linked to or conjugated to a therapeutic agent.

Chimeric antibodiesIs a recombinant protein comprising the variable domains of heavy and light antibody chains, including the Complementarity Determining Regions (CDRs) of antibodies derived from a species, preferably rodent antibodies, more preferably murine antibodies, while the constant domains of the antibody molecules are derived from the constant domains of human antibodies. For veterinary applications, the constant domains of the chimeric antibodies may be derived from constant domains of other species, such as primates, cats or dogs.

Humanized antibodiesIs a recombinant protein in which the CDRs of an antibody from a species, such as a murine antibody, are transferred from the variable heavy and variable light chains of the murine antibody into the human heavy and light chain variable domains (framework regions). The constant domains of the antibody molecules are derived from the constant domains of human antibodies. In some cases, modifications may be made, for example, from the original murine, rodent, non-human animalThe corresponding residues of the long animal or other antibody replace specific residues of the framework regions of the humanized antibody, particularly those residues that touch or are near the CDR sequences.

Human antibodiesAre, for example, antibodies obtained from transgenic mice that have been "genetically engineered" to produce human antibodies in response to antigen challenge. In this technique, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for various antigens, and mice can be used to generate human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al, NatureGenet.7:13(1994), Lonberg et al, Nature368:856(1994), and Taylor et al, int.Immun.6:579 (1994). Fully human antibodies can also be constructed by genetic or chromosomal transfection methods as well as phage display techniques, all of which are known in the art. See, e.g., McCafferty et al, Nature 348:552-553(1990) which describes the in vitro production of human antibodies and fragments thereof from immunoglobulin variable domain gene libraries from non-immunized donors. In this technique, human antibody variable domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting said properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for a review of which see, for example, Johnson and Chiswell, Current Opinion in Structural Biology3:5564-571 (1993). Human antibodies can also be produced from in vitro activated B cells. See, U.S. Pat. nos. 5,567,610 and 5,229,275, the examples of each of which are incorporated herein by reference in their entirety.

Therapeutic agentsAre useful atoms for the treatment of diseasesA molecule or compound. Examples of therapeutic agents include, but are not limited to, antibodies, antibody fragments, immunoconjugates, drugs, cytotoxic agents, pro-apoptotic agents, toxins, nucleases (including dnazymes and rnases), hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radionuclides, oligonucleotides, interfering RNAs, sirnas, RNAi, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides, or combinations thereof.

ImmunoconjugatesIs an antibody, antigen-binding antibody fragment, antibody complex, or antibody fusion protein conjugated to a therapeutic agent. Conjugation may be covalent or non-covalent. Preferably, the conjugation is covalent. When the therapeutic agent is a drug, the resulting immunoconjugate is an antibody-drug conjugate or ADC.

As used herein, the termAntibody fusion proteinsIs a recombinantly produced antigen-binding molecule in which one or more natural antibodies, single chain antibodies or antibody fragments are linked to another moiety such as a protein or peptide, toxin, cytokine, hormone, or the like. In certain preferred embodiments, the fusion protein may comprise two or more identical or different antibodies, antibody fragments, or single chain antibodies fused together, which may bind to the same epitope, different epitopes on the same antigen, or different antigens.

ImmunomodulatorAre therapeutic agents that alter, inhibit or stimulate the body's immune system when present. Typically, the immunomodulator used stimulates immune cells to proliferate or be activated in an immune response cascade, such as macrophages, dendritic cells, B cells and/or T cells. However, in some cases, the immunomodulator may inhibit proliferation or activation of immune cells. An example of an immunomodulator as described herein is a cytokine, which is a soluble small protein of about 5kDa to 20kDa that is released by one cell population (e.g., primed T-lymphocytes) upon contact with a specific antigen, and which acts as an intercellular mediator between cells. As will be appreciated by the skilled artisan, examples of cytokines include lymphokines, monokines, leukokinesCertain interleukins and interferons are examples of cytokines that stimulate the proliferation of T cells or other immune cells.

CPTIs an abbreviation for camptothecin, and as used in this application, CPT denotes camptothecin itself or an analogue or derivative of camptothecin, such as SN-38. The structures of camptothecin and some of its analogs are given below in scheme 1, formula 1, wherein the numbers are assigned and the rings are labeled with the letters a-E.

Chart 1

anti-Trop-2 antibodies

Various embodiments relate to the use of antibodies or fragments thereof that bind to Trop-2. In a particularly preferred embodiment, the anti-Trop-2 antibody may be a humanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785, incorporated herein by reference in its entirety) that includes the light chain CDR sequences CDR1(KASQDVSIAVA, SEQ ID NO:1), CDR2(SASYRYT, SEQ ID NO:2) and CDR3(QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO: 6).

The RS7 antibody is mouse IgG produced by crude membrane preparation of human primary squamous cell lung cancer₁. (Stein et al, Cancer Res.50:1330,1990) RS7 antibody recognizes the 46-48kDa glycoprotein, which is characterized as cluster 13. (Stein et al, int.J. cancer Supp.8:98-102,1994) the antigen was named EGP-1 (epithelial glycoprotein-1), but also called Trop-2.

Trop-2 is a type I transmembrane protein and has been cloned from human cells (Fornaro et al, Int J Cancer 1995; 62:610-8) and mouse cells (Sewedy et al, Int J Cancer 1998; 75: 324-30). The amino acid sequence of human Trop-2 is well known (see, e.g., NCBI catalog No. P09758.3). In addition to its role as a tumor-associated calcium signaling mediator (Ripani et al, Int J Cancer 1998; 76:671-6), expression of human Trop-2 was shown to be essential for tumorigenesis and colon Cancer cell invasion, which can be effectively reduced with polyclonal antibodies directed against the extracellular domain of Trop-2 (Wang et al, Mol Cancer Ther 2008; 7: 280-5).

Increasing interest in Trop-2 as a therapeutic target for solid cancers (Cubas et al, BiochimBiophys Acta 2009; 1796:309-14) is confirmed by further reports that document the clinical significance of over-expressed Trop-2 in breast Cancer (Huang et al, Clin Cancer Res 2005; 11:4357-64), Colorectal Cancer (Ohmachi et al, Clin Cancer Res 2006; 12: 3057-63; Fang et al, Int J Coloracal Dis 2009; 24:875-84) and oral squamous cell carcinoma (Fong et al, model Pathol 2008; 21: 186-91). Recent evidence that prostate basal cells expressing high levels of Trop-2 are rich in vitro and in vivo stem-like activity is particularly noteworthy (Goldstein et al, Proc Natl Acad Sci USA 2008; 105: 20882-7).

Flow cytometry and immunohistochemical staining studies have shown that RS7 mabs detect antigens on a variety of tumor types with limited binding to normal human tissues (Stein et al, 1990). Trop-2 is predominantly expressed by carcinomas such as lung, stomach, bladder, breast, ovary, uterus and prostate cancer. Localization and therapeutic studies using radiolabeled murine RS7MAb in animal models have demonstrated tumor targeting and therapeutic efficacy (Stein et al, 1990; Stein et al, 1991).

Strong RS7 staining has been demonstrated in tumors from lung, breast, bladder, ovary, uterus, stomach and prostate. (Stein et al, int.j. cancer 55:938,1993) cases of lung cancer include squamous cell carcinoma and adenocarcinoma. (Stein et al, int.j. cancer 55:938,1993) both cell types stained strongly, indicating that RS7 antibody does not distinguish between histological classes of non-small cell lung cancer.

RS7MAb internalizes rapidly into target cells (Stein et al, 1993). The internalization rate constants of the RS7MAb are between those of two other rapid internalization mabs, which has proven useful for ADC production. There is ample evidence (supra) that internalization of ADC is a prerequisite for antitumor activity. (Pastan et al, Cell 47:641,1986) internalization of drug ADC has been described as a major factor in anti-tumor efficacy. (Yang et al, Proc. nat' l Acad. Sci. USA 85:1189,1988) therefore, RS7 antibodies exhibit several important properties for therapeutic applications.

Although hRS7 antibodies are preferred, other anti-Trop-2 antibodies are known and/or are publicly available and may be used in the ADCs of the present invention in alternative embodiments. Although humanized or human antibodies are preferred for reduced immunogenicity, in alternative embodiments, chimeric antibodies may be used. As discussed below, methods of antibody humanization are well known in the art and can be used to convert available murine or chimeric antibodies into humanized forms.

anti-Trop-2 antibodies are commercially available from a number of sources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417 (Lifesspan BioSciences, Inc., Seattle, WA); 10428-MM01, 10428-MM02, 10428-R001, 10428-R030(Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, CA); sc-376181, sc-376746, Santa Cruz Biotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals, Littleton, CO); ab79976 and ab89928(Cambridge,MA).。

Other anti-Trop-2 antibodies are disclosed in the patent literature. For example, U.S. publication No. 2013/0089872 discloses anti-Trop-2 antibodies K5-70 (accession number FERM BP-11251), K5-107 (accession number FERM BP-11252), K5-116-2-1 (accession number FERM BP-11253), T6-16 (accession number FERM BP-11346) and T5-86 (accession number FERM BP-11254), deposited at International Patent organization Depositry, Tsukuba, Japan. U.S. Pat. No. 5,840,854 discloses anti-Trop-2 monoclonal antibody BR110(ATCC No. HB11698). U.S. Pat. No. 7,420,040 discloses anti-Trop-2 antibodies produced by the hybridoma cell line AR47A6.4.2, deposited with the IDAC under accession number 141205-05 (International depositability Authority of Canada, Winnipeg, Canada). U.S. patent No. 7,420,041 discloses an anti-Trop-2 antibody produced by the hybridoma cell line ar52a301.5, deposited with IDAC under accession number 141205-03. U.S. publication No. 2013/0122020 discloses anti-Trop-2 antibodies 3E9, 6G11, 7E6, 15E2, 18B 1. Hybridomas encoding representative antibodies are deposited with the American Type Culture Collection (ATCC) under accession numbers PTA-12871 and PTA-12872. ADCPF06263507, which comprises an anti-5T 4 (anti-Trop-2) antibody linked to the tubulin inhibitor monomethyl uracil F (MMAF), is commercially available from Pfizer, Inc. (Groton, CT) (see, e.g., Sapra et al, 2013, Mol cancer Ther 12: 38-47). U.S. Pat. No. 8,715,662 discloses anti-Trop-2 antibodies produced by the hybridoma deposited with AID-ICLC (Genoa, Italy) under accession numbers PD 08019, PD 08020 and PD 08021. U.S. patent application publication No. 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 and KM 4590. U.S. Pat. No. 8,309,094(Wyeth) discloses antibodies a1 and A3 identified by the sequence listing. The example section of each patent or patent application cited above in this paragraph is incorporated herein by reference. Non-patent publication Lipinski et al, (1981, ProcNatl. Acad Sci USA, 78:5147-50) discloses anti-Trop-2 antibodies 162-25.3 and 162-46.2.

Many anti-Trop-2 antibodies are known in the art and/or are publicly available. As discussed below, methods of making antibodies against known antigens are routine in the art. Methods of producing humanized, human or chimeric antibodies are also known. One of ordinary skill in the art, reading this disclosure, will be able to obtain and use the properties of the anti-Trop-2 antibodies in the ADCs of the invention.

The use of antibodies against targets associated with Trop-2 for immunotherapeutic agents other than ADCs has been disclosed. Mouse anti-Trop-1 IgG2a antibody Ebenolizumab (edrecolomab)Have been used in therapyColorectal Cancer, although murine antibodies are not suitable for human clinical use (Baeuuerle and Gires, 2007, Br. J Cancer 96: 417-. Low dose subcutaneous administration of epilomab was reported to induce a humoral immune response against vaccine antigens (baeuuerle and Gires, 2007). Adalimumab (adetsumab) (MT201), a fully human anti-Trop-1 antibody, has been used for metastatic breast cancer and early prostate cancer and has been reported to act through ADCC and CDC activities (Baeuerle and Gires, 2007). MT110, a single chain anti-Trop-1/anti-CD 3 bispecific antibody construct, has reported efficacy against ovarian cancer (Baeuerle and Gires, 2007). Proloxinium (Proxinium), an immunotoxin comprising an anti-Trop-1 single-chain antibody fused to Pseudomonas exotoxin, has been tested in head and neck and bladder cancers (Baeuuerle and Gires, 2007). None of these studies contain any disclosure of the use of anti-Trop-2 ADCs, particularly in patients resistant to treatment with checkpoint inhibitor antibodies.

Camptothecin conjugates

Non-limiting methods and compositions for making ADCs comprising camptothecin therapeutic agents linked to an antibody or antigen-binding antibody fragment are described below. In a preferred embodiment, the solubility of the drug is enhanced by placing a defined polyethylene glycol (PEG) moiety (i.e., a PEG containing a defined number of monomeric units) between the drug and the antibody, where the PEG defined is a low molecular weight PEG, preferably containing from 1 to 30 monomeric units, more preferably from 1 to 12 monomeric units.

Preferably, the first linker is attached to the drug at one end and may be terminated with an acetylene or azide group at the other end. The first linker may comprise a defined PEG moiety having an azide or acetylene group at one end and a different reactive group at the other end, such as a carboxylic acid or hydroxyl group. The bifunctional defined PEG may be attached to an amine group of an amino alcohol and the hydroxyl group of the latter may be attached to a hydroxyl group on the drug in the form of a carbonate. Alternatively, the non-azide (or acetylene) moiety of said defined bifunctional PEG is optionally linked to the N-terminus of an L-amino acid or polypeptide, whose C-terminus is linked to the amino group of an amino alcohol, and the hydroxyl group of the latter is linked to the hydroxyl group of the drug, respectively in the form of a carbonate or carbamate.

A second linker comprising an antibody coupling group and a reactive group complementary to the azide (or acetylene) group of the first linker, acetylene (or azide), can be reacted with the drug- (first linker) conjugate by an acetylene-azide cycloaddition reaction to provide a final bifunctional drug product useful for conjugation to a disease-targeting antibody. The antibody coupling group is preferably a thiol or thiol-reactive group.

The following provides a method for selectively regenerating the 10-hydroxy group in the presence of C-20 carbonate in the preparation of drug-linker precursors involving CPT analogs such as SN-38. For reactive hydroxyl groups in drugs such as the phenolic hydroxyl group in SN-38, other protecting groups can also be used, such as t-butyldimethylsilyl or t-butyldiphenylsilyl, and these are deprotected by tetrabutylammonium fluoride prior to attachment of the derivatized drug to the antibody coupling moiety. Alternatively, the 10-hydroxy group of the CPT analog is protected as an ester or carbonate other than "BOC" so that the bifunctional CPT is conjugated to the antibody without prior deprotection of the protecting group. The protecting group is readily deprotected at physiological pH conditions after administration of the bioconjugate.

In acetylene-azide coupling, known as "click chemistry", the azide moiety may be on L2 and the acetylene moiety on L3. Alternatively, L2 may contain acetylene and L3 contains azide. "click chemistry" refers to a copper (+1) catalyzed cycloaddition reaction between an acetylene moiety and an azide moiety (Kolb HC and Sharpless KB, Drug Discov Today 2003; 8:1128-37), although alternative forms of click chemistry are known and can be used. Click chemistry is performed in aqueous solution at near neutral pH conditions and is therefore suitable for drug conjugation. Click chemistry has the advantage that it is chemoselective and complements other well-known conjugation chemistries, such as thiol-maleimide reactions.

Although the present application focuses on the use of antibodies or antibody fragments as targeting moieties, one skilled in the art will recognize that where the conjugate comprises an antibody or antibody fragment, another type of targeting moiety may be substituted, such as an aptamer, avidity multimer, affibody, or peptide ligand.

An exemplary preferred embodiment relates to conjugates of drug derivatives and antibodies of general formula 2,

MAb-[L2]-[L1]-[AA]_m-[A’]-Drug (2)

wherein the MAb is a disease-targeting antibody; l2 is a component of a cross-linker comprising an antibody-coupling moiety and one or more acetylene (or azide) groups; l1 comprises a defined PEG with an azide (or acetylene) at one end, which is complementary to the acetylene (or azide) moiety in L2, and a reactive group such as a carboxylic acid or hydroxyl group at the other end; AA is L-amino acid; m is an integer having a value of 0,1, 2,3 or 4; a' is a further spacer selected from ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol or substituted or unsubstituted ethylenediamine. The L amino acid of 'AA' is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. If the A' group contains a hydroxyl group, it is attached to the hydroxyl group or amino group of the drug in the form of a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A' is ethanolamine derived from the substitution of an L-amino acid wherein the carboxylic acid group of the amino acid is partially substituted with a hydroxymethyl group. A' may be derived from any of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

In the case of the conjugate of the preferred embodiment of formula 2, m is 0, A' is L-valinol, and the drug is exemplified by SN-38. The structure obtained is shown in formula 3.

In another example of the conjugate of the preferred embodiment of formula 2, m is 1 and is represented by derivatized L-lysine, A' is L-valinol, and the drug is exemplified by SN-38. The structure is shown in formula 4.

In this embodiment, an amide bond is first formed between an amino acid, such as the carboxylic acid of lysine, and the amino group of valinol using an orthogonal protecting group for the lysine amino group. The protecting group on the N-terminus of lysine was removed, leaving the protecting group on the lysine side chain intact, and the N-terminus was coupled to the carboxyl group on the PEG defined, and at the other end to the azide (or acetylene). The hydroxyl group of valinol is then attached to the 20-chloroformate derivative of 10-hydroxy protected SN-38 and this intermediate is coupled to an L2 component carrying an antibody binding moiety involved in click cycloaddition chemistry and a complementary acetylene (or azide) group. Finally, the lysine side chain and the protecting group on SN-38 are removed to give the product of this example, as shown in formula 3.

While not wishing to be bound by theory, the small MW SN-38 product produced after intracellular proteolysis, valinol-SN-38 carbonate, has an additional pathway to release intact SN-38 through intramolecular cyclization involving the amino group of valinol and the carbonyl group of the carbonate.

In another preferred embodiment, A' of formula 2 is A-OH, wherein A-OH is a collapsible moiety, such as 4-aminobenzyl alcohol or a C substituted at the benzyl position₁-C₁₀Alkyl radicalA group-substituted 4-aminobenzyl alcohol, and the latter is linked by its amino group to an L-amino acid or a polypeptide comprising up to 4L-amino acid moieties; wherein the N-terminus is linked to a cross-linking agent that terminates in an antibody binding group.

An example of a preferred embodiment is given below, wherein the a-OH embodiment of a ' of formula (2) is derived from a substituted 4-aminobenzyl alcohol and ' AA ' consists of a single L-amino acid, wherein, in formula (2), m ═ 1 and the drug is exemplified by SN-38. This structure is represented by the following formula (formula 5, referred to as MAb-CLX-SN-38). The single amino acid of AA is selected from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The substituent R on the 4-aminobenzyl alcohol moiety (A' in the A-OH embodiment) is hydrogen or an alkyl group selected from C1-C10 alkyl groups.

A particularly preferred embodiment of MAb-CLX-SN-38 of formula 5, wherein the single amino acid AA is L-lysine and R ═ H, and the drug is exemplified by SN-38 (formula 6; designated MAb-CL 2A-SN-38). This structure differs from the linker MAb-CL2-SN-38 in that a single lysine residue replaces the Phe-Lys dipeptide seen in the CL2 linker. The Phe-Lys dipeptide was designed as a cathepsin B cleavage site for lysosomal enzymes, which is thought to be important for intracellular release of bound drugs. Surprisingly, despite the elimination of the cathepsin cleavage site, ADCs comprising CL2A linkers were at least as effective and could be more effective in vivo than those comprising CL2 linkers.

Other embodiments are possible in the context of 10-hydroxy containing camptothecins such as SN-38. In the example of SN-38 as a drug, the more reactive 10-hydroxy group of the drug is derivatized such that the 20-hydroxy group is not affected. In formula 2, a' is a substituted ethylenediamine. An example of this embodiment is represented by the following formula '7', wherein the phenolic hydroxyl group of SN-38 is derivatized as a carbamate with substituted ethylene diamine, wherein another amine of the diamine is derivatized as a carbamate with 4-aminobenzyl alcohol and the amino group of the latter is linked to a Phe-Lys dipeptide. In this structure (formula 7), R and R' are independently hydrogen or methyl. When R ═ R' ═ methyl, it is known as MAb-CL17-SN-38 or MAb-CL 2E-SN-38.

In certain embodiments, the AA comprises a polypeptide moiety, preferably a di-, tri-or tetrapeptide, which is cleavable by an intracellular peptidase. Examples are as follows: Ala-Leu, Leu-Ala-Leu and Ala-Leu-Ala-Leu (SEQ ID NO:20) (Trouet et al, 1982). Another example is the Phe-Lys moiety, which is cleavable by lysosomal cathepsins.

In a preferred embodiment, the L1 component of the conjugate contains a defined polyethylene glycol (PEG) spacer having 1-30 repeating monomer units. In a further preferred embodiment, the PEG is a defined PEG having 1-12 repeating monomer units. The introduction of PEG may include the use of commercially available heterobifunctional PEG derivatives. The heterobifunctional PEG may contain an azide or acetylene group. Examples of heterobifunctional defined PEGs containing 8 repeating monomer units (where 'NHS' is a succinimidyl group) are given in formula 8 below:

in a preferred embodiment, L2 has multiple acetylene (or azide) groups in the range of 2 to 40, but preferably 2 to 20, more preferably 2 to 5, and a single antibody binding moiety.

Representative SN-38 conjugates of antibodies containing multiple drug molecules and a single antibody binding moiety are shown below. The 'L2' component of this structure attaches to 2 acetylene groups, resulting in the attachment of two azido-appended SN-38 molecules. Binding to MAb is expressed as succinimide.

Wherein the R residues are:

in a preferred embodiment, when the bifunctional drug contains a thiol-reactive moiety as the antibody binding group, a thiolating reagent is used to generate a thiol on the antibody at the lysine group of the antibody. Methods for introducing thiol groups onto antibodies by modification of the lysine groups of MAbs are well known in the art (Wong, Chemistry of protein conjugation and cross-linking, CRC Press, Inc., Boca Raton, FL (1991), pages 20-22). Alternatively, a slight reduction of the interchain disulfide bond on an antibody using a reducing agent such as Dithiothreitol (DTT) (Willner et al, bioconjugateCHEm.4:521-527(1993)) can produce 7 to 10 thiols on the antibody; this has the advantage of incorporating multiple drug moieties in the interchain regions of the MAb remote from the antigen binding region. In a more preferred embodiment, SN-38 is linked to a reduced disulfide sulfhydryl group resulting in the formation of an antibody-SN-38 ADC in which 6 SN-38 moieties are covalently attached per antibody molecule. Other methods of providing cysteine residues for attachment of drugs or other therapeutic agents are known, such as the use of cysteine engineered antibodies (see U.S. patent No. 7,521,541, the examples section of which is incorporated herein by reference).

In an alternative preferred embodiment, the chemotherapeutic moiety is selected from the group consisting of Doxorubicin (DOX), epirubicin, morpholino doxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolinyl-doxorubicin (2-PDOX), Pro-2PDOX, CPT, 10-hydroxycamptothecin, SN-38, topotecan, lurtotcan (lurtotecan), 9-aminocamptothecin, 9-nitrocamptothecin, taxane, geldanamycin, ansamycin, and epothilone. In a more preferred embodiment, the chemotherapeutic moiety is SN-38. Preferably, in the conjugates of the preferred embodiments, the antibody is linked to at least one chemotherapeutic moiety; preferably to 1 to about 12 chemotherapeutic moieties; most preferably with from about 6 to about 12 chemotherapeutic moieties.

Additionally, in a preferred embodiment, linker component 'L2' comprises a thiol group that reacts with a thiol-reactive residue introduced at one or more lysine side chain amino groups of the antibody. In such cases, the antibody is pre-derivatized with a thiol-reactive group such as maleimide, vinyl sulfone, bromoacetamide, or iodoacetamide by procedures well described in the art.

In the context of this work, a method has surprisingly been found by which CPT drug-linkers can be prepared, wherein CPT additionally has a 10-hydroxy group. The method includes, but is not limited to, protecting the 10-hydroxy group as a tert-Butoxycarbonyl (BOC) derivative, followed by preparation of the penultimate intermediate of the drug-linker conjugate. Typically, removal of the BOC group requires treatment with a strong acid such as trifluoroacetic acid (TFA). Under these conditions, the CPT 20-O-linker carbonate containing the protecting group to be removed is also susceptible to cleavage, thereby producing unmodified CPT. Indeed, as demonstrated in the art, the rationale for using mild removable methoxytrityl (MMT) protecting groups for the lysine side chains of the linker molecule is just to avoid this possibility (Walker et al, 2002). It was found that the phenolic BOC protecting groups can be selectively removed by carrying out the reaction for a short period of time, optimally 3 to 5 minutes. Under these conditions, the main product is the ` BOC ` with the 10-hydroxyl position removed, while the carbonate in the ` 20 ` position is intact.

An alternative approach involves protecting the 10-hydroxyl position of the CPT analogue with a group other than 'BOC' so that the final product is ready for conjugation to the antibody without the need to deprotect the 10-OH protecting group. The 10-hydroxy protecting group that converts 10-OH to a phenolic carbonate or ester is readily deprotected by esterase either by physiological pH conditions or after administration of the conjugate in vivo. He et al have described that phenolic carbonates in the 10 position are removed more rapidly than versatic esters in the 20 position of 10-hydroxycamptothecin under physiological conditions. (He et al, Bioorganic)&Medicinal Chemistry 12:4003-4008 (2004)). The 10-hydroxy protecting group on SN-38 can be 'COR', where R can be a substituted alkyl, such as "N (CH)₃)₂-(CH₂)_n- ", wherein n is 1 to 10, and wherein the terminal amino group is optionally in the form of a quaternary salt for enhanced water solubility, or a simple alkyl residue, such as" CH₃-(CH₂)_n- ", where n is 0 to 10, or it may be an alkoxy moiety, such as" CH₃-(CH₂) N-O- ", wherein N is 0-10, or" N (CH)₃)₂-(CH₂)_n-O- ", wherein n is 2-10, or" R ")₁O-(CH₂-CH₂-O)_n-CH₂-CH₂-O- ", wherein R₁Is ethyl or methyl and n is an integer having a value of 0 to 10. If the final derivative is a carbonate, these 10-hydroxy derivatives can be readily prepared by treatment with the chloroformate of the selected reagent. Typically, camptothecin containing a 10-hydroxy group, such as S N-38, is treated with a molar equivalent of chloroformate in dimethylformamide using triethylamine as the base. Under these conditions, the 20-OH position is not affected. To form the 10-O-ester, the acid chloride of the selected reagent is used.

In a preferred method of preparing a conjugate of a drug derivative and an antibody of formula 2, wherein the descriptors L2, L1, AA and A-X are as described in the previous section, a bifunctional drug moiety [ L2 ] is first prepared]-[L1]-[AA]_m-[A-X]-a drug, followed by conjugation of a bifunctional drug moiety to an antibody (herein indicated as "MAb").

In a preferred method of preparing a conjugate of a drug derivative and an antibody of general formula 2, wherein the descriptors L2, L1, AA and a-OH are as described in the previous section, the bifunctional drug moiety is prepared by first linking the a-OH to the C-terminus of AA via an amide bond, and then coupling the amine terminus of AA to the carboxylic acid group of L1. If AA is absent (i.e., m ═ 0), a — OH is directly attached to L1 through an amide bond. Cross-linking agent [ L1]-[AA]_m-[A-OH]To the hydroxy or amino group of the drug, and then to the L1 moiety by reaction via click chemistry between the azide (or acetylene) and acetylene (or azide) groups in L1 and L2.

In one embodiment, the antibody is a monoclonal antibody (MAb). In other embodiments, the antibody may be a monovalent and/or multispecific MAb. The antibody may be a murine, chimeric, humanized or human monoclonal antibody, and the antibody may be intact, fragment (Fab, Fab', F (ab))₂、F(ab’)₂) Or a subfragment (single chain construct) form, or is of IgG1, IgG2a, IgG3, IgG4, IgA isotype or a sub-molecule thereof.

In a preferred embodiment, the antibody binds to an antigen or epitope of an antigen, most preferably Trop-2, expressed on cancer or malignant cells. The cancer cell is preferably a cell from a hematopoietic tumor, carcinoma, sarcoma, melanoma, or glioma. Preferred malignant tumors to be treated according to the present invention are malignant solid tumors or hematopoietic tumors.

In a preferred embodiment, the intracellularly cleavable moiety can be cleaved upon internalization into the cell upon binding of the MAb-drug conjugate to its receptor.

Checkpoint inhibitor antibodies

Studies of checkpoint inhibitor antibodies for cancer therapy have resulted in unprecedented response rates in cancers previously thought to be resistant to cancer treatment (see, e.g., Ott and Bhardwaj,2013, Frontiers in immunology 4: 346; Menzies and Long,2013, Ther Adv Med Oncol5: 278-85; pardol, 2012, Nature Reviews12: 252-. Therapy with antagonistic checkpoint blocking antibodies against CTLA-4, PD-1 and PD-L1 is one of the most promising new approaches to immunotherapy of cancer and other diseases. In contrast to most anticancer agents, checkpoint inhibitors do not target tumor cells directly, but rather target lymphocyte receptors or their ligands to enhance the endogenous antitumor activity of the immune system. (pardol, 2012, Nature Reviews12: 252-264) because such antibodies act primarily by modulating the immune response to diseased cells, tissues or pathogens, they can be used in combination with other therapeutic modalities, such as antibody-drug conjugates (ADCs), to enhance the anti-tumor effect of the ADCs.

Programmed cell death protein 1(PD-1, also known as CD279) encodes a cell surface membrane protein of the immunoglobulin superfamily that is expressed in B cells and NK cells (Shinohara et al, 1995, Genomics 23: 704-6; Blank et al, 2007, Cancer immunological Immunol 56: 739-45; Finger et al, 1997, Gene 197: 177-87; Pardol, 2012, Nature Reviews12: 252-. anti-PD 1 antibodies have been used to treat melanoma, non-small cell lung Cancer, bladder Cancer, prostate Cancer, colorectal Cancer, head and neck Cancer, triple negative breast Cancer, leukemia, lymphoma, and renal cell carcinoma (Topalian et al, 2012, N Engl J Med366: 2443-54; Lipson et al, 2013, Clin Cancer Res19: 462-8; Berger et al, 2008, Clin Cancer Res14: 3044-51; Gildener-Leapman et al, 2013, Oral Oncol 49: 1089-96; Menzies and Long,2013, Ther Adv Med Oncol5: 278-85).

Exemplary anti-PD 1 antibodies include palimumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and pidilizumab (CT-011, CURETECH LTD.). The anti-PD 1 antibody can be prepared, for example, from(AB137132)、(EH12.2H7, RMP1-14) and AFFYMETRIXYEBIOSCIENCE (J105, J116, MIH4) are commercially available.

Programmed cell death 1 ligand 1(PD-L1, also known as CD274) is a ligand for PD-1 and is found on activated T cells, B cells, bone marrow cells, and macrophages. Complexes of PD-1 and PD-L1 inhibit proliferation of CD8+ T cells and reduce immune responses (Topalian et al, 2012, N Engl J Med366: 2443-54; Brahmer et al, 2012, N Eng JMed366: 2455-65). anti-PDL 1 antibody has been used to treat non-small cell lung Cancer, melanoma, colorectal Cancer, renal cell carcinoma, pancreatic Cancer, gastric Cancer, ovarian Cancer, breast Cancer, and hematological malignancies (Brahmer et al, N Eng J Med366: 2455-65; Ott et al, 2013, Clin Cancer Res19: 5300-9; Radvanyi et al, 2013, Clin Cancer Res19: 5541; Menzies and Long,2013, Ther Adv Med Oncol5: 278-85; Berger et al, 2008, Clin Cancer Res14: 13044-51).

Exemplary anti-PDL 1 antibodies include MDX-1105 (MERAREX), DOVALUMAb (MEDI4736, MEDIMMUNE), ATTRIBUMAb (MPDL3280A, GENENTECH) and BMS-936559 (BRISTOL-MYERSSQUIBB). anti-PDL 1 antibodies are also commercially available, for example, from AFFYMETRIX EBIOSCIENCE (MIH 1).

Cytotoxic T lymphocyte antigen 4(CTLA-4, also known as CD152) is also a member of the immunoglobulin superfamily that is expressed exclusively on T cells only. CTLA-4 is used to inhibit T cell activation, and has been reported to inhibit helper T cell activity and enhance regulatory T cell immunosuppressive activity (pardol, 2012, Nature Reviews12: 252-. anti-CTL 4A antibodies have been used in clinical trials for the treatment of melanoma, prostate Cancer, small cell lung Cancer, non-small cell lung Cancer (Robert and Ghiringhelli, 2009, Oncoloist 14: 848-61; Ott et al, 2013, Clin Cancer Res19: 5300; Weber,2007, Oncoloist 12: 864-72; Wada et al, 2013, J Transl Med 11: 89).

Exemplary anti-CTLA 4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). The anti-PD 1 antibody can be derived, for example, from(AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H) and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205, MA 1-35914). Ipilimumab recently received FDA approval for the treatment of metastatic melanoma (Wada et al, 2013, J trans Med 11: 89).

These and other known checkpoint inhibitor antibodies can be used in combination with IMMU-132. In preferred embodiments, IMMU-132, alone or in combination, is effective in treating cancer patients refractory to checkpoint inhibitor antibodies alone.

General antibody technology

Techniques for making monoclonal antibodies against virtually any target antigen are well known in the art. For example, seeAnd Milstein, Nature 256:495(1975), and Coligan et al (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, Vol.1, pp.2.5.1-2.6.7 (John Wiley&Sons 1991). Briefly, monoclonal antibodies can be obtained by the following method: injecting a composition comprising an antigen into a mouse, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma culture. One of ordinary skill will recognize that when an antibody is administered to a human subject, the antibody will bind to a human antigen.

Mabs can be isolated and purified from hybridoma cultures by a variety of established techniques. The separation techniques include protein a or protein G agarose affinity chromatography, size exclusion chromatography, and ion exchange chromatography. See, e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. See also Baines et al, "purification of immunoglobulin G (IgG),", METHODS IN murine BIOLOGY, Vol.10, pp.79-104 (the Humana Press, Inc.1992).

After the initial production of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.

The skilled artisan will recognize that the claimed methods and compositions can utilize any of a variety of antibodies known in the art. The antibodies used are commercially available from a variety of known sources. For example, a variety of antibody-secreting hybridoma cell lines are available from the american type culture collection (ATCC, Manassas, VA). A number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited with the ATCC and/or have disclosed variable region sequences and are useful in the claimed methods and compositions. See, e.g., U.S. patent nos. 7,312,318; 7,282,567, respectively; 7,151,164, respectively; 7,074,403, respectively; 7,060,802, respectively; 7,056,509, respectively; 7,049,060, respectively; 7,045,132, respectively; 7,041,803, respectively; 7,041,802, respectively; 7,041,293, respectively; 7,038,018, respectively; 7,037,498, respectively; 7,012,133, respectively; 7,001,598, respectively; 6,998,468, respectively; 6,994,976, respectively; 6,994,852, respectively; 6,989,241, respectively; 6,974,863, respectively; 6,965,018, respectively; 6,964,854, respectively; 6,962,981, respectively; 6,962,813, respectively; 6,956,107, respectively; 6,951,924, respectively; 6,949,244, respectively; 6,946,129, respectively; 6,943,020, respectively; 6,939,547, respectively; 6,921,645, respectively; 6,921,645, respectively; 6,921,533, respectively; 6,919,433, respectively; 6,919,078, respectively; 6,916,475, respectively; 6,905,681, respectively; 6,899,879, respectively; 6,893,625, respectively; 6,887,468, respectively; 6,887,466, respectively; 6,884,594, respectively; 6,881,405, respectively; 6,878,812, respectively; 6,875,580, respectively; 6,872,568, respectively; 6,867,006, respectively; 6,864,062, respectively; 6,861,511, respectively; 6,861,227, respectively; 6,861,226, respectively; 6,838,282, respectively; 6,835,549, respectively; 6,835,370, respectively; 6,824,780, respectively; 6,824,778, respectively; 6,812,206, respectively; 6,793,924, respectively; 6,783,758, respectively; 6,770,450, respectively; 6,767,711, respectively; 6,764,688, respectively; 6,764,681, respectively; 6,764,679, respectively; 6,743,898, respectively; 6,733,981, respectively; 6,730,307, respectively; 6,720,155, respectively; 6,716,966, respectively; 6,709,653, respectively; 6,693,176, respectively; 6,692,908, respectively; 6,689,607, respectively; 6,689,362, respectively; 6,689,355, respectively; 6,682,737, respectively; 6,682,736; 6,682,734, respectively; 6,673,344, respectively; 6,653,104, respectively; 6,652,852, respectively; 6,635,482, respectively; 6,630,144, respectively; 6,610,833, respectively; 6,610,294, respectively; 6,605,441, respectively; 6,605,279, respectively; 6,596,852, respectively; 6,592,868, respectively; 6,576,745, respectively; 6,572, respectively; 856; 6,566,076, respectively; 6,562,618, respectively; 6,545,130, respectively; 6,544,749, respectively; 6,534,058, respectively; 6,528,625, respectively; 6,528,269, respectively; 6,521,227, respectively; 6,518,404, respectively; 6,511,665, respectively; 6,491,915, respectively; 6,488,930, respectively; 6,482,598, respectively; 6,482,408, respectively; 6,479,247, respectively; 6,468,531, respectively; 6,468,529, respectively; 6,465,173, respectively; 6,461,823, respectively; 6,458,356, respectively; 6,455,044, respectively; 6,455,040,6,451,310; 6,444,206, respectively; 6,441,143, respectively; 6,432,404, respectively; 6,432,402, respectively; 6,419,928, respectively; 6,413,726, respectively; 6,406,694, respectively; 6,403,770, respectively; 6,403,091, respectively; 6,395,276, respectively; 6,395,274, respectively; 6,387,350, respectively; 6,383,759, respectively; 6,383,484, respectively; 6,376,654, respectively; 6,372,215, respectively; 6,359,126, respectively; 6,355,481, respectively; 6,355,444, respectively; 6,355,245, respectively; 6,355,244, respectively; 6,346,246, respectively; 6,344,198, respectively; 6,340,571, respectively; 6,340,459, respectively; 6,331,175, respectively; 6,306,393, respectively; 6,254,868, respectively; 6,187,287; 6,183,744, respectively; 6,129,914, respectively; 6,120,767, respectively; 6,096,289, respectively; 6,077,499; 5,922,302, respectively; 5,874,540; 5,814,440, respectively; 5,798,229, respectively; 5,789,554, respectively; 5,776,456; 5,736,119, respectively; 5,716,595, respectively; 5,677,136, respectively; 5,587,459, respectively; 5,443,953, 5,525,338, the example sections of which are each incorporated herein by reference. These are merely exemplary and a variety of other antibodies and hybridomas thereof are known in the art. One skilled in the art will recognize that antibody sequences or antibody-secreting hybridoma cells directed against virtually any disease-associated antigen can be obtained by simply searching the ATCC, NCBI, and/or USPTO databases for antibodies directed against selected disease-associated targets of interest. The antigen binding domain of the cloned antibody may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell, and used for protein production using standard techniques well known in the art. The isolated antibody can be conjugated to a therapeutic agent, such as camptothecin, using the techniques disclosed herein.

Chimeric and humanized antibodies

A chimeric antibody is a recombinant antibody in which the variable regions of a human antibody have been replaced with, for example, the variable regions of a mouse antibody, including the Complementarity Determining Regions (CDRs) of a mouse antibody. Chimeric antibodies exhibit reduced immunogenicity and enhanced stability when administered to a subject. Methods for constructing chimeric antibodies are well known in the art (e.g., Leung et al, 1994, Hybridoma13: 469).

Chimeric monoclonal antibodies can be humanized by transferring the mouse CDRs from the variable heavy and variable light chains of a mouse immunoglobulin into the corresponding variable domains of a human antibody. The mouse Framework Region (FR) in the chimeric monoclonal antibody may also be replaced with a human FR sequence. To maintain the stability and antigen specificity of the humanized monoclonal, one or more human FR residues may be replaced with mouse counterpart residues. The humanized monoclonal antibodies can be used for therapeutic treatment of a subject. Techniques for generating humanized monoclonal antibodies are well known in the art. (see, e.g., Jones et al, 1986, Nature,321: 522; Riechmann et al, Nature,1988,332: 323; Verhoeyen et al, 1988, Science,239: 1534; Carter et al, 1992, Proc. Nat' lAcad. Sci.USA,89: 4285; Sandhu, Crit. Rev. Biotech.,1992,12: 437; Tempest et al, 1991, Biotechnology 9: 266; Singer et al, J.Immun.,1993,150: 2844.)

Other embodiments may relate to non-human primate antibodies. For example, general techniques for generating therapeutically useful antibodies in baboons can be seen in Goldenberg et al, WO 91/11465(1991) and Losman et al, int.J. cancer46:310 (1990). In another embodiment, the antibody can be a human monoclonal antibody. Such antibodies can be obtained from transgenic mice that have been genetically engineered to produce specific human antibodies in response to antigen challenge, as discussed below.

Human antibodies

Methods for producing fully human antibodies using combinatorial approaches or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al, 2004, New Microbiol.27: 315-28; Conrad and Scheller,2005, comb. chem. high through Screen.8: 117-26; Brekke and Loset,2003, curr. Opin. Phamacol.3: 544-50; each incorporated herein by reference). The fully human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and to function as substantially endogenous human antibodies in vivo. In certain embodiments, the claimed methods and procedures may use human antibodies produced by such techniques.

In one alternative, phage display technology can be used to generate human antibodies (e.g., Dantas-Barbosa et al, 2005, Genet. mol. Res.4:126-40, incorporated herein by reference). Human antibodies can be produced by normal humans or by humans exhibiting specific disease states such as cancer (Dantas-Barbosa et al, 2005). The advantage of constructing human antibodies from diseased individuals is that the circulating antibody repertoire may be biased towards antibodies directed against disease-associated antigens.

In one non-limiting example of this approach, Dantas-Barbosa et al (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Generally, total RNA is obtained from circulating blood lymphocytes (supra). Recombinant fabs were cloned from mu, gamma and kappa chain antibody libraries and inserted into phage display libraries (supra). RNA was converted to cDNA and used to prepare Fab cDNA libraries using specific primers for the heavy and light chain immunoglobulin sequences (Marks et al, 1991, J.mol.biol.222:581-97, incorporated herein by reference). Library construction was performed according to Andris-Widhopf et al (2000, phase Display Laboratory Manual, Barbas et al (eds.), 1 st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 9.1 to 9.22, incorporated herein by reference). The final Fab fragments were digested with restriction endonucleases and inserted into the phage genome to make phage display libraries. Such libraries can be screened by standard phage display methods. The skilled artisan will recognize that this technique is merely exemplary and that any known method for making and screening human antibodies or antibody fragments by phage display may be used.

In another alternative, transgenic animals that have been genetically engineered to produce human antibodies can be used to produce antibodies against essentially any immunogenic target using standard immunization protocols as discussed above. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet.7:13(1994), Lonberg et al, Nature368:856(1994), and Taylor et al, int.Immun.6:579 (1994). Non-limiting examples of such systems are available from Abgenix (Fremont, CA)(e.g., Green et al, 1999, J.Immunol. methods 231:11-23, incorporated herein by reference), in which a mouse antibody gene has been inactivated and replaced with a functional human antibody gene, while the remainder of the mouse immune system remains intact.

Transgenic mice were transformed with germline-configured YACs (yeast artificial chromosomes) containing portions of the human IgH and Ig κ loci, including most of the variable region sequences along with accessory genes and regulatory sequences. The repertoire of human variable regions can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. Immunising with target antigensHuman antibodies will be produced by a normal immune response, which can be harvested and/or produced by standard techniques as discussed above. A variety of strains of genetically engineered mice are available, each capable of producing a different class of antibodies. Human antibodies produced transgenically have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al, 1999). The skilled artisan will recognize that the claimed compositions and methods are not limited to useThe system, but can utilize any transgenic animal that has been genetically engineered to produce human antibodies.

Production of antibody fragments

Some embodiments of the claimed methods and/or compositions may relate to antibody fragments. Such antibody fragments may be obtained, for example, by pepsin or papain digestion of intact antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of the antibody with pepsin to provide a 5S fragment, designated F (ab')₂. The fragment can be further cleaved using a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from disulfide cleavage to produce 3.5S Fab' monovalent fragmentsAnd (3) fragment. Alternatively, enzymatic cleavage using pepsin yields two monovalent Fab fragments and one Fc fragment. Exemplary methods for producing antibody fragments are disclosed in U.S. Pat. nos. 4,036,945; U.S. Pat. nos. 4,331,647; nisonoff et al, 1960, arch, biochem, biophysis, 89: 230; porter,1959, biochem.j.,73: 119; edelman et al, 1967, METHODS IN Enzymology, p 422 (Academic Press); and Coligan et al (eds., 1991), Current Protocols IN IMMUNOLOGY, (John Wiley)&Sons).

Other methods of cleaving antibodies, such as isolating heavy chains to form monovalent light-heavy chain fragments, further cleaving fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen recognized by the intact antibody. For example, the Fv fragment comprises V_HAnd V_LAssociation of chains. This association may be non-covalent, as described in Inbar et al, 1972, proc.nat' l.acad.sci.usa,69: 2659. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or crosslinked by chemicals such as glutaraldehyde. See Sandhu,1992, Crit. Rev. Biotech, 12: 437.

Preferably, the Fv fragment comprises V linked by a peptide linker_HAnd V_LAnd (3) a chain. These single-chain antigen binding proteins (scFv) were constructed by construction to contain the coding V_HAnd V_LStructural genes of the DNA sequences of the domains are prepared, which are linked by oligonucleotide linker sequences. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E.coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFv are well known in the art. See Whitlow et al, 1991, Methods: A company to Methods enzymology2: 97; bird et al, 1988, Science,242: 423; U.S. Pat. nos. 4,946,778; pack et al, 1993, Bio/Technology,11: 1271; and Sandhu,1992, crit.rev.biotech, 12: 437.

Another form of antibody fragment is a single domain antibody (dAb), sometimes referred to as a single chain antibody. Techniques for generating single domain antibodies are well known in the art (see, e.g., Cossins et al, Protein expression and purification,2007,51: 253-59; Shuntao et al, Molecunol 2006,43: 1912-19; Tanha et al, J.biol.chem.2001,276: 24774-. Other types of antibody fragments may comprise one or more Complementarity Determining Regions (CDRs). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region of RNA from antibody-producing cells. See Larrick et al, 1991, Methods: A company to Methods in Enzymology2: 106; ritter et al (eds.), 1995, MonoC NAL ANTIBODIES: prediction, ENGINEERING AND CLINICALAPPLICATION, pp 166-179 (Cambridge University Press); birch et al (eds.), 1995, MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pp.137-185 (Wiley-Liss, Inc.)

Antibody variants

In certain embodiments, the sequence of the antibody, such as the Fc portion of the antibody, can be altered to optimize a physiological characteristic of the conjugate, such as half-life in serum. Methods of replacing amino acid sequences in proteins are widely known in the art, such as by site-directed mutagenesis (e.g., Sambrook et al, Molecular Cloning, A laboratory manual, 2 nd edition, 1989). In preferred embodiments, the modification may involve the addition or removal of one or more glycosylation sites in the Fc sequence (e.g., U.S. patent No. 6,254,868, the examples section of which is incorporated herein by reference). In other preferred embodiments, specific amino acid substitutions in the Fc sequence may be made (e.g., Hornick et al, 2000, JNuclMed41: 355-62; Hinton et al, 2006, JImmunol 176: 346-56; Petkova et al, 2006, IntImmunol 18: 1759-69; U.S. Pat. No. 7,217,797, each of which is incorporated herein by reference).

Target antigens and exemplary antibodies

In a preferred embodiment, antibodies are used which recognize and/or bind to antigens which are expressed at high levels on target cells and are predominantly or exclusively expressed on target cellsExpressed on diseased cells but not on normal tissue. More preferably, the antibody is internalized rapidly upon binding. An exemplary rapidly internalizing antibody is the LL1 (anti-CD 74) antibody, with an internalization rate of about 8X 10 per cell per day⁶An antibody molecule (e.g., Hansen et al, 1996, Biochem J.320: 293-300). Thus, a "rapid internalization" antibody may be one that internalizes at a rate of about 1 × 10 for each cell per day⁶To about 1X 10⁷Exemplary antibodies for use in the claimed compositions and methods may include MAb having the properties described above for the treatment of, e.g., cancer, include, but are not limited to, LL or RFB (anti-CD), trastuzumab (hA, anti-CD), rituximab (anti-CD), atozumab (GA101, anti-CD), lanborrelizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS (anti-epithelial glycoprotein-1 (EGP-1, also known as TRT-2)), PAM or KC (all anti-adhesion proteins), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66 or CEACAM), MN-15 or MN-3 (anti-ACAM), Mu-9 (anti-colon-specific antigen-p), Immun 31 (anti-fetoprotein), R (anti-IGF-1R), A (anti-CD), MTP-72, such as anti-ACAABCA, anti-CEMTB, anti-CTAB (anti-CTLA), anti-epithelial cell glycoprotein A-1 (anti-CTLA-VEGF), anti-VEGF-4), anti-TNF-2), anti-epithelial cell glycoprotein-glycoprotein (anti-EGF), anti-VEGF-protein, anti-VEGF-protein, anti-VEGF-TNF-VEGF, anti-TNF-protein, anti-TNF-protein, anti-TNF-protein, anti-TNF-National patent No. 7,282,567), hA20 (U.S. patent No. 7,251,164), hA19 (U.S. patent No. 7,109,304), hmimu-31 (U.S. patent No. 7,300,655), hLL1 (U.S. patent No. 7,312,318), hLL2 (U.S. patent No. 7,074,403), hMu-9 (U.S. patent No. 7,387,773), hL243 (U.S. patent No. 7,612,180), hMN-14 (U.S. patent No. 6,676,924), hMN-15 (U.S. patent No. 7,541,440), hR1 (U.S. patent application 12/772,645), hRS7 (U.S. patent No. 7,238,785), hMN-3 (U.S. patent No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application 11/983,372 deposited as ATCC PTA-4405 and PTA-4406), and D2/B (WO 2009/130575), the text of each of the listed patents or applications is incorporated herein by reference with respect to the drawings and examples section. In a particularly preferred embodiment, the antibody is hRS 7. One of ordinary skill will recognize that in certain embodiments, antibodies directed to TAAs other than Trop-2 may be used in combination with anti-Trop-2 antibodies.

Other useful antigens that can be targeted include carbonic anhydrase IX, B7, CCL19, CCL21, CSAP, HER-2/neu, BrE3, CD1, CD1A, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16 (e.g. C2B 16, hA 16, 1F 16 MAb), CD16, CD40 16, CD16, IFN-16, TNF-P-16, TNF-P-16, TNF-P, TNF-16, TNF-P-16, TNF-P, TNF-induced TNF-16, TNF-receptor, TNF-16, TNF-induced receptor, TNF-induced TNF-16, TNF-receptor, TNF-16, TNF-induced receptor, TNF-induced protein, TNF-induced receptor, TNF-induced protein, TNF-receptor, TNF-induced receptor, TNF-3617, TNF-induced TNF-receptor, TNF-induced TNF-16, TNF-receptor, TNF-16, TNF-receptor, TNF-receptor, TNF-16, TNF-16, TNF-receptor, TNF-16, TNF-36.

A comprehensive analysis of suitable antigen (cluster assignment, or CD) targets on hematopoietic malignant cells as demonstrated by flow cytometry and as a guide for suitable antibodies for selective drug-conjugated immunotherapy is Craig and Foon, Blood, pre-published on line at 1/15 of 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigen consists of five different glycoproteins with similar structures, CD66a-e, which are encoded by the carcinoembryonic antigen (CEA) gene family members BCG, CGM6, NCA, CGM1, and CEA, respectively. These CD66 antigens (e.g., CEACAM6) are expressed primarily in granulocytes, normal epithelial cells of the gut, and tumor cells of various tissues. Also included are Cancer testis antigens as suitable targets for Cancer, such as NY-ESO-1 (Theurilat et al, int.J.cancer 2007; 120(11):2411-7), and CD79a in myeloma leukemia (Kozlov et al, Cancer Genet.cytogene.2005; 163(1):62-7) and B-cell diseases, and CD79B for non-Hodgkin's lymphoma (Poison et al, Blood110(2): 616-. Many of the aforementioned antigens are disclosed in U.S. provisional application No. 60/426,379 entitled "Use of Multi-specific, Non-covalent Compounds for Targeted Delivery of Therapeutics", filed on 15.11.2002. Cancer stem cells, considered to be a more treatment-resistant population of precursor malignant cells (Hill and Perris, J.Natl.cancer Inst.2007; 99:1435-40), with antigens that can target certain Cancer types, such as prostate Cancer (Maitland et al, Ernst Schering Foundation.Sympos.Proc.2006; 5:155-79), non-small cell lung Cancer (Donnenberg et al, J.Control Release 2007; 122(3):385-91), and CD133 in glioblastoma (Beier et al, Cancer Res.2007; 67(9): 4010-5); and colorectal Cancer (Dalerba et al, Proc. Natl. Acad. Sci. USA 2007; 104 (24)) 10158-63), pancreatic Cancer (Li et al, Cancer Res.2007; 67(3):1030-7), and CD44 in head and neck squamous cell carcinoma (Prince et al, Proc. Natl. Acad. Sci. USA 2007; 104 (3)) 973-8). Another useful target for breast cancer therapy is the LIV-1 antigen described by Taylor et al (biochem. J.2003; 375: 51-9). The CD47 antigen is another useful target for Cancer stem cells (see, e.g., Naujokat et al, 2014, Immunotherapy 6: 290-308; Goto et al, 2014, Eur J Cancer50: 1836-46; Unenue, 2013, Proc Natl Acad Sci USA 110: 10886-7).

For multiple myeloma therapies, suitable targeting antibodies have been described for example for CD38 and CD138(Stevenson, Mol Med 2006; 12(11-12): 345-; 346; Tassone et al, Blood 2004; 104(12):3688-96), CD74(Stein et al, supra), CS1(Tai et al, Blood 2008; 112(4):1329-37) and CD40(Tai et al, 2005; cancer Res.65(13): 5898-; 5906).

Macrophage Migration Inhibitory Factor (MIF) is an important regulator of innate and adaptive immunity and apoptosis. CD74 has been reported to be the endogenous receptor for MIF (Leng et al, 2003, J Exp Med 197: 1467-76). The therapeutic effect of antagonist anti-CD 74 antibodies on MIF-mediated intracellular pathways is useful in the treatment of a wide range of disease states, such as cancers of the bladder, prostate, breast, lung, colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al, 2004, BMCCancer 12: 34; Shachar and Haran,2011, Leuk Lymphoma 52: 1446-54); autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (Morand Leech,2005, Front Biosci 10: 12-22; Shachar and Haran,2011, Leuk Lymphoma 52: 1446-54); kidney diseases such as renal allograft rejection (Lan,2008, Nephron expnephrol.109: e 79-83); and many inflammatory diseases (Meyer-Siegler et al, 2009, Mediators inflamepub, 2009, 3 months, 22 days; Takahashi et al, 2009, Respir Res 10: 33; milnacumab (hLL1) is an exemplary anti-CD 74 antibody with therapeutic use for the treatment of MIF-mediated diseases.

anti-TNF- α antibodies are known in the art and can be used to treat immune diseases such as autoimmune diseases, immune dysfunction (e.g., graft-versus-host disease, organ transplant rejection) or Diabetes known antibodies to TNF- α include the human antibody CDP571(Ofei et al, 2011, diabets 45:881-85), the murine antibodies MTNFAI, M2TNFAI, M3TNFABI, M302B and M303(Thermo Scientific, Rockford, IL), infliximab (Centocor, Malvern, PA), trastuzumab (UCB, Brussels, Belgium), and adalimumab (Abbott, Abbott Park, IL), these and many other known anti-TNF- α antibodies can be used in the claimed methods and compositions.

In another preferred embodiment, antibodies that internalize rapidly are used and then are re-expressed, processed and presented on the cell surface, allowing the cell to continuously take up and increase circulating conjugate. An example of the most preferred antibody/antigen pair is LL1, anti-CD 74MAb (invariant chain, class II specific chaperone protein, Ii) (see, e.g., U.S. Pat. No. 3,6,653,104; 7,312,318; the respective examples section is incorporated herein by reference). The CD74 antigen is highly expressed on B-cell lymphomas, including multiple myeloma and leukemias, certain T-cell lymphomas, melanoma, colon, lung and kidney cancers, glioblastoma, and certain other cancers (Ong et al, Immunology 98:296-302 (1999)). A review of the use of CD74 antibody in Cancer is contained in Stein et al, Clin Cancer res.2007, 9/15; 13(18Pt 2):5556s-5563s, which are incorporated herein by reference.

Diseases that are preferably treated with anti-CD 74 antibodies include, but are not limited to, non-hodgkin's lymphoma, hodgkin's disease, melanoma, lung cancer, kidney cancer, colon cancer, glioblastoma multiforme, histiocytoma, myeloid leukemia, and multiple myeloma. CD74 antigen is expressed on the surface of target cells in short succession, then internalizes and re-expresses the antigen, enabling the targeted LL1 antibody to be internalized with any chemotherapeutic moiety it carries. This allows high and therapeutic concentrations of LL 1-chemotherapeutic drug conjugate to accumulate within such cells. Internalized LL 1-chemotherapeutic drug conjugates circulate through the lysosome and endosomes, and the chemotherapeutic moiety is released in the active form within the target cell.

Bispecific and multispecific antibodies

Bispecific antibodies are useful in many biomedical applications. For example, bispecific antibodies with binding sites for tumor cell surface antigens and for T cell surface receptors can direct T cells to lyse specific tumor cells. Bispecific antibodies that recognize the CD3 epitope on gliomas and T cells have been successfully used to treat brain tumors in human patients (Nitta et al, Lancet. 1990; 355: 368-371). Preferred bispecific antibodies are anti-CD 3X anti-CD 19 antibodies. In alternative embodiments, the anti-CD 3 antibody or fragment thereof may be linked to an antibody or fragment directed against another B cell-associated antigen, such as anti-CD 3X anti-Trop-2, anti-CD 3X anti-CD 20, anti-CD 3X anti-CD 22, anti-CD 3X anti-HLA-DR, or anti-CD 3X anti-CD 74. In certain embodiments, the techniques and compositions disclosed herein for ADC therapy may be used in combination with bispecific or multispecific antibodies. For example, anti-Trop-2 x anti-CD 3 bsAb may be administered before, simultaneously with, or after anti-Trop-2 ADC.

Numerous methods for producing bispecific or multispecific antibodies are known, as disclosed, for example, in U.S. Pat. No. 7,405,320, the examples section of which is incorporated herein by reference. Bispecific antibodies can be produced by a quadruple hybridoma method which involves fusing two different hybridomas each producing a monoclonal antibody that recognizes a different antigenic site (Milstein and Cuello, Nature, 1983; 305: 537-540).

Another approach to generate bispecific antibodies is to chemically tether two different monoclonal antibodies using a heterobifunctional crosslinker (Staerz et al, Nature, 1985; 314: 628-631; Perez et al, Nature, 1985; 316: 354-356). Bispecific antibodies can also be generated by reducing each of the two parent monoclonal antibodies to the corresponding half-molecule, then mixing them and allowing reoxidation to obtain a hybrid structure (Staerz and Bevan, Proc Natl Acad Sci US A.1986; 83: 1453-. Another alternative involves chemically cross-linking two or three separately purified Fab' fragments using appropriate linkers. (see, for example, European patent application 0453082).

Other methods include by transferring different selectable marker genes into each parental hybridoma via a retrovirus-derived shuttle vector (DeMonte et al, Proc Natl Acad Sci U S A.1990,87: 2941-2945); or transfection of hybridoma cell lines with expression plasmids containing the heavy and light chain genes of different antibodies to improve the efficiency of hybridoma production.

Can convert the same source V into_HAnd V_LThe domains are joined to a peptide linker of appropriate composition and length (typically consisting of more than 12 amino acid residues) to form a single chain fv (scfv) with binding activity. Methods of making scfvs are disclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405, the respective example sections of which are incorporated herein by reference. Reducing the peptide linker length to less than 12 amino acid residues prevents V on the same chain_HAnd V_LDomain pairing and forcing V_HAnd V_LThe domains pair with complementary domains on other strands, resulting in the formation of functional multimers. V conjugated to a linker between 3 and 12 amino acid residues_HAnd V_LThe polypeptide chains of the domains form mainly dimers (called diabodies). Using a linker between 0 and 2 amino acid residues favors the formation of trimers (called triabodies) and tetramers (called tetrabodies), but in addition to the linker length, the exact mode of oligomerization appears to depend on the composition and V-domain orientation (V-domain)_H-linker-V_LOr V_L-linker-V_H)。

These techniques for producing multispecific or bispecific antibodies present various difficulties in terms of low technical yields, required purification, low stability or high labor costs. More recently, a technique known as "dock and lock" (DNL) has been utilized to produce virtually any desired combination of antibodies, antibody fragments, and other effector molecules (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111; and 8,034,352, the respective examples of which are incorporated herein by reference in their entirety). The technology utilizes complementary protein binding domains, called Anchoring Domains (AD) and Dimerization and Docking Domains (DDD), that bind to each other and allow assembly of complex structures ranging from dimers, trimers, tetramers, pentamers, and hexamers. These form stable complexes in high yield without extensive purification. The DNL technique allows the assembly of monospecific, bispecific or multispecific antibodies. Any technique known in the art for making bispecific or multispecific antibodies may be utilized in practicing the claimed methods of the invention.

In various embodiments, a conjugate as disclosed herein can be part of a composite multispecific antibody. Such antibodies may contain two or more different antigen binding sites with different specificities. Multispecific complexes may bind different epitopes of the same antigen, or may bind two different antigens.

^TMDOCK-AND-LOCK

In a preferred embodiment, the bivalent or multivalent antibody is formed as DOCK-AND-The composite (see, for example,U.S. patent nos. 7,521,056; 7,527,787, respectively; 7,534,866, respectively; 7,550,143, respectively; 7,666,400, respectively; 7,858,070, respectively; 7,871,622, respectively; 7,906,121, respectively; 7,906,118, respectively; 8,163,291, respectively; 7,901,680, respectively; 7,981,398, respectively; 8,003,111 and 8,034,352, the respective examples of which are incorporated herein by reference. ) Generally, the technology exploits the specific and high affinity binding interactions that occur between the Dimerization and Docking Domain (DDD) sequences of the regulatory (R) subunits of cAMP-dependent Protein Kinases (PKA) and the Anchoring Domain (AD) sequences derived from any of a variety of AKAP proteins (bailie et al, febsletters.2005; 579:3264. Wong and Scott, nat. rev. mol. cell biol.2004; 5:959). The DDD and AD peptides can be linked to any protein, peptide, or other molecule. Because DDD sequences spontaneously dimerize and bind to AD sequences, this technique allows for the formation of complexes between any selected molecule that can be linked to either DDD or AD sequences.

Albeit standardThe complex comprises trimers with two DDD linker molecules linked to one AD linker molecule, but variations of the complex structure allow for the formation of dimers, trimers, tetramers, pentamers, hexamers and other multimers. In some embodiments of the present invention, the substrate is,a complex may comprise two or more antibodies, antibody fragments or fusion proteins that bind to the same antigenic determinant or two or more different antigens.The complex may also comprise one or more other effectors, such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases, e.g., anti-tumor ribonucleases, inhibitory oligonucleotides, e.g., siRNA, antigens or xenoantigens, polymers, e.g., PEG, enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, or any other molecule or aggregateAnd (3) a body.

In 1968, PKA was first isolated from rabbit skeletal muscle, which plays a key role in one of the most thoroughly studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunit (Walsh et al, J.biol.chem.1968; 243: 3763). The structure of the holoenzyme consists of two catalytic subunits that are maintained in an inactive form by the R subunit (Taylor, J.biol.chem.1989; 264: 8443). The isoenzymes of PKA were found to have two types of R subunits (RI and RII), and each type hasAndisoform (Scott, Pharmacol. Ther.1991; 50: 123). Thus, the four isoforms of the PKA regulatory subunit areAndthe R subunit only separates as a stable dimer and shows that the dimerization domain consists of the first 44 amino terminal residues of RII α (Newson et al, nat. struct.biol.1999, 6222.) As discussed below, analogous portions of the amino acid sequences of other regulatory subunits are involved in dimerization and docking, which are located near the N-terminus of the regulatory subunits, respectively

Over 50 AKAP with diverse structures, localized to various subcellular sites including plasma membrane, actin cytoskeleton, nucleus, mitochondria and endoplasmic reticulum, have been identified since the first AKAP microtubule-associated protein-2 was characterized in 1984 (Lohmann et al, Proc. Natl. Acad. Sci. USA.1984; 81:6723), in species ranging from yeast to humans (Wong and Scott, nat. Rev. mol. CellBiol.2004; 5):959). The AD used in AKAP for PKA is an amphipathic helix with 14 to 18 residues (Carr et al, J.biol.chem.1991; 266: 14188). The amino acid sequence of AD varies quite between individual AKAPs, with reported binding affinities for RII dimers ranging from 2nM to 90nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003; 100: 4445). AKAP binds only the dimeric R subunit. For humanIn other words, AD binds to a hydrophobic surface formed by 23 amino-terminal residues (Collidge and Scott, trends cell biol.1999; 6: 216). Thus, a personBoth the dimerization domain and the AKAP binding domain of (A) are located within the same N-terminal 44 amino acid sequence (Newlon et al, nat. struct. biol. 1999; 6: 222; Newlon et al, EMBO J. 2001; 20:1651), which is referred to herein as DDD.

We have developed a platform technology that utilizes DDD of the human PKA regulatory subunit and AD of AKAP as a pair of superior linker modules for docking any two entities (hereinafter referred to as A and B) into a non-covalent complex that can be further locked into DNL by introducing cysteine residues in critical positions of DDD and AD to facilitate disulfide bond formation^TMAnd (c) a complex. The general approach for this approach is as follows. Entity a is constructed by linking the DDD sequence to a precursor of a, resulting in a first component referred to hereinafter as a. Since the DDD sequence will effect the spontaneous formation of dimers, A will be represented by a₂And (4) forming. Entity B is constructed by ligating the AD sequence to a precursor of B, resulting in a second component hereinafter referred to as B. a is₂The dimerization motif of DDD contained in (a) will create a docking site for binding to the AD sequence contained in (b), thereby promoting a₂Easily associate with b to form a₂b is a binary trimeric complex. This binding event is irreversible by means of a subsequent reaction of covalent immobilization of the two entities via disulfide bridges, which reaction takes place extremely efficiently on the basis of the principle of effective local concentration, since the initial binding interaction should take placeReactive thiol groups disposed on both DDD and AD are approached (Chmura et al, proc.natl.acad.sci.usa.2001; 98:8480) for site-specific ligation. By using various combinations of linkers, adapter modules, and precursors, a wide variety of DNLs of different stoichiometry can be generated and used^TMConstructs (see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400).

Linking DDD and AD by functional groups remote from the two precursors, it is also expected that such site-specific linking retains the original activity of the two precursors. This approach is modular in nature and potentially applicable to site-specifically and covalently linking a variety of substances, including peptides, proteins, antibodies, antibody fragments, and other effector moieties with a variety of activities. Almost any protein or peptide can be incorporated using the method of constructing fusion proteins that conjugate the effectors of AD and DDD described in the examples belowIn the construct. However, this technique is not limiting and other conjugation methods may be used.

Various methods are known for preparing fusion proteins, including nucleic acid synthesis, hybridization, and/or amplification to produce synthetic double-stranded nucleic acids encoding the fusion proteins of interest. Such double-stranded nucleic acids can be inserted into expression vectors for the production of fusion proteins by standard Molecular biology techniques (see, e.g., Sambrook et al, Molecular Cloning, A laboratory and, 2 nd edition, 1989). In such preferred embodiments, the AD and/or DDD moieties may be linked to the N-terminus or C-terminus of the effector protein or peptide. However, the skilled artisan will recognize that the site at which the AD or DDD moiety is attached to the effector moiety may vary depending on the chemical nature of the effector moiety and the moiety in the effector moiety that is involved in its physiological activity. Site-specific attachment of various effector moieties can be performed using techniques known in the art, such as using divalent crosslinking reagents and/or other chemical conjugation techniques.

In various embodimentsThe antibody or antibody fragment may be incorporated into the DNL by, for example, attaching a DDD or AD moiety to the C-terminal portion of the antibody heavy chain^TMIn the complex, as described in detail below. In a more preferred embodiment, a DDD or AD moiety, more preferably an AD moiety, can be attached to the C-terminus of the light chain of an antibody (see, e.g., U.S. patent application Ser. No. 13/901,737 filed on 24.5.13.5.9, the examples section of which is incorporated herein by reference)

Structural functional relationships in AD and DDD moieties

For different types of DNL^TMConstructs, different AD or DDD sequences can be used. Exemplary DDD and AD sequences are provided below.

The skilled artisan will recognize that DDD1 and DDD2 are protein kinase A-based humansDDD sequence of isotype. However, in alternative embodiments, the DDD and AD moieties may be protein kinase A-based humansDDD sequences and corresponding AKAP sequences in the form as exemplified in DDD3, DDD3C, and AD3 below.

In other alternative embodiments, inFor example, there are only 4 variants of the human PKA DDD sequence, corresponding to the DDD portions of PKA RI α, RII α, RI β and RII β. the RII α DDD sequenceColumns are the basis for the above disclosed DDD1 and DDD2 four human PKA DDD sequences are shown below the DDD sequence represents residues 1-44 of RII α, residues 1-44 of RII β, residues 12-61 of RI α and residues 13-66 of RI β (note that the sequence of DDD1 is slightly altered compared to the human PKA RII α DDD moiety.)

The structural functional relationship of the AD and DDD domains has been studied. (see, e.g., Burns-Hamuro et al, 2005, Protein Sci 14: 2982-92; Carr et al, 2001, JBiol Chem 276: 17332-38; Alto et al, 2003, Proc Natl Acad Sci USA100: 4445-50; Hundsrucker et al, 2006, Biochem J396: 297: 306; Stokka et al, 2006, Biochem J400: 493-99; Gold et al, 2006, Mol Cell24: 383-95; Kinderman et al, 2006, Mol Cell24: 397-408, the entire text of each of which is incorporated herein by reference.)

Allotype of antibody

The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and a decreased duration of therapeutic response (Baert et al, 2003, NEnglJMed 348: 602-08). The extent to which a therapeutic antibody induces an immune response in a host can be determined, in part, by the allotype of the antibody (Stickler et al, 2011, Genes and immune 12: 213-21). Antibody allotypes are associated with amino acid sequence variants at specific positions in the constant region sequence of an antibody. The allotype of IgG antibodies containing heavy chain gamma-type constant regions was designated the Gm allotype (1976, J Immunol 117: 1056-59).

The most common allotype for the common IgG1 human antibody is G1m1(Stickler et al, 2011, Genes and similarity 12: 213-21). However, the G1m3 allotype is also frequently present in Caucasians (supra). It has been reported that when administered to non-G1 m1(nG1m1) recipients such as G1m3 patients, the G1m1 antibody contains an allotypic sequence (supra) that tends to induce an immune response. non-G1 m1 alloantibodies were not as immunogenic when administered to G1m1 patients (supra).

The human G1m1 allotype comprises the amino acid aspartic acid at Kabat position 356 and leucine at Kabat position 358 in the CH3 sequence of heavy chain IgG 1. The nG1m1 allotype comprises the amino acid glutamic acid at Kabat position 356 and methionine at Kabat position 358. Both G1m1 and nG1m1 allotypes contain a glutamic acid residue at Kabat position 357, and these allotypes are sometimes referred to as DEL and EEM allotypes. Non-limiting examples of heavy chain constant region sequences for the G1m1 and nG1m1 allotypes are shown for the exemplary antibodies rituximab (SEQ ID NO:18) and veltuzumab (SEQ ID NO: 19).

Rituxib single-antibody heavy chain variable region sequence (SEQ ID NO:18)

Vituzumab heavy chain variable region (SEQ ID NO:19)

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variants that are characteristic of IgG allotypes and their effect on immunogenicity. They reported that the G1m3 allotype was characterized by an arginine residue at Kabat position 214, compared to the lysine residue at Kabat 214 in the G1m17 allotype. The nG1m1,2 allotype is characterized by a glutamic acid at Kabat position 356, a methionine at Kabat position 358, and an alanine at Kabat position 431. The G1m1,2 allotype is characterized by an aspartic acid at Kabat position 356, a leucine at Kabat position 358 and a glycine at Kabat position 431. In addition to the heavy chain constant region sequence variants, Jefferis and Lefranc (2009) report allotypic variants in the kappa light chain constant region, wherein the Km1 allotype is characterized by a valine at Kabat position 153 and a leucine at Kabat position 191, the Km1,2 allotype is characterized by an alanine at Kabat position 153 and a leucine at Kabat position 191, and the Km3 allotype is characterized by an alanine at Kabat position 153 and a valine at Kabat position 191.

For therapeutic antibodies, veltuzumab and rituximab are humanized and chimeric IgG1 antibodies, respectively, against CD20 that are useful for treating a variety of hematological malignancies. Table 1 compares the allotypic sequences of rituximab and veltuzumab. As shown in table 1, rituximab (G1m17,1) is a DEL allotype IgG1 with additional sequence variation of lysine in rituximab versus arginine in veltuzumab at Kabat position 214 (heavy chain CH 1). It has been reported that veltuzumab is less immunogenic than rituximab in a subject (see, e.g., Morchhauser et al, 2009, JClin Oncol 27: 3346-53; golden enberg et al, 2009, Blood 113: 1062-70; Robak and Robak,2011, BioDrugs 25:13-25), and this effect has been attributed to the difference between humanized and chimeric antibodies. However, the allotypic differences between EEM and DEL allotypes may also explain the lower immunogenicity of veltuzumab.

TABLE 1 allotype of rituximab versus veltuzumab

To reduce the immunogenicity of a therapeutic antibody in an individual of nG1m1 genotype, it is desirable to select an allotype of the antibody that corresponds to the G1m3 allotype, characterized by an arginine on Kabat 214; and nG1m1,2 null allotypes characterized by a glutamic acid at Kabat position 356, a methionine at Kabat position 358, and an alanine at Kabat position 431. Surprisingly, it was found that repeated subcutaneous administration of the G1m3 antibody did not result in a significant immune response over a long period of time. In an alternative embodiment, the human IgG4 heavy chain, which is isotypic to G1m3, has an arginine on Kabat 214, a glutamic acid on Kabat 356, a methionine on Kabat 359, and an alanine on Kabat 431. Since immunogenicity appears to be at least partially related to the residues at those positions, the use of the human IgG4 heavy chain constant region sequence for therapeutic antibodies is also a preferred embodiment. The combination of the G1m3 IgG1 antibody and the IgG4 antibody may also be useful for therapeutic administration.

High affinity multimers

In certain embodiments, the binding moieties described herein can comprise one or more high affinity multimeric sequences. High affinity multimers are a class of binding proteins that are somewhat similar to antibodies in their affinity and specificity for various target molecules. They were developed from human extracellular receptor domains by in vitro exon shuffling and phage display. (Silverman et al, 2005, nat. Biotechnol.23: 1493-94; Silverman et al, 2006, nat. Biotechnol.24: 220). The resulting multidomain protein may comprise a plurality of independent binding domains that may exhibit improved affinity (in some cases, sub-nanomolar) and specificity compared to a single epitope binding protein. In various embodiments (supra), high affinity multimers can be linked to, for example, DDD and/or AD sequences for use in the claimed methods and compositions. Additional details regarding methods of constructing and using high affinity multimers are disclosed, for example, in U.S. patent application publication nos. 20040175756, 20050048512, 20050053973, 20050089932, and 20050221384, the respective examples sections of which are incorporated herein by reference.

Phage display

Certain embodiments of the claimed compositions and/or methods can relate to binding peptides and/or peptidomimetics of various target molecules, cells, or tissues. Binding peptides can be identified by any method known in the art including, but not limited to, phage display technology. Various methods of phage display and techniques for generating various peptide populations are well known in the art. For example, U.S. Pat. nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods of making phage libraries. Phage display technology involves the genetic manipulation of phages so that small peptides can be expressed on their surface (Smith and Scott, 1985, Science 228: 1315-; 1317; Smith and Scott,1993, meth.enzymol.21: 228-; 257). In addition to peptides, larger protein domains such as single chain antibodies can also be displayed on the surface of phage particles (Arap et al, 1998, Science 279: 377-380).

Targeting amino acid sequences that are selective for a given organ, tissue, cell type or target molecule can be isolated by panning (Pasqualini and Ruoslahti, 1996, Nature380: 364-. Briefly, a phage library containing putative targeting peptides is administered to an intact organism or to an isolated organ, tissue, cell type, or target molecule, and a sample containing bound phage is collected. Phage that bind the target can be eluted from the target organ, tissue, cell type, or target molecule and then amplified by growing it in the host bacteria.

In certain embodiments, the phage can be propagated in the host bacteria between rounds of panning. Instead of by phage lysis, the bacteria may secrete multiple phage copies displaying a particular insert. If desired, the amplified phage may be re-exposed to the target organ, tissue, cell type or target molecule and collected for additional rounds of panning. Multiple rounds of panning may be performed until a population of selective or specific binders is obtained. The amino acid sequence of the peptide can be determined by sequencing the DNA corresponding to the targeted peptide insert in the phage genome. The identified targeting peptides can then be generated as synthetic peptides by standard protein chemistry techniques (Arap et al, 1998, Smith et al, 1985).

In some embodiments, a subtractive approach may be used to further reduce background phage binding. The purpose of the subtraction is to remove from the library phage that bind to targets other than the target of interest. In an alternative embodiment, the phage library can be pre-screened against a control cell, tissue or organ. For example, tumor binding peptides can be identified after pre-screening the library against control normal cell lines. After depletion, the library may be screened against the molecule, cell, tissue or organ of interest. Other methods of subtractive schemes are known and can be used in the practice of the claimed methods, such as disclosed in U.S. patents 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, the targeting moiety used may be an aptamer. Methods for constructing and determining binding characteristics of aptamers are well known in the art. Such techniques are described, for example, in U.S. patent nos. 5,582,981, 5,595,877, and 5,637,459, the respective examples sections of which are incorporated herein by reference. Methods of making and screening for aptamers that bind to a particular target of interest are well known, for example, U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163, the respective examples sections of which are incorporated herein by reference.

Aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific for the same target. Generally, a minimum of about 3 nucleotides, preferably at least 5 nucleotides, is necessary to achieve specific binding. Aptamers of sequences shorter than 10 bases may be feasible, although aptamers of 10, 20, 30 or 40 nucleotides may be preferred.

Aptamers can be isolated, sequenced and/or amplified or synthesized as conventional DNA or RNA molecules. Alternatively, the aptamer of interest may comprise a modified oligomer. Any hydroxyl group typically present in aptamers can be replaced by, for example, phosphonate, phosphate, protected with standard protecting groups, or activated to make additional linkages to other nucleotides, or can be conjugated to a solid support. One or more phosphodiester bonds may be replaced by alternative linking groups, for example P (O) O by P (O) S, P (O) NR₂P (O) R, P (O) OR', CO OR CNR₂A substitution wherein R is H or alkyl (1-20C) and R' is alkyl (1-20C); in addition, the group may be linked to an adjacent nucleotide through O or S. Not all of the oligomersThe linkages must all be the same.

Affibody and Fynomer

Affinity bodies are commercially available from Affibody AB (Solna, Sweden.) the affinity bodies are small proteins that function as antibody mimetics and are used to bind target molecules are developed by combinatorial genetic engineering on the α helix Protein scaffold (Nord et al, 1995, Protein Eng 8: 601-8; Nord et al, 1997, Nat Biotechnol 15: 772-77.) the affinity body design is based on a triple helix bundle structure comprising the IgG binding domain of Protein A (Nord et al, 1995; 1997). after randomizing the thirteen amino acids involved in the Fc binding activity of bacterial Protein A, affinity bodies with broad binding affinity can be generated (Nord et al, 1995; 1997). after which the PCR-amplified library is cloned into a phagemid vector for screening against any known phage display antigen using standard phage display techniques (e.g.phage display screening against phage display libraries; e.g.expressing phage display, 89: Natquarl, 364. and Nat.159. for target antigens, Nat.159. 92. Nat. 92. Nat Biotechnol. 159. 1997).

Has proven to be specific for HER2/neu¹⁷⁷Lu tag affibodies target HER2 expressing xenografts in vivo (Tolmachev et al, 2007, Cancer Res67: 2773-82). Although nephrotoxicity due to accumulation of low molecular weight radiolabeled compounds was initially a problem, reversible binding to albumin reduces renal accumulation, enabling radionuclide-based therapy with labeled affibodies (supra).

The feasibility of using radiolabeled affibodies for in vivo tumor imaging has recently been demonstrated (Tolmachev et al, 2011, Bioconjugate Chem 22: 894-902). Conjugation of maleimide-derived NOTA to anti-HER 2 affibodies and use¹¹¹In was radiolabeled (supra). Administration to mice bearing HER2 expressing DU-145 xenografts followed by gamma camera imaging allowing visualization of the xenograftsAnd (same as above).

Fynomer may also bind to the target antigen with similar affinity and specificity as the antibody. Fynomer is based on the human FynSH3 domain as a scaffold for assembling binding molecules. The Fyn SH3 domain is a fully human 63 amino acid protein that can be produced in high yields in bacteria. Fynomers can be linked together to produce a multispecific binding protein having affinity for two or more different antigen targets. Fynomer is commercially available from COVAGEN AG (Zurich, Switzerland).

The skilled artisan will recognize that affibodies or fynomers may be used as targeting molecules in the practice of the claimed methods and compositions.

Immunoconjugates

In certain embodiments, a cytotoxic drug or other therapeutic or diagnostic agent may be covalently linked to an antibody or antibody fragment to form an immunoconjugate. In some embodiments, a drug or other agent may be linked to the antibody or fragment thereof via a carrier moiety. The carrier moiety may be attached to, for example, a reduced SH group and/or a carbohydrate side chain. The carrier moiety may be linked to the hinge region of the reduced antibody component via disulfide bond formation. Alternatively, such reagents may be linked using heterobifunctional crosslinkers such as N-succinyl 3- (2-pyridyldithio) propionate (SPDP). Yu et al, int.J. cancer 56:244 (1994). General techniques for such conjugation are well known in the art. See, e.g., Wong, CHEMISTRY OFPROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); updalacis et al, "Modification of Antibodies by Chemical Methods,", MONOCLONAL ANTIBODIES: PRINCIPLES AND application, Birch et al (eds.), page 187-230 (Wiley-Liss, Inc.1995); price, "Production AND Characterization of Synthetic Peptide-derived nucleotides,", MonoC nal ANTIBODIES: Production, ENGINEERING AND CLINICALAPPLICATION, Ritter et al (eds.), pages 60-84 (Cambridge University Press 1995). Alternatively, the carrier moiety may be conjugated via a carbohydrate moiety in the Fc region of the antibody.

Methods for conjugating functional groups to antibodies via the carbohydrate moiety of the antibody are well known to those skilled in the art. See, e.g., Shih et al, int.J. cancer 41:832 (1988); shih et al, int.J.cancer46: 1101 (1990); and Shih et al, U.S. Pat. No. 5,057,313, the examples section of which is incorporated herein by reference. The general method involves reacting an antibody having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine functional group. This reaction produces an initial schiff base (Schiffbase) (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

If the antibody component of the ADC is an antibody fragment, the Fc region may not be present. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full-length antibody or antibody fragment. See, e.g., Leung et al, J.Immunol.154:5919 (1995); U.S. patent nos. 5,443,953 and 6,254,868, examples of which are incorporated herein by reference in their entirety. Engineered carbohydrate moieties are used to link therapeutic or diagnostic agents.

An alternative method of linking the carrier moiety to the targeting molecule involves the use of click chemistry reactions. Click chemistry was originally conceived as a method for rapidly producing complex substances by joining small subunits together in a modular fashion. (see, e.g., Kolb et al, 2004, Angew Chem Int Ed40: 3004-31; Evans,2007, Aust J Chem 60: 384-95.) various forms of click chemistry reactions are known in the art, such as the Huisgen (Huisgen)1, 3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al, 2002, J Organic Chem 67:3057-64), which is commonly referred to as a "click reaction". Other alternatives include cycloaddition reactions such as Diels-Alder reactions (Diels-Alder), nucleophilic substitution reactions (especially for small strained rings such as epoxy and aziridine compounds), carbonyl chemistry formation reactions of urea compounds, and reactions involving alkynes in carbon-carbon double bonds such as mercapto-alkyne reactions.

The azide alkyne huisis root cycloaddition reaction catalyzes the reaction of the terminal alkyne group attached to the first molecule using a copper catalyst in the presence of a reducing agent. In the presence of a second molecule comprising an azide moiety, the azide reacts with the activated alkyne to form a1, 4-disubstituted 1,2, 3-triazole. The copper-catalyzed reaction is carried out at room temperature and is sufficiently specific that purification of the reaction product is generally not required. (Rostovstev et al, 2002, Angew Chem IntEd 41: 2596; Tornoe et al, 2002, J Org Chem 67: 3057.) the azide and alkyne functionalities are substantially inert to biomolecules in aqueous media, allowing the reaction to proceed in complex solutions. The triazoles formed are chemically stable and do not undergo enzymatic cleavage, making the click chemistry products highly stable in biological systems. Although copper catalysts are toxic to living cells, copper-based click chemistry reactions can be used in vitro to form ADCs.

Copper-free click reactions have been proposed for covalent modification of biomolecules. (see, e.g., Agard et al, 2004, JAm Chem Soc126: 15046-47.) the copper-free reaction uses ring strain instead of a copper catalyst to promote the [3+2] azide-alkyne cycloaddition reaction (supra). For example, cyclooctyne is an 8-carbocyclic ring structure comprising an internal alkyne bond. The closed ring structure induces substantial bond angle distortion of the acetylene, which reacts readily with azide groups to form triazoles. Thus, cyclooctyne derivatives can be used for copper-free click reactions (supra).

Ning et al reported another copper-free click reaction. (2010, Angew Chem Int Ed 49:3065-68), involving a tension-promoted alkyne-nitrone cycloaddition. To solve the problem of the slow reaction rate of the original cyclooctyne, an electron withdrawing group is adjacent to the triple bond (supra). Examples of such substituted cyclooctynes include difluorinated cyclooctyne, 4-dibenzocyclooctyne alcohol, and azacyclooctyne (supra). An alternative copper-free reaction involves a tension-promoted alkyne-nitrone cycloaddition to give an N-alkylated isoxazoline (supra). This reaction is reported to have particularly fast reaction kinetics and is used in a one-pot three-step protocol for site-specific modification of peptides and proteins (supra). Nitrones are prepared by condensation of the appropriate aldehyde with N-methylhydroxylamine, and the cycloaddition reaction is carried out in a mixture of acetonitrile and water (supra). These and other known click chemistry reactions can be used to attach carrier moieties to antibodies in vitro.

Agard et al (2004, J Am Chem Soc126:15046-47) demonstrated that recombinant glycoproteins expressed in CHO cells resulted in the bioconjugation of the corresponding N-azidoacetylsialic acid into the carbohydrate of the glycoprotein in the presence of fully acetylated N-azidoacetylmannosamine. The azido-derivatized glycoprotein reacts specifically with biotinylated cyclooctyne to form a biotinylated glycoprotein, while the control glycoprotein without the azido moiety remains unlabeled (supra). Lamghlin et al (2008, Science 320: 664-. The azido-derivatized glycans were reacted with a Difluorocyclooctyne (DIFO) reagent, allowing for in vivo visualization of the glycans.

Diels-Alder reactions have also been used to label molecules in vivo. Rossin et al (2010, Angew Chemint Ed 49:3375-78) reported tumor-localized anti-TAG 72(CC49) antibodies carrying a trans-cyclooctene (TCO) reactive moiety in combination with¹¹¹Yield between In-labeled tetrazine DOTA derivatives was 52% In vivo. TCO-labeled CC49 antibody was administered to mice bearing colon cancer xenografts, followed by injection 1 day later¹¹¹In-labeled tetrazine probes (supra). The reaction of the radiolabeled probe with tumor-localized antibodies resulted in significant radioactive localization in the tumor, as demonstrated by SPECT imaging of live mice three hours after injection of the radiolabeled probe, with a tumor to muscle ratio of 13:1 (supra). The results confirm the in vivo chemical reaction of TCO with tetrazine labeled molecules.

Bioincorporation of antibody labeling techniques using a labeling moiety are further disclosed in U.S. patent No. 6,953,675 (the examples section of which is incorporated herein by reference). Such "landscaped" antibodies are prepared to have reactive ketone groups at the glycosylation sites. The method involves an expression cell transfected with an expression vector encoding an antibody having one or more N-glycosylation sites in the CH1 or vk domain in a medium comprising a ketone derivative of a sugar or sugar precursor. The ketone derivatized saccharides or precursors include N-levulinyl mannosamine and N-levulinyl fucose. The beautified antibody is then reacted with a reagent comprising a ketone-reactive moiety, such as a hydrazide group, a hydrazine group, an hydroxylamine group, or a thiosemicarbazide group, to form a labeled targeting molecule. Exemplary agents for attachment to the beautified antibody include chelating agents, such as DTPA; large drug molecules, such as doxorubicin-dextran; and acyl-hydrazide containing peptides. Beautification techniques are not limited to the generation of antibodies comprising ketone moieties, but may alternatively be used to introduce click chemistry reactive groups such as nitrones, azides, or cyclooctynes onto antibodies or other biomolecules.

Modification of click chemistry reactions is suitable for in vitro or in vivo use. The reactive targeting molecule may be formed by chemical conjugation or by biological incorporation. Targeting molecules, such as antibodies or antibody fragments, can be activated with an azido moiety, a substituted cyclooctyne or alkyne group, or a nitrone moiety. Where the targeting molecule comprises an azido or nitrone group, the corresponding targetable construct will comprise a substituted cyclooctyne or alkyne group, or vice versa. As discussed above, such activated molecules can be made by metabolic incorporation in living cells.

Alternatively, methods of chemically conjugating such moieties to biomolecules are well known in the art, and any such known method may be used. General methods of ADC formation are disclosed, for example, in U.S. patent nos. 4,699,784; 4,824,659; 5,525,338, respectively; 5,677,427, respectively; 5,697,902, respectively; 5,716,595, respectively; 6,071,490, respectively; 6,187,284, respectively; 6,306,393, respectively; 6,548,275, respectively; 6,653,104, respectively; 6,962,702, respectively; 7,033,572, respectively; 7,147,856, respectively; and 7,259,240, the respective examples section of which is incorporated herein by reference.

Preferred conjugation schemes are based on thiol-maleimide, thiol-vinylsulfone, thiol-bromoacetamide or thiol-iodoacetamide reactions that are readily carried out at neutral or acidic pH. This eliminates the need for higher pH conditions for conjugation, which would be necessary, for example, when using active esters. Additional details of exemplary conjugation schemes are described below in the examples section.

Therapeutic treatment

In another aspect, the invention relates to a method of treating a subject comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate (ADC) as described herein, such as IMMU-132. Preferably, the subject has a Trop-2 positive cancer that is resistant to treatment with the checkpoint inhibitor antibody. The ADC may be administered once or repeatedly depending on the disease state and tolerance of the conjugate, and may also be used optimally in combination with other therapeutic modalities such as surgery, external radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy, interfering RNA therapy, gene therapy, and the like. Each combination will be appropriate for the tumor type, stage, patient condition and prior therapy, and other factors considered by the administering physician.

As used herein, the term "subject" refers to any animal (i.e., vertebrates and invertebrates), including but not limited to mammals, including humans. The term is not intended to be limited to a particular age or gender. Thus, adult and newborn subjects, as well as fetuses, whether male (male) or female (female), are encompassed by this term. The dosages given herein are for humans, but may be adjusted to the size of other mammals, as well as children, depending on body weight or square meter size.

In a preferred embodiment, therapeutic conjugates comprising anti-TROP-2 antibodies, such as hRS7MAb, can be used to treat carcinomas, such as esophageal, pancreatic, lung, gastric, colorectal, bladder, breast, ovarian, uterine, renal, and prostate cancers, as disclosed in U.S. patent nos. 7,238,785, 7,517,964, and 8,084,583, the examples of which are incorporated herein by reference in their entirety. The hRS7 antibody is a humanized antibody comprising the light chain Complementarity Determining Region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:1), CDR2(SASYRYT, SEQ ID NO:2) and CDR3(QQHYITPLT, SEQ ID NO:3), and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO: 6).

In a preferred embodiment, the antibody for use in the treatment of a human disease is a human or humanized (CDR grafted) version of the antibody; although murine and chimeric versions of the antibody may also be used. IgG molecules of the same species as the delivery agent are most preferred for minimizing immune responses. This is particularly important when considering repeated treatments. For humans, human or humanized IgG antibodies are less likely to produce an anti-IgG immune response from the patient. Antibodies such as hLL1 and hLL2 internalize rapidly upon binding to an internalizing antigen on the target cell, meaning that the chemotherapeutic drug carried is also internalized into the cell rapidly. However, antibodies with slower internalization rates can also be used to administer selective therapies.

In a preferred embodiment, more efficient incorporation into cells can be achieved by using multivalent, multispecific or multivalent, monospecific antibodies. Examples of such bivalent and bispecific antibodies can be found in U.S. patent nos. 7,387,772; 7,300,655, respectively; 7,238,785, respectively; and 7,282,567, the respective examples of which are incorporated herein by reference in their entirety. These multivalent or multispecific antibodies are particularly preferred when targeting multiple antigen targets and even multiple epitopes of the same antigen target, but often circumvent antibody targeting and adequate binding of immunotherapy to cancer and infectious organisms (pathogens) due to insufficient expression or availability of a single antigen target on the cell and pathogen. By targeting multiple antigens or epitopes, the antibodies show higher binding to the target and retention time, thus providing higher saturation with the drugs targeted in the present invention.

In another preferred embodiment, the therapeutic agent used in combination with the camptothecin conjugates of the invention can comprise one or more isotopes. Radioisotopes useful for treating diseased tissue include, but are not limited to-¹¹¹In、¹⁷⁷Lu、²¹²Bi、²¹³Bi、²¹¹At、⁶²Cu、⁶⁷Cu、⁹⁰Y、¹²⁵I、¹³¹I、³²P、³³P、⁴⁷Sc、¹¹¹Ag、⁶⁷Ga、¹⁴²Pr、¹⁵³Sm、¹⁶¹Tb、¹⁶⁶Dy、¹⁶⁶Ho、¹⁸⁶Re、¹⁸⁸Re、¹⁸⁹Re、²¹²Pb、²²³Ra、²²⁵Ac、⁵⁹Fe、⁷⁵Se、⁷⁷As、⁸⁹Sr、⁹⁹Mo、¹⁰⁵Rh、¹⁰⁹Pd、¹⁴³Pr、¹⁴⁹Pm、¹⁶⁹Er、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²²⁷Th and²¹¹pb. the therapeutic radionuclide preferably has a decay energy In the range of 20keV to 6,000keV, preferably In the range of 60keV to 200keV for auger emitters, 100keV to 2,500keV for β emitters, and 4,000keV to 6,000keV for α emitters the maximum decay energy of available β particle emitting nuclides is preferably 20keV to 5,000keV, more preferably 100keV to 4,000keV, and most preferably 500keV to 2,500keV, radionuclides that substantially decay with auger emitting particles are also preferred, e.g., Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, and Ir-192. the decay energy of useful β emitting particles is preferably selected from<1,000keV, more preferably<100keV, and most preferably<Radionuclides that substantially decay with the production of α particles are also preferred, including but not limited to Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227, and Fm-255 useful α particle emitting radionuclides preferably have decay energies of 2,000keV to 10,000keV, more preferably 3,000keV to 8,000keV, and most preferably 4,000keV to 7,000keV¹¹C、¹³N、¹⁵O、⁷⁵Br、¹⁹⁸Au、²²⁴Ac、¹²⁶I、¹³³I、⁷⁷Br、^113mIn、⁹⁵Ru、⁹⁷Ru、¹⁰³Ru、¹⁰⁵Ru、¹⁰⁷Hg、²⁰³Hg、^121mTe、^122mTe、^125mTe、¹⁶⁵Tm、¹⁶⁷Tm、¹⁶⁸Tm、¹⁹⁷Pt、¹⁰⁹Pd、¹⁰⁵Rh、¹⁴²Pr、¹⁴³Pr、¹⁶¹Tb、¹⁶⁶Ho、¹⁹⁹Au、⁵⁷Co、⁵⁸Co、⁵¹Cr、⁵⁹Fe、⁷⁵Se、²⁰¹Tl、²²⁵Ac、⁷⁶Br、¹⁶⁹Yb, and the like.

Radionuclides and other metals can be delivered, for example, using a chelating group attached to an antibody or conjugate. Macrocyclic chelators such as NOTA, DOTA and TETA are used with a variety of metals and radiometals, most particularly with the radionuclides gallium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the size of the ring to the metal of interest. Other cyclic chelates may be used, such as for complexation²²³Macrocyclic polyethers of Ra.

Therapeutic agents for use in combination with the camptothecin conjugates described herein also include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins (epidophyllotoxins), taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotic agents, antiangiogenic and proapoptotic agents, particularly doxorubicin, methotrexate, paclitaxel, other camptothecins, as well as others from these and other classes of anticancer agents, and the like. Other cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON 'S PHARMACEUTICAL SCIENCES, 19 th edition (MackPublishing Co.1995) and GOODMAN AND GILMAN' S THEPHEAMAGOLOGICAL BASIS OF THERAPEUTIC, 7 th edition (MacMillan Publishing Co.1985) and revisions OF these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those skilled in the art.

Exemplary drugs that are useful include, but are not limited to: 5-fluorouracil, afatinib, aplidine (aplidine), azalipine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, Cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecin, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinicoxib (dinicib), docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrroline doxorubicin (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin nucleotide, Epirubicin glucuronide, erlotinib, estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogen receptor binding agent, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5' -O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flazapine, fotattinib, ginetidine (ganetespib), GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, ifosfamide, imatinib, L-asparaginase, lapatinib, lenalidomide, leucovorin, M-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, and 6-mercaptopurine, Methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, noviban, lenatinib, nilotinib, nitrosourea, olaparib, plicamycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozotocin, SU11248, sunitinib, tamoxifen, temozolomide, antiplatin, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uramustine, vartanib, vinorelbine, vinblastine, vincristine, vinca alkaloids, and ZD 1839. Such agents may be part of the conjugates described herein, or may be administered in combination with the conjugates before, simultaneously with, or after the conjugates.

Therapeutic agents that can be used with camptothecin conjugates can also comprise a toxin conjugated to a targeting moiety. Toxins that may be used in this regard include ricin, abrin, ribonuclease (rnase), dnase I, staphylococcal enterotoxin-a, pokeweed antiviral protein, gelonin, diphtheria toxin, pseudomonas exotoxin and pseudomonas endotoxin. (see, e.g., Pastan et al, Cell (1986),47:641, and Sharkey and Goldenberg, CACACANCErJClin.2006, months 7-8; 56(4): 226-43.) additional toxins suitable for use herein are known to those of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Another class of therapeutic agents may include one or more immunomodulators the immunomodulators used may be selected from cytokines, stem cell growth factors, lymphotoxins, hematopoietic factors, Colony Stimulating Factors (CSF), Interferons (IFNs), erythropoietin, thrombopoietin and combinations thereof particularly useful are lymphotoxins such as Tumor Necrosis Factor (TNF), hematopoietic factors such as Interleukins (IL), colony stimulating factors such as granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF), interferons such as interferon- α, interferon- β, interferon-gamma or interferon-lambda, and stem cell growth factors such as "S1 factor" included among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone, parathyroid hormone, thyroxine, insulin, relaxin, pinoresinogen, glycoprotein hormones such as FSH, thyroid hormone (TSH), and also as TNF-7, TNF-IL-7, TNF-gamma-TNF-gamma-or TNF-gamma-TNF-gamma-or a combination thereof, and the like, and the cytokines such as IL-7, the growth hormone-7, TNF-7, TNF-7, TNF-gamma-7, TNF-gamma-TNF, TNF-TNF, and the like, and TNF-7, and TNF-IL-7, and the like, and the recombinant IL-7, and TNF-7, and the like, and TNF-7, and the IL-7, and the like, and the mouse, and TNF-7, and the like, and TNF-7, and TNF-IL-7, and TNF-7, and the like, and IL-7, and the recombinant IL-7, and TNF-7, and the like, and IL-7, and the like, and the use of the recombinant IL-7.

Chemokines used include RANTES, MCAF, MIP1- α, MIP1- β and IP-10.

In certain embodiments, the therapeutic agent used in combination with the anti-Trop-2 ADC is a microtubule inhibitor, such as a vinca alkaloid, a taxane, a maytansinoid, or an auristatin. Exemplary known microtubule inhibitors include paclitaxel, vincristine, vinblastine, methamine, epothilone, docetaxel, discodermolide (discodermolide), combestin (combestin), podophyllotoxin, CI-980, phenylahistins, eleutherobin, curcins, 2-methoxyestradiol, E7010, methoxybenzenesulfonamide, vinorelbine, vinflunine, vindesine, dolastatin, halichondrin (spongistatin), lisoprotein (rhizoxin), tasedotin (tasidotin), halichondrins (halichondrins), hemiasterins (cryptophycin)52, MMAE, and eribulin mesylate (butilin mesylate).

In an alternative embodiment, the therapeutic agent to be used in combination with an ADC is a PARP inhibitor, such as olaparib, talapanib (BMN-673), rupalab, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983, or 3-aminobenzamide.

In another alternative, the therapeutic agent used in combination with the ADC is a Bruton's tyrosine kinase inhibitor, such as ibrutinib (PCI-32765), PCI-45292, CC-292(AVL-292), ONO-4059, GDC-0834, LFM-A13, or RN 486.

In another alternative, the therapeutic agent used in combination with the ADC is a PI3K inhibitor, such as Idelalisib, Wortmannin (Wortmannin), desmethoxychloromycetin (demethoxyviridin), perifosine (perifosine), PX-866, IPI-145 (Duvelsibu), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid (Palomid)529, GSK1059615, ZSK 474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136, or LY 292.

One of ordinary skill in the art will recognize that the ADCs of the present invention comprising camptothecin conjugated to an antibody or antibody fragment may be used alone or in combination with one or more other therapeutic agents, such as a second antibody, a second antibody fragment, a second immunoconjugate, a radionuclide, a toxin, a drug, a chemotherapeutic agent, radiation therapy, a chemokine, a cytokine, an immunomodulator, an enzyme, a hormone, an oligonucleotide, an RNAi or an siRNA. Such additional therapeutic agents may be administered alone, in combination with, or linked to the antibody-drug ADC of the invention.

Formulation and administration

Suitable routes of administration for the conjugates include, but are not limited to, oral, parenteral, subcutaneous, rectal, transmucosal, enteral, intramuscular, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection. The preferred route of administration is parenteral. Alternatively, the compound may be administered locally rather than systemically, e.g., via direct injection of the compound into a solid tumor.

The ADC may be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the ADC is combined in a mixture with a pharmaceutically suitable excipient. Sterile phosphate buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. See, for example, Ansel et al, PHARMACEUTICAL DOSAGEFORMS AND DRUG DELIVERY SYSTEMS, 5 th edition (Lea AND Febiger1990) AND Gennaro (eds.), REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition (MackPublishing Company 1990) AND revisions thereof.

In a preferred embodiment, the ADC is formulated in Good's biological buffer (pH 6-7) using a buffer selected from the group consisting of: n- (2-acetylamino) -2-aminoethanesulfonic Acid (ACES); n- (2-acetamido) iminodiacetic acid (ADA); n, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES); 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES); 2- (N-morpholino) ethanesulfonic acid (MES); 3- (N-morpholino) propanesulfonic acid (MOPS); 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO); and piperazine-N, N' -bis (2-ethanesulfonic acid) [ Pipes ]. More preferably the buffer is MES or MOPS, preferably at a concentration in the range of 20mM to 100mM, more preferably about 25 mM. Most preferred is 25mM MES, pH 6.5. The formulation may also contain 25mM trehalose and 0.01% v/v polysorbate 80 as excipients, with the final buffer concentration modified to 22.25mM due to the addition of the excipient. The preferred method of storage is as a lyophilized formulation of the conjugate is storage at a temperature in the range of-20 ℃ to 2 ℃, most preferably at 2 ℃ to 8 ℃.

The ADC may be formulated for intravenous administration via, for example, bolus infusion, slow infusion, or continuous infusion. Preferably, the antibodies of the invention are infused over a period of less than about 4 hours, and more preferably over a period of less than about 3 hours. For example, the first 25mg to 50mg may be infused over 30 minutes, preferably even over 15 minutes, and the remainder over the next 2 to 3 hours. Where subcutaneous administration is desired, the antibody may be concentrated, for example as disclosed in U.S. patent No. 9,180,205, the examples section of which is incorporated herein by reference. Subcutaneous injections may be administered as 1ml, 2ml or 3ml injections, which may be administered at a single site or at two or more sites. Each injection typically contains 1.5mg/kg to 4mg/kg of concentrated ADC.

Injectable formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control the duration of action of the therapeutic conjugate. Controlled release formulations can be prepared by complexing or adsorbing ADC with a polymer. For example, biocompatible polymers include poly (ethylene-co-vinyl acetate) matrices and polyanhydride copolymer matrices of stearic acid dimer and sebacic acid. Sherwood et al, Bio/Technology 10:1446 (1992). The rate of release of ADC from such matrices depends on the molecular weight of the ADC, the amount of ADC within the matrix and the size of the dispersed particles. Saltzman et al, Biophys.J.55:163 (1989); sherwood et al, supra. Other solid dosage forms are described in the following documents: ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5 th edition (Lea AND Febiger1990) AND Gennaro (eds.), REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition (Mack Publishing Company 1990) AND revisions thereof.

In general, the dosage of ADC administered to a person will vary depending on factors such as the patient's age, weight, height, sex, general medical condition and past medical history. It may be desirable to provide the recipient with a dose of ADC in the range of about 1mg/kg to 24mg/kg as a single intravenous infusion, although lower or higher doses may also be administered as the case may be. For example, the dose may be 1mg/kg to 20mg/kg for a 70kg patient, e.g., 70mg to 1,400mg, or 41mg/m for a 1.7-m patient²To 824mg/m². The dosage may be repeated as needed, for example once a week for 4 to 10 weeks, once a week for 8 weeks, or once a week for 4 weeks. It may also be given less frequently, such as once every week for several months, or once a month or quarterly for many months, when needed in maintenance therapy. Preferred dosages may include, but are not limited to, 1mg/kg, 2mg/kg, 3mg/kg. 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 22mg/kg and 24 mg/kg. Any amount in the range of 1mg/kg to 24mg/kg may be used. The dose is preferably administered multiple times, once or twice weekly. A minimum dose regimen of 4 weeks, more preferably 8 weeks, more preferably 16 weeks or more may be used. The administration regimen may comprise once or twice weekly administration at a cycle selected from the group consisting of: (i) weekly; (ii) every other week; (iii) treatment for one week, followed by rest for two, three or four weeks; (iv) treatment for two weeks, followed by a rest for one, two, three or four weeks; (v) treatment for three weeks followed by a rest of one, two, three, four or five weeks; (vi) treatment for four weeks, followed by a rest of one, two, three, four or five weeks; (vii) treatment for five weeks, followed by a rest of one, two, three, four or five weeks; (viii) every month. The cycle may be repeated 4,6, 8, 10, 12, 16 or 20 or more times.

Alternatively, the ADC may be administered as one dose every 2 or 3 weeks, for a total of at least 3 doses. Alternatively, twice weekly for 4-6 weeks. If the dosage is reduced to about 200mg/m²To 300mg/m²(340 mg per dose for 1.7-m patients, or 4.9mg/kg for 70kg patients), then it can be administered once or even twice weekly for 4 to 10 weeks. Alternatively, the time course of administration may be reduced, i.e. every 2 or 3 weeks for 2 to 3 months. However, it has been determined that even higher doses can be administered even by slow intravenous infusion, such as 12mg/kg once weekly or once every 2 to 3 weeks, for repeated dosing cycles. The dosing schedule can optionally be repeated at other time intervals, and the doses can be administered by various parenteral routes with appropriate adjustment of the dose and schedule.

In preferred embodiments, the ADC is useful for the treatment of cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More specific examples of such cancers are described below and include: squamous cell cancer (e.g., epithelial squamous cell cancer), Ewingsarcoma (Ewingsarcoma), Wilms' tumor (Wilms tumor), astrocytoma, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, stomach or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumor, medullary thyroid cancer, thyroid differentiation cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, anal cancer, penile cancer, and head and neck cancer. The term "cancer" includes primary malignant cells or tumors (e.g., tumors in which cells do not migrate to a site other than the site of the original malignant disease or tumor in the subject) and secondary malignant cells or tumors (e.g., tumors resulting from metastasis, which is the migration of malignant cells or tumor cells to a secondary site different from the site of the original tumor).

Other examples of cancer or malignancy include, but are not limited to: acute childhood lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myelogenous leukemia, adult hodgkin's lymphoma, adult lymphocytic lymphoma, adult non-hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignancy, anal carcinoma, astrocytoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain stem glioma, brain tumor, breast carcinoma, renal pelvis and ureter carcinoma, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cervical carcinoma, child (primary) hepatocellular carcinoma, Childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial blastoma, childhood hodgkin's disease, childhood hodgkin's lymphoma, childhood hypothalamic and optic pathway glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-hodgkin's lymphoma, childhood pineal and supratentorial primitive neuroectodermal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood optic and hypothalamic glioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine islet cell carcinoma, endometrial carcinoma, ependymoma, epithelial carcinoma, esophageal carcinoma, neuroblastoma, cervical carcinoma, cervical, Ewing's Sarcoma and related tumors, exocrine pancreatic cancer, extracranial blastoma, gonadal ectoblastoma, extrahepatic cholangiocarcinoma, ocular cancer, female breast cancer, Gaucher's Disease, gallbladder cancer, gastric cancer, gastrointestinal benign tumors, gastrointestinal tumors, blastoma, gestational trophoblastic carcinoma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's Sarcoma, renal cancer, laryngeal cancer, labial oral cancer, hepatic cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, primary metastatic squamous neck cancer, metastatic primary cervical cancer, metastatic cervical cancer, and metastatic cervical cancer, Metastatic squamous neck cancer, multiple myeloma/plasma cell neoplasm, myelodysplastic syndrome, myelogenous leukemia, myeloproliferative disorders, cancer of the nasal and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, primary occult metastatic squamous neck cancer, oropharyngeal cancer, osteosarcoma/malignant fibrosarcoma, osteosarcoma/malignant fibrous histiocytoma of the skeleton, epithelial carcinoma of the ovary, embryoblastoma of the ovary, low malignant potential tumors of the ovary, pancreatic carcinoma, proteinemia of the lesion, polycythemia vera, parathyroid carcinoma, penile carcinoma, pheochromocytoma, pituitary tumor, primary central nervous system lymphoma, primary hepatic carcinoma, prostate carcinoma, pancreatic carcinoma, colorectal carcinoma, pancreatic carcinoma, cervical, Rectal cancer, renal cell carcinoma, carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcomatosis sarcoma, Sezary Syndrome (Sezary Syndrome), skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous neck cancer, gastric cancer, supratentorial primary neuroectodermal and pineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastoma, ureter and renal pelvis cell carcinoma, ureter cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, vulval cancer, waldenstrom macroglobulinemia, wilms' tumor, and any other hyperproliferative disease located in the upper column organ system other than neoplasms.

The methods and compositions described and claimed herein are useful for treating malignant or pre-malignant conditions and for preventing the development of neoplastic or malignant states, including but not limited to those disorders described above. Such use is indicated in conditions known or suspected of prior progression to neoplasia or cancer, particularly where non-neoplastic cell growth consists of hyperproliferation, metaplasia, or most particularly the occurrence of dysplasia (for a review of such abnormal growth conditions, see robblins and Angell, Basic pathology, 2 nd edition, w.b. saunders co., philidelphia, pages 68-79 (1976)).

Dysplasia is often a precursor to cancer and is found primarily in the epithelium. It is the most disordered form of nonneoplastic cell growth, involving loss of individual cell consistency and cell structure orientation. In the presence of chronic stimuli or inflammation, dysplasia characteristically occurs. Dysplastic disorders that may be treated include, but are not limited to: anhidrotic ectodermal dysplasia, anterior dysplasia, asphyxiative thoracic dysplasia, atrial-digital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroaortical dysplasia, clavicular cranial dysplasia, congenital ectodermal dysplasia, cranial diaphyseal dysplasia, cranial metaphyseal dysplasia, dentinal dysplasia, diaphyseal dysplasia, ectodermal dysplasia, enamel dysplasia, cerebral eyeball dysplasia, hemiepiphyseal dysplasia, multiple epiphyseal dysplasia, punctate epiphyseal dysplasia, epithelial dysplasia, facial-digital dysplasia, familial fibrous dysplasia of the jaw, familial white-fold dysplasia, fibrous-muscular dysplasia, fibrous-fibrous dysplasia, exuberant dysplasia, etc, Hereditary renal retinal dysplasia, perspirant ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenia thymic dysplasia, mammary dysplasia, mandibular facial dysplasia, metaphyseal dysplasia, Mondini dyplasia (Mondini dyssplasia), single bone fibrodysplasia, mucous epithelium dysplasia, multiple epiphyseal dysplasia, ocular ear spondylodysplasia, ocular tooth dysplasia, ocular vertebral dysplasia, odontogenic dysplasia, ocular mandibular dysplasia, periapical cementosclerotic dysplasia, bony fibrous dysplasia, pseudochondrodysplasia spondylosis, retinal dysplasia, septal-ocular dysplasia, spondyloepiphyseal dysplasia, and ventricular radial dysplasia.

Additional preneoplastic disorders that may be treated include, but are not limited to, benign hyperproliferative disorders (e.g., benign tumors, fibrocystic pathologies, tissue hypertrophy, intestinal polyps or adenomas, and abnormal hyperplasia of the esophagus), leukoplakia, keratosis, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.

In a preferred embodiment, the methods of the invention are used to inhibit the growth, development and/or metastasis of cancer, particularly the cancers listed above.

Other hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, the progression and/or metastasis of malignant diseases and related disorders, such as leukemia (including acute leukemia; e.g., acute lymphocytic leukemia, acute myelogenous leukemia [ including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia ]) and chronic leukemia (e.g., chronic myelogenous [ myelogenous ] leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and related disorders, Chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small-cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that can be treated with ADC include acute and chronic immune thrombocytopenia, dermatomyositis, sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, glandular syndrome, bullous pemphigoid, juvenile diabetes, allergic purpura, post-streptococcal infection nephritis, erythema nodosum, takayasu arteritis, ANCA-associated vasculitis, Edison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, vasculitis obliterans, sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, rheumatoid arthritis, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tuberculosis of the spinal cord, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis and fibrositis.

Medicine box

Various embodiments can be directed to kits containing components suitable for treating diseased tissue in a patient. An exemplary kit can contain at least one ADC or other targeting moiety as described herein. If the composition containing the components for administration is not formulated for delivery through the digestive tract, such as by oral delivery, a device capable of delivering the kit components by some other route may be included. One type of device for applications such as parenteral delivery is a syringe, which is used to inject a composition into a subject. Inhalation devices may also be used.

The kit components may be packaged together or divided into two or more containers. In some embodiments, the container may be a vial containing a sterile lyophilized formulation of the composition suitable for reconstitution. The kit may also contain one or more buffers suitable for reconstituting and/or diluting other reagents. Other containers that may be used include, but are not limited to, bags, trays, boxes, tubes, and the like. The kit components may be aseptically packaged and maintained within the containers. Another component that may be included is instructions provided to the user of the cartridge.

Examples

The following examples illustrate various embodiments of the present invention without limiting its scope.

Example 1 Generation and use of anti-Trop-2-SN-38 antibody-drug conjugates

Humanized RS7(hRS7) anti-Trop-2 antibodies are produced as described in U.S. patent No. 7,238,785, the figures and examples of which are incorporated herein by reference. According to U.S. patent 7,999,083, SN-38 linked to a CL2A linker was generated and conjugated to hRS7 (anti-Trop-2), hPAM4 (anti-MUC 5ac), hA20 (anti-CD 20), or hMN-14 (anti-CEACAM 5) antibodies (examples 10 and 12 of this patent are incorporated herein by reference). The conjugation scheme results in a ratio of about 6 SN-38 molecules attached per antibody molecule.

Immunocompromised athymic nude mice (females) with subcutaneous human pancreatic or colon tumor xenografts were treated or not treated with specific CL2A-SN-38 conjugates or control conjugates. The therapeutic efficacy of the specific conjugate was observed. In the Capan1 pancreatic tumor model, specific CL2A-SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4 (anti-MUC-5 ac), and hMN-14 (anti-CEACAM 5) antibodies showed better efficacy than the control hA20-CL2A-SN-38 conjugate (anti-CD 20) and untreated controls (not shown). Similarly, in the BXPC3 model of human pancreatic cancer, the specificity hRS7-CL2A-SN-38 showed better therapeutic efficacy than control treatment (not shown).

Example 2 therapeutic use of anti-Trop-2 ADC (Saxizumab-gaulthikazene) in patients refractory to checkpoint inhibitor therapy

SUMMARY

IMMU-132 (Saxizumab govitegam, also known as hRS7-CL2A-SN-38) showed promising therapeutic results in phase II trials of metastatic triple negative breast cancer and other cancer patients who underwent extensive pretreatment (clinical trials. gov, NCT01631552) and expressed high levels of Trop-2. This novel Trop-2 targeted humanized antibody is conjugated to 7.6 moles of SN-38 (the active form of irinotecan) through the CL2A linker discussed above, and is less glucuronidated in vivo than irinotecan, resulting in a significant reduction in the incidence of diarrhea in patients treated with this agent.

Surprisingly, IMMU-132 was highly effective in patients who had previously relapsed from or shown to be resistant to many standard anticancer therapies, including the parent compound irinotecan. A novel class of anti-cancer agents, known as checkpoint inhibitors, includes antibodies or other inhibitors against cytotoxic T lymphocyte antigen 4(CTLA-4), programmed cell death protein (PD-1), and programmed cell death ligand 1 (PD-L1). As described herein, IMMU-132 exhibits surprising and unexpected efficacy against tumors that are relapsed/refractory to checkpoint inhibitors and other anti-cancer agents.

The following data summarizes the results of an exemplary case study of patients (255-.

Cancer: TNBC (patient progressed after PD-L1 treatment)

Optimal response: partial response, confirmation (shrinkage 54%)

Initial dose of IMMU-132: 10mg/kg

Treatment number of IMMU-132: 40+

Time of progression: 12.4+ month (not reached)

And (4) finishing the research: continue the treatment

Medical historyThe patient was a 47 year old female, she was first diagnosed with a 1.8cm lump in the right breast 10 months in 2007. She received a right mastectomy and sentinel lymph node biopsy (no malignancy in the node). Tumors were determined to be negative for ER, PR, and Her-2 (i.e., TNBC). She continued with 4 cycles of AC (doxorubicin and cyclophosphamide) treatment followed by weekly low dose paclitaxel treatment (x 12). 10 months 2008, CT studies showed lung nodules and breast enlargement nodules not previously noted; the nodule is a cancer. She underwent local surgery to remove nodules and then underwent XRT (radiation therapy) 3 months prior to 2009. At 6 months 2010, CT recorded recurrent lung and bone involvement. The patient selects for use(bevacizumab),(onartuzumab, anti-hepatocyte growth factor) andclinical trial of (paclitaxel), she remained until 3 months 2013 with a complete response, but eventually progressed to peripheral lung nodules and nodules in the AP (main pulmonary artery) window. She started using PD-L1 antibody (MPDL3280A) for 6 cycles in 9/2014, but made progress in 1/2015. She was then referred to the IMMU-132 trial.

IMMU-132 treatmentPatients started treatment at a dose of 10mg/kg starting on 6 days 2/2015 and continued to receive the dose without reducing or delaying the planned time course, now the 40+ dose.

Results

The patient initially presented 3 target lesions (1 in the lung with 2 lymph nodes in the chest), totaling a diameter equal to 60mm, and in addition non-target lesions (another lymph node in the chest) (FIG. 2). At the first assessment (day 7/4/2015), the sum of target lesion diameters decreased by 33% for the patient, consistent with RECIST 1.1 partial response. Confirmed follow-up CT performed after about 5 weeks (5 months and 18 days 2015) showed improved response with an overall reduction of 48% (fig. 3, fig. 4). CT showed the patient continued to respond partially, with a shrinkage of 50%, on day 19, month 7, 2015. CT for 9/13/2015 showed sustained PR with shrinkage of 52%, and CT for 15/11/15 showed shrinkage of 54%. The latest CT performed 2, 25 days in 2016 showed continued PR with a shrinkage of 46%.

A summary of CT results is shown in figure 1. The baseline CT scan is shown in fig. 2 with the top row having an axial image and the bottom row having a sagittal image (arrows indicate tumors). A direct comparison of target lesions is shown in figures 3 and 4. Figure 3 shows target lesions 1 and 2 in the same plane. The baseline (1 month, 2015, 29 days) is shown at the top. Significant contraction of L1 and L2 was evident in the second response assessment (5 months and 19 days 2015), shown at the bottom. Fig. 4 shows an equivalent image of target lesion 3 with baseline at the top and second response assessment at the bottom. After not having been previously treated with the checkpoint inhibitor, the patient is continuing treatment and continues to display a partial response to IMMU-132.

Immunohistochemical analysis of Trop-2 expression in patient tumors showed positive expression with a 2+ staining level (data not shown).

ToxicityBy the date of the report, the most serious adverse effect was G3 hypophosphatemia (no correlation), with a possible associated toxicity of G2 neutropenia 2015, 4 months and 2 days; dose 6), fatigue (G1), nausea (G1), maculopapular rash (G1), alopecia (G2) and rhinorrhea (G1).

These results demonstrate that IMMU-132 is very effective in patients who have previously relapsed from or are resistant to checkpoint inhibitor therapy. At therapeutic doses of IMMU-132, patients only show controlled toxicity. These results show the utility of IMMU-132 for Trop-2 positive cancers such as Triple Negative Breast Cancer (TNBC).

Example 3 ADCC Activity of anti-Trop-2 ADC

ADCC activity of various hRS7-ADC conjugates was determined compared to hRS7IgG (not shown). PBMCs were purified from blood purchased from the new jersey blood center. The Trop-2 positive human pancreatic cancer cell line (BxPC-3) was used as the target cell line with an effector to target ratio of 100: 1. hRS7IgG mediated ADCC was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and reduced and capped hRS 7-NEM. All were used at 33.3 nM.

The overall activity was very low but significant (not shown). The hRS7IgG had a specific cleavage of 8.5%, which was not significantly different from hRS 7-Pro-2-PDox. Both were significantly superior to hLL2 control and hRS7-NEM and saxizumab gavaticam (P <0.02, two-tailed t-test). There was no difference between hRS7-NEM and saxizumab gavaticam.

Example 4 summary of the efficacy of anti-Trop-2-SN-38 ADCs against various epithelial cancers in vivo

The objective of this study was to evaluate the efficacy of SN-38-anti-Trop-2 (hRS7) ADCs against several human solid tumor types and to assess their tolerance to mice and monkeys, which had similar tissue cross-reactivity to hRS7 as humans. Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2-humanized antibody hRS 7. ADC stability, binding and cytotoxicity were characterized in vitro. Efficacy was tested in five different human solid tumor-xenograft models expressing Trop-2 antigen. Toxicity was assessed in mice and cynomolgus monkeys.

hRS7 conjugates of two SN-38 derivatives in drug replacement (about 6), cell binding (K)_dAbout 1.2nmol/L), cytotoxicity (IC)₅₀About 2.2nmol/L) and in vivo serum stability (t ^ er)_1/2About 20 hours). Exposure of cells to ADC demonstrated that the signaling pathway led to PARP cleavage, but differences from free SN-38 in p53 and p21 upregulation were noted. With Calu-3 (P.ltoreq.0.05), Capan-1 (P) when compared to the non-targeted control ADC<0.018)、BxPC-3(P<0.005) and COLO 205 tumors (P)<0.033) produced significant antitumor effects in mice by non-toxic doses of saxizumab gavatikukan. Mice tolerated a dose of 2X 12mg/kg (SN-38 equivalents) with only a transient increase in ALT and AST liver enzyme levels. Cynomolgus monkeys infused at 2x 0.96mg/kg showed only a short drop in blood cell counts, but importantly, these values were not below the normal range.

In summary, anti-Trop-2 hRS7-CL2A-SN-38 ADCs provide significant and specific anti-tumor effects against a range of human solid tumor types. At clinically relevant doses, it was well tolerated in monkeys, with tissue Trop-2 expression similar to that in humans.

Introduction to

Successful irinotecan treatment in solid tumor patients is limited, in large part due to the low conversion of CPT-11 prodrug to active SN-38 metabolite. Others have examined non-targeted forms of SN-38 as a means of bypassing this transformation need and passively delivering SN-38 to tumors. We covalently conjugated SN-38 to the humanized anti-Trop-2 antibody hRS 7. The antibody-drug conjugates have specific anti-tumor effects in a range of subcutaneous human cancer xenograft models including non-small cell lung cancer, pancreatic cancer, colorectal cancer, and squamous cell lung cancer, all at non-toxic doses (e.g., ≦ 3.2mg/kg cumulative SN-38 equivalent doses). Trop-2 is widely expressed in many epithelial cancers, but also in some normal tissues, so dose escalation studies were performed in cynomolgus monkeys to assess the clinical safety of the conjugate. Monkeys tolerated 24mg SN-38 equivalents/kg with only slight reversible toxicity. Given its tumor targeting and safety profile, saxilizumab govitegran provides significant improvements in the management of solid tumors in response to irinotecan.

Materials and methods

Cell lines, antibodies and chemotherapeutic agentsAll human cancer cell lines used in this study were purchased from the american type culture collection. These include Calu-3 (non-small cell lung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205 (colon adenocarcinoma), Capan-1 and BxPC-3 (pancreatic carcinoma) and PC-3 (prostate adenocarcinoma). Humanized RS7IgG and control humanized anti-CD 20(hA20 IgG, vetuzumab) and anti-CD 22(hLL2 IgG, epratuzumab) antibodies were prepared by immunolamedics, inc. Irinotecan (20mg/mL) was obtained from Hospira, inc.

SN-38 ADCs and in vitro aspectsThe synthesis of CL2-SN-38 has been described previously (Mo on et al, 2008, JMed Chem51: 6916-26). Conjugation to hRS7I gG and serum stability were performed as described (Moon et al, 2008, JMed Chem51: 6916-26; Govindan et al, 2009, Clin Chem Res 15: 6052-61). The preparation of CL2A-SN-38(m.w.1480) and its hRS7 conjugate and stability, binding and cytotoxicity studies were performed as described in the previous examples.

In vivo therapeutic studyFor all animal studies, the dose of SN-38 ADC and irinotecan is shown as SN-38 equivalents. Based on an average SN-38/IgG substitution ratio of 6, 20-g mice were administered a dose of 500. mu.g ADC (25mg/kg) containing 0.4mg/kg of SN-38. IrinotecanThe dose is also shown as SN-38 equivalents (i.e., 40mg irinotecan/kg corresponds to 24mg/kg of SN-38).

NCr female athymic (nu/nu) mice, 4 to 8 weeks old, and male Swiss-Webster (Swiss-Webster) mice, 10 weeks old, were purchased from Taconcec farm. Tolerance studies were performed in cynomolgus monkeys (Macacafascicularis; 2.5kg to 4kg male and female).

Animals were implanted subcutaneously with different human cancer cell lines. Tumor Volume (TV) was determined by 2-dimensional measurement using calipers, the volume being defined as: l x w²And/2, wherein L is the longest dimension of the tumor and w is the shortest dimension. At the beginning of the treatment, the size of the tumor was 0.10cm³And 0.47cm³In the meantime. The treatment regimen, dose and number of animals in each experiment are described in the results. Lyophilized saxizumab govitikang and control ADC were reconstituted and diluted in sterile saline as needed. All agents were administered intraperitoneally (0.1mL) except for intravenous irinotecan. The dosing regimen was influenced by our previous investigation in which ADC was administered once every 4 days or twice a week for different lengths of time (Moon et al, 2008, J Med Chem51: 6916-26; Govindan et al, 2009, Clin Chem Res 15: 6052-61). This dosing frequency reflects in vitro serum half-life considerations of the conjugate to allow for more continuous exposure to the ADC.

Statistics ofGrowth curves are shown as a percentage change of the initial TV over time. Statistical analysis of tumor growth was based on area under the curve (AUC). A profile of individual tumor growth was obtained by linear curve modeling. Prior to statistical analysis of growth curves, the f-test was used to determine homogeneity of variance between groups. Statistical significance between various treatment groups and controls was assessed using the two-tailed t-test in addition to the saline control, in which the single-tailed t-test was used (significance at P ≦ 0.05). Statistical comparisons of AUC were performed only at the time the first animal in the group was euthanized as a result of progression.

Pharmacokinetics and biodistribution-to be¹¹¹In-radiolabeled Saxizumab gavietin and hRS7IgG were injected into the tapeWith subcutaneous SK-MES-1 tumor (about 0.3 cm)³) In nude mice. One group of 20. mu. Ci (250. mu.g protein) intravenous injections¹¹¹In-Saxizumab gavelikukan, while the other group received 20. mu. Ci (250. mu.g protein)¹¹¹In-hRS7 IgG. At different time points, mice (5 per time point) were anesthetized, exsanguinated by intracardiac puncture, and then euthanized. Tumors and various tissues were removed, weighed, and counted by gamma scintillation to determine the percent injected dose per gram of tissue (% ID/g). The third group is administered¹¹¹In-Saxizumab govitikang 250 μ g of unlabelled Saxizumab govitikang was injected 3 days before injection and necropsy was performed as well. After determining homogeneity of variance using the f-test, the two-tailed t-test was used to compare the saxizumab govitegradn and hRS7IgG uptake. Pharmacokinetic analysis of blood clearance was performed using WinNonLin software (design Corp.).

Swiss-Webster mouse and cynomolgus monkey toleranceBriefly, mice were divided into 4 groups, each group receiving 2-mL intraperitoneal sodium acetate buffer control or 3 different doses of saxizumab gavoriconazole (4mg/kg, 8mg/kg or 12mg/kg SN-38) on days 0 and 3, followed by blood and serum collection as described in the results. Succinimizumab govitikang was administered at 2 different doses to cynomolgus monkeys (3 males and 3 females; 2.5kg to 4.0 kg). The results describe the dose, number and number of monkey bleeds used to evaluate possible hematologic toxicity and serum chemistry.

Results

Stability and efficacy of hRS7-SN-38SN-38 was conjugated to hRS7IgG using two different linkages (not shown). The first linkage is referred to as CL2-SN-38 and has been previously described (Moon et al, 2008, JMed Chem51: 6916-26; Govindan et al, 2009, Clin Chem Res 15: 6052-61). Changes in the synthesis of CL2 were used to remove the phenylalanine moiety within the linker to produce a CL2A linker. This variation simplifies synthesis but does not affect conjugation results (e.g., both CL2-SN-38 and CL2A-SN-38 incorporate about 6 SN-38 per IgG molecule). Side-by-side comparison of serum stability, antigen binding or lack of in vitro cytotoxicityThere were significant differences. This result is surprising because the phenylalanine residue in CL2 is part of the designed cleavage site for cathepsin B (lysosomal protease).

To confirm that the change in SN-38 linker from CL2 to CL2A did not affect in vivo efficacy, Saxizumab govitegnac and hRS7-CL2-SN-38 were compared twice weekly using 0.4mg or 0.2mg/kg SN-38 for 4 weeks in mice bearing COLO 205 (not shown) or Capan-1 tumor (not shown), respectively, and wherein the starting tumor size was 0.25cm in both studies³. Comparison with untreated control (comparison with saline in COLO 205 model, AUC_{14 days}，P<0.002; comparison of saline, AUC in the Capan-1 model_{21 days}P<0.001), and the non-targeted anti-CD 20 control ADC, hA20-CL2A-SN-38 (AUC in COLO-205 model, AUC)_{14 days}，P<0.003; AUC in the Capan-1 model_{35 days}：P<0.002) compared, both the saxizumab gavatica and the CL2-SN-38 conjugate significantly inhibited tumor growth. At the end of the C apan-1 model study (day 140), 50% of the mice treated with Saxizumab govitikon were tumor free and 40% of the hRS7-CL2-SN-38 mice were tumor free, while only 20% of the animals treated with hA20-ADC showed no obvious signs of disease. The CL2A linker yielded higher efficacy compared to CL2 (not shown).

Mechanism of actionIn vitro cytotoxicity studies demonstrated that Saxizumab govitegam has an IC in the nmol/L range for several different solid tumor lines₅₀Values (table 2). IC of free SN-38₅₀Lower than in all cell lines. Although there was no clear correlation between Trop-2 expression and sensitivity to saxizumab gavatikukan, the IC of ADC with free SN-38 in cells expressing higher Trop-2₅₀The ratio is lower, which likely reflects the increased ability to internalize the drug when more antigen is present.

SN-38 is known to activate several signaling pathways in cells, leading to apoptosis (e.g., Cusack et al, 2001, Cancer Res 61: 3535-40; Liu et al, 2009, Cancer Lett 274: 47-53; Lagadec et al, 2008)Br J Cancer 98: 335-44). Our preliminary study examined early signaling events in vitro (p 21)^{Waf1 /Cip1}And p53) and 1 late apoptotic event [ cleavage by poly-ADP-ribose polymerase (PARP)]Expression of 2 proteins involved in (1) (not shown). In BxPC-3, SN-38 results in p21^Waf1/Cip1The expression increased 20-fold (not shown), whereas the saxizumab govitikang resulted in only a 10-fold increase (not shown), which is consistent with the high activity of free SN-38 in this cell line (table 2). However, as compared to free SN-38, Saxizumab gavitegam gavit causes p in Calu-3^21Waf1/Cip1Expression was increased more than 2-fold (not shown).

A greater difference between the saxizumab govittacin-mediated and free SN-38-mediated signaling events was observed in p53 expression (not shown). In BxPC-3 and Calu-3, p53 was upregulated with free SN-38 until 48 hours, whereas Saxizumab gavoriconazole upregulated p53 within 24 hours (not shown). Furthermore, p53 expression was higher in cells exposed to ADC in both cell lines compared to SN-38 (not shown). Interestingly, although hRS7IgG was on p21^Waf1/Cip1Expression had no significant effect, but it did induce up-regulation of p53 in BxPC-3 and Calu-3, but only after 48 hours of exposure (not shown). In the case of later apoptotic events, lysis of PARP was evident in both cell lines when incubated with SN-38 or conjugate (not shown). The presence of PARP cleaved at 24 hours in BxPC-3 was higher (not shown), which is associated with high expression of p21 and its lower IC₅₀And (4) correlating. The higher degree of lysis of free SN-38 compared to ADC is consistent with the results of cytotoxicity studies.

Efficacy of hRS7-SN-38Since Trop-2 is widely expressed in several human cancers, studies were performed in several different human cancer models, starting with hRS7-CL2-SN-38 linkage, but later with a conjugate linked to CL 2A-linkage. Nude mice bearing Calu-3 dosed with 0.04mg SN-38/kg hRS7-CL2-SN-38 every 4 days had significantly improved response compared to animals dosed with equal amounts of non-targeted hLL2-CL2-SN-38 (TV ═ 0.14 ± 0.22cm, respectively) for 4 cycles³Comparison 0.80 ±. + -.)0.91cm³；AUC_{42 days}P<0.026; not shown). When the dose was increased to 0.4mg/kg SN-38, a dose response was observed (not shown). At this higher dose level, all mice given the specific hRS7 conjugate were "cured" within 28 days and remained tumor-free at day 147 until the end of the study, while tumors grew again in animals treated with irrelevant ADCs (specificity versus irrelevant, AUC)_{98 days}: p ═ 0.05). In mice receiving a mixture of hRS7IgG and SN-38, tumors progressed on day 56>4.5 times (TV is 1.10 + -0.88 cm)³；AUC_{For 56 days}P<0.006 vs. hRS7-CL2-SN-38) (not shown).

Efficacy in human colon tumor xenografts (COLO 205) and pancreatic tumor xenografts (Capan-1) was also examined. Animals bearing COLO 205 tumors (not shown)http:// clincancerres.aacrjournals.org/content/17/10/3157.long-F3) In (b), hRS7-CL2-SN-38(0.4mg/kg, q4dx8) prevented tumor growth over a 28 day treatment period, with significantly smaller tumors (TV ═ 0.16 ± 0.09cm, respectively) compared to control anti-CD 20 ADC (hA20-CL2-SN-38) or hRS7IgG (0.4mg/kg, q4dx8)³、1.19±0.59cm³And 1.77. + -. 0.93cm³；AUC_{28 days}P<0.016)。

TABLE 2 Trop-2 cytotoxic expression of SN-38 and hRS7-SN-38 in vitro in various solid tumor lines

The MTD of irinotecan (24mg SN-38/kg, q2dx5) was as effective in COLO 205 cells as hRS7-CL2-SN-38 because mouse serum could convert irinotecan to SN-38 more efficiently than human serum (Morton et al, 2000, Cancer Res60:4206-10), but the SN-38 dose (2,400 μ g accumulated) of irinotecan was 37.5-fold higher than the conjugate (64 μ g total).

Animals bearing Capan-1 (not shown) did not show a significant response to irinotecan alone when administered at a dose of SN-38 equivalent to the hRS7-CL2-SN-38 conjugate (e.g., on day 35, mean tumor size was 0.04. + -. 0.05cm in animals administered 0.4mg SN-38/kghRS7-SN-38³In contrast, the mean tumor size was 1.78. + -. 0.62cm in animals treated with irinotecan administered with 0.4mg/kg SN-38³；AUC_{35 days}P<0.001; not shown). When irinotecan doses were increased 10-fold to 4mg/kg SN-38, the response was improved, but still not as significant as the conjugates at the 0.4mg/kg SN-38 dose level (TV 0.17. + -. 0.18 cm)³Comparison 1.69. + -. 0.47cm³，AUC_{For 49 days}P<0.001) (not shown). Equivalent doses of non-targeted hA20-CL2-SN-38 also had significant anti-tumor effects compared to irinotecan treated animals, but the specific hRS7 conjugate was clearly superior to the unrelated ADC (TV 0.17 ± 0.18 cm)³Contrast 0.80. + -. 0.68cm³，AUC_{For 49 days}P<0.018) (not shown).

The study of the saxizumab gaviticon ADC was then extended to 2 other human epithelial cancer models. Among mice bearing BxPC-3 human pancreatic tumors (not shown), control mice treated with saline or an equivalent amount of non-targeted hA20-CL2A-SN-38 (TV ═ 0.24 ± 0.11cm, respectively)³Comparison 1.17. + -. 0.45cm³And 1.05. + -. 0.73cm³；AUC_{21 days}P<0.001) or control mice treated with irinotecan at 10-fold higher SN-38 equivalent doses (TV 0.27 ± 0.18cm, respectively)³Comparison is made at 0.90. + -. 0.62cm³；AUC_{25 days}P<0.004) (not shown), again, saxilizumab gavatikan significantly inhibited tumor growth. Interestingly, in mice bearing SK-MES-1 human squamous cell lung tumor treated with 0.4mg/kg ADC (not shown), tumor growth inhibition was superior to saline or unconjugated hRS7IgG (TV 0.36 ± 0.25cm, respectively)³Comparison 1.02. + -. 0.70cm³And 1.30. + -. 1.08cm³；AUC_{28 days}，P<0.043), but not hA20-CL2A-SN-38 or irinotecan, provides conjugation with specific hRS7-SN-38The same antitumor effect of the compound (not shown). In all murine studies, hRS7-SN-38 ADC was well tolerated in weight loss (not shown).

Biodistribution of Saxizumab gavitikangUsing the corresponding¹¹¹In-labeled substrate, comparison of the biodistribution of either Saxizumab govitegradn or unconjugated hRS7IgG In mice bearing SK-MES-1 human squamous cell lung carcinoma xenografts (not shown). Pharmacokinetic analysis was performed to determine clearance of saxizumab govitikang relative to unconjugated hRS7 (not shown). Clearance of the ADC was faster than an equivalent amount of unconjugated hRS7, and the ADC exhibited a shorter half-life and average residence time of about 40%. Nevertheless, this had little effect on tumor uptake (not shown). Although there was a significant difference between the 24 hour and 48 hour time points, by 72 hours (peak uptake) the amount of both agents in the tumor was similar. Among normal tissues, differences between liver and spleen were most prominent (not shown). Saxizumab gavittacobra hRS7IgG (not shown) in liver 24 hours after injection>2 times. In contrast, in the spleen, there was 3-fold more parent hRS7IgG to pizza plus gavitikang (not shown) at peak uptake (48 hour time point). Uptake and clearance in the remaining tissues typically reflect differences in blood concentration (not shown).

Since twice weekly doses were given for treatment, the examination was at injection¹¹¹Tumor uptake In a group of animals that received first a pre-dose of 0.2mg/kg (250 μ g protein) of hRS7ADC 3 days before the In-labeled antibody. In predosed mice, compared to animals not receiving the predose¹¹¹Tumor uptake of In-saxizumab gavelutin was significantly reduced at each time point (e.g., at 72 hours, tumor uptake at predosed 12.5% + -3.8% ID/g versus 25.4% + -8.1% ID/g In animals not given predose; P ═ 0.0123; not shownhttp:// clincancerres.aacrjournals.org/content/17/10/3157.long-F4). The predose had no significant effect on blood clearance or tissue uptake (not shown). These studies suggest that in many tumor models, the reduction in tumor size can be achieved by previous dosesThe tumor of the heterotypic antibody increased, which might explain why the specificity of the therapeutic response could be reduced with increasing ADC dose and why no further dose escalation was indicated.

Tolerance of saxizumab govitikang in swiss-weibert mice and cynomolgus monkeysSwiss-Webster mice tolerated 2 doses of each of 4mg, 8mg and 12mg SN-38/kg of Saxizumab govitegradn within 3 days with minimal transient weight loss (not shown). No hematopoietic toxicity occurred and serum chemistry showed only elevated aspartate aminotransferase (AST, not shown) and alanine aminotransferase (ALT, not shown). AST elevation above normal levels in all 3 treatment groups (not shown) 7 days post-treatment: (>298U/L), the largest proportion of mice in the 2X 8mg/kg group. However, most animals were within the normal range 15 days after treatment. ALT levels were also above the normal range within 7 days of treatment (>77U/L) (not shown), and there was evidence of normalization on day 15. The livers from all these mice showed no histological evidence of tissue damage (not shown). With respect to renal function, only glucose and chloride levels were slightly elevated in the treatment group. At 2X 8mg/kg, 5 of 7 mice had slightly elevated glucose levels (ranging from 273mg/dL to 320mg/dL with an upper normal limit of 263mg/dL), which returned to normal 15 days after injection. Also, chloride levels were slightly elevated, 116mmol/L to 127mmol/L (upper limit of normal range 115mmol/L) in the 2 highest dose groups (57% of mice in the 2X 8mg/kg group and 100% of mice in the 2X 12mg/kg group), and remained elevated for up to 15 days after injection. This may also indicate gastrointestinal toxicity, as most chloride is obtained by intestinal absorption; however, at termination, there was no histological evidence of tissue damage in any organ system examined (not shown).

Because mice do not express Trop-2 target antigen of hRS7 in normal tissues, a more appropriate model is needed to determine the potential of hRS7 conjugates for clinical use. Immunohistological studies revealed binding in various tissues of human and cynomolgus monkey (breast, eye, gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas, parathyroid gland, prostate, salivary gland, skin, thymus, thyroid, tonsil, ureter and urinary tract; not shown). Based on this cross-reactivity, tolerance studies were performed in monkeys.

The group receiving 2X 0.96mg SN-38/kg of Saxizumab govitegam had no significant clinical events after infusion and after the end of the study. Weight loss did not exceed 7.3% and returned to acclimatized weight on day 15. Transient decreases were not noted in most of the blood count data (not shown), but values were not below the normal range. No outliers were found in serum chemistry. Histopathology of animals at necropsy on day 11 (8 days after the last injection) showed microscopic changes in hematopoietic organs (thymus, mandible and mesenteric lymph nodes, spleen and bone marrow), gastrointestinal organs (stomach, duodenum, jejunum, ileum, caecum, colon and rectum), female reproductive organs (ovary, uterus and vagina) and injection sites. These changes ranged from minimal to moderate, and were completely reversed at the end of the recovery period (day 32) in all tissues except in the thymus and gastrointestinal tract, with a tendency to complete recovery at time points thereafter (not shown).

Of the 2X 1.92mg SN-38/kg dose levels of the conjugate, 1 died due to gastrointestinal complications and myelosuppression, and the other animals within this group showed similar but more severe adverse events as the 2X 0.96mg/kg group (not shown). These data indicate that the dose limiting toxicity is the same as irinotecan; namely, intestinal tract and hematology. Thus, the MTD of Saxizumab govitegam is between 2X 0.96mg and 2X 1.92mg SN-38/kg, which represents a human equivalent dose of 2X 0.3 to 0.6mg/kg SN-38.

Discussion of the related Art

Trop-2 is a protein expressed on many epithelial tumors, including lung, breast, colorectal, pancreatic, prostate, and ovarian cancers, making it a potentially important target for the delivery of cytotoxic agents (Ohmachi et al, 2006, Clin Cancer Res 12: 3057-63; Fong et al, 2008, Br J Cancer 99: 1290-95; Cubas et al, 2009, Biochim biophysis Acta 1796: 309-14). When bound to Trop-2, the RS7 antibody internalizes (Shih et al, 1995, Cancer Res 55:5857s-63s), which enables direct intracellular delivery of cytotoxins.

SN-38 is a potent topoisomerase-I inhibitor, IC in several cell lines₅₀Values are in the nanomolar range. It is the active form of the prodrug irinotecan, useful for the treatment of colorectal cancer, and also active in lung, breast and brain cancers. We conclude that directly targeted SN-38 in ADC form would be a significantly improved therapeutic over CPT-11 by overcoming the latter's low and patient-varying biotransformation to active SN-38 (Mathijssen et al, 2001, Clinccancer Res 7: 2182-94).

The Phe-Lys peptide inserted in the original CL2 derivative allows for possible cleavage by cathepsin B. To simplify the synthesis, phenylalanine was eliminated in CL2A, thus eliminating the cathepsin B cleavage site. Interestingly, the product had a better defined chromatogram than the broad spectrum obtained with CL2 (not shown), but more importantly, this change had no effect on the binding or stability of the conjugate and surprisingly produced a small increase in potency in the side-by-side test.

In vitro cytotoxicity of hRS7ADC on a range of solid tumor cell lines consistently had IC in the nmol/L range₅₀The value is obtained. However, cells exposed to free SN-38 demonstrated lower IC compared to ADC₅₀The value is obtained. This difference between free SN-38 and conjugated SN-38 has also been reported for ENZ-2208(Sapra et al, 2008, Clin Cancer Res14: 1888-96; Zhao et al, 2008, bioconjugate Chem19:849-59) and NK012(Koizumi et al, 2006, Cancer Res 66: 10048-56)). ENZ-2208 uses branched PEG to attach about 3.5 to 4 SN-38 molecules per PEG, while NK012 is a micellar nanoparticle containing 20 wt% SN-38. With our ADC, as Trop-2 expression levels in tumor cells increase, this difference (i.e., the ratio of the potency of free SN-38 over conjugated SN-38) decreases, suggesting an advantage for targeted drug delivery. In terms of in vitro serum stability, both the CL2-SN-38 and the CL2A-SN-38 form of hRS7-SN-38 produce t ^ er/standard of about 20 hours_1/2This is in contrast to ENZ-2208Reported short t ^ er of 12.3 minutes_1/2(Zhao et al, 2008, bioconjugg Chem19:849-59) but similar to 57% release of SN-38 from NK012 after 24 hours under physiological conditions (Koizumi et al, 2006, Cancer Res 66: 10048-56).

Treatment of tumor-bearing mice with hRS7-SN-38 (with CL2-SN-38 or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumor models. In 4 of these, tumor regression was observed, and in the case of Calu-3, all mice receiving the highest dose of hRS7-SN-38 were tumor-free at the end of the study. Unlike humans, irinotecan is converted very efficiently to SN-38 by plasma esterases in mice with conversions greater than 50% and produces greater efficacy in mice than in humans (Morton et al, 2000, Cancer Res60: 4206-10; Furman et al, 1999, J Clin Oncol17: 1815-24). hRS7-SN-38 was significantly better at controlling tumor growth when irinotecan was administered at SN-38 levels more than 10-fold or comparable. Irinotecan was comparable in efficacy to hRS7-SN-38 only when administered at an MTD of 24mg/kg q2dx5(37.5 times greater SN-38). In patients, we would expect this advantage to be even more favorable for the saxizumab govitikang, since the biotransformation of irinotecan would be greatly reduced.

We also show in some cell lines expressing antigen, such as SK-MES-1, that using antigen-binding ADCs cannot guarantee a better therapeutic response than non-binding unrelated conjugates. This is not an unusual or unexpected finding. Indeed, the aforementioned non-binding SN-38 conjugates enhance therapeutic activity when compared to irinotecan, and therefore unrelated IgG-SN-38 conjugates are expected to have some activity. This is related to the fact that tumors have immature leaky vessels that allow macromolecules to pass better than normal tissues (Jain,1994, Sci Am 271: 58-61). With our conjugate, 50% of the SN-38 was released within about 13 hours when the pH was lowered to a level that mimics lysosomal levels (e.g., pH 5.3 at 37 ℃; data not shown), while at neutral pH the release rate was reduced by nearly 2-fold. If an unrelated conjugate enters the acidic tumor microenvironment, some local release of SN-38 is expected. Other factors such as tumor physiology and innate sensitivity to drugs will also play a role in defining this "baseline" activity. However, specific conjugates with longer residence times will have enhanced potency over this baseline response, as long as there is sufficient antigen to capture the specific antibody. Biodistribution studies in the SK-MES-1 model also show that if tumor antigens become saturated due to continuous dosing, the tumor uptake of specific conjugates decreases, which yields similar therapeutic results to those found with unrelated conjugates.

Although a direct comparison between our reported reports of ADCs and other SN-38 delivery agents is challenging, some general observations can be made. In our treatment study, the highest individual dose was 0.4mg/kg SN-38. In the Calu-3 model, only 4 injections were given in 20g mice for a total cumulative dose of 1.6mg/kg SN-38 or 32. mu.g SN-38. Multiple studies of ENZ-2208 were performed with its MTD of 10mg/kg × 5 (Sapra et al, 2008, Clin Cancer Res14: 1888-96; Pastorini et al, 2010, Clin Cancer Res 16:4809-21) and preclinical studies of NK012 involved its MTD of 30mg/kg × 3(Koizumi et al, 2006, Cancer Res 66: 10048-56). Thus, significant antitumor effects were obtained with hRS7-SN-38 at 30-fold and 55-fold fewer SN-38 equivalents than the doses reported in ENZ-2208 and NK012, respectively. Even less than 10-fold hRS7ADC (0.04mg/kg), significant antitumor effects were observed without presenting lower doses of ENZ-2208, and efficacy was lost when NK012 doses were reduced 4-fold to 7.5mg/kg (Koizumi et al, 2006, cancer res 66: 10048-56). Normal mice showed no acute toxicity, with a cumulative dose of 24mg/kg SN-38(1,500mg/kg conjugate) at 1 week, indicating a higher MTD. Thus, tumor-bearing animals are effectively treated with 7.5 to 15 times lower amounts of SN-38 equivalent.

Biodistribution studies revealed that saxizumab gavelutinkang has similar tumor uptake as the parent hRS7IgG, but clearance rates were significantly faster, with liver uptake 2-fold higher, probably due to the hydrophobic nature of SN-38. As ADC is cleared by the liver, it is expected that hepatic and gastrointestinal toxicity will be dose limiting. Although mice have evidence of elevated liver transaminase, gastrointestinal toxicity is best mild with only a brief loss of body weight and no abnormalities in histopathological examination. Interestingly, no hematologic toxicity was noted. However, monkeys show the same toxicity profile as expected for irinotecan, with gastrointestinal and hematologic toxicity being dose limiting.

Since Trop-2 recognized by hRS7 is not expressed in mice, it is important to perform toxicity studies in monkeys that have Trop-2 tissue expression similar to humans. Monkeys tolerated 0.96 mg/kg/dose (approximately 12 mg/m) ²) With mild and reversible toxicity, extrapolated to about 0.3 mg/kg/dose (about 11 mg/m)²) Human dose of (a). In phase I clinical trial of NK012, solid tumor patients tolerated 28mg/m every 3 weeks²SN-38 of (1), wherein grade 4 neutropenia acts as a dose-limiting toxicity (DLT; Hamaguchi et al, 2010, Clin Cancer Res16: 5058-66). Similarly, phase I clinical trials with ENZ-2208 revealed that dose-limiting febrile neutropenia, recommending 10mg/m administration every 3 weeks²Or 16mg/m if G-CSF is administered to the patient²(Kurzrock et al, AACR-NCI-EORTC International conference on Molecular Targets and Cancer Therapeutics; 2009, 11 months 15-19; Boston, MA; Poster No C216; Patnaik et al, AACR-NCI-EORTC International conference on Molecular Targets and Cancer Therapeutics; 2009, 11 months 15-19; Boston, MA; Poster No C221). Since monkeys tolerate 22mg/m²The cumulative human equivalent dose of (a), it appears that even though hRS7 bound to many normal tissues, the MTD of a single treatment of hRS7ADC may be similar to other non-targeted SN-38 agents. Indeed, the specificity of the anti-Trop-2 antibody appears to have no effect in defining DLT, as the toxicity profile is similar to that of irinotecan. More importantly, if anti-tumor activity can be achieved in humans, as in mice responding to a human equivalent dose of only 0.03mg SN-38 equivalents/kg/dose, a clinically significant anti-tumor response can be achieved.

In summary, in combination with an in vivo human cancer xenograft model in mice, toxicology studies in monkeys indicate that this Trop-2-targeting ADC is an effective therapeutic agent in several tumors of different epithelial origin.

Example 5 cell binding assay for anti-Trop-2 antibodies

Two different murine monoclonal antibodies against human Trop-2 were obtained for ADC conjugation. Initially 162-46.2 was purified from hybridomas (ATCC, HB-187) grown in roller bottles. Second antibody MAB650 from R&D Systems (Minneapolis, MN). To compare binding, Trop-2 positive human gastric cancer NCI-N87 was used as a target. Cells (1.5X 10) were plated the day before the binding assay⁵/well) were plated into 96-well plates. The next morning, dose/response curves were generated with 162-46.2, MAB650 and murine RS7(0.03nM to 66 nM). These primary antibodies were incubated with the cells at 4 ℃ for 1.5 hours. The wells were washed and an anti-mouse-HRP secondary antibody was added to all wells for 1 hour at 4 ℃. The wells are washed again and then the luminogenic substrate is added. Plates were read using an Envision plate reader and values were reported as relative luminescence units.

All three antibodies have similar Ks_DValue, K for RS7_DThe value was 0.57nM for 162-46.2, K_DThe value was 0.52nM and for MAB650, K_DThe value was 0.49 nM. However, when comparing 162-46.2 with maximum binding of MAB650 (B)_max) Maximum binding to RS7 (B)_max) They were reduced by 25% and 50%, respectively, compared to RS7 (B of RS7)_MaxB of 11,250, 162-46.2_Max8,471, and B of MAB650_Max6,018) indicating different binding characteristics.

Example 6 cytotoxicity of anti-Trop-2 ADC (MAB650-SN-38)

New anti-Trop-2 ADCs were prepared with SN-38 and MAB650, yielding an average drug-to-antibody substitution ratio of 6.89. Cytotoxicity assays were performed using two different human pancreatic cancer cell lines (BxPC-3 and Capan-1) and a human triple negative breast cancer cell line (MDA-MB-468) as targets to compare MAB650-SN-38 and saxizumab gavitikang ADC.

One day prior to addition of ADC, cells were harvested from tissue culture and plated into 96-well plates. The following day cells were exposed to the drug in the range of 3.84X 10^-12To 2.5X 10^-7M of Saxizumab govitegradn, MAB650-SN-38 and free SN-38. Unconjugated MAB650 was used at protein equivalent doses as a control for MAB 650-SN-38. The plates were incubated at 37 ℃ for 96 hours. After this incubation period, MTS substrate was added to all plates and color was read at half hour intervals until untreated cells reached an OD of about 1.0_492nm. Growth inhibition was measured as percent growth relative to untreated cells using Microsoft Excel and Prism software (non-linear regression to generate sigmoidal dose response curves that yield IC₅₀-a value.

Saxizumab gavoriconazole and MAB650-SN-38 have similar growth inhibition, IC₅₀Values in the low nM range, typical for SN-38-ADCs in these cell lines (not shown). Saxizumab gavitegam ADC showed IC in human Capan-1 pancreatic cancer cell line (not shown)₅₀3.5nM, in contrast, for MAB650-SN-38ADC, IC₅₀4.1nM and for free SN-38, IC₅₀Was 1.0 nM. Saxizumab gavitegam ADC shows IC in human BxPC-3 pancreatic cancer cell line (not shown)₅₀2.6nM, in contrast, for MAB650-SN-38ADC, IC₅₀3.0nM and for free SN-38, IC₅₀Was 1.0 nM. Saxizumab gavitegam ADC showed IC in human NCI-N87 gastric cancer cell line (not shown)₅₀3.6nM, in contrast, for MAB650-SN-38ADC, IC₅₀4.1nM and for free SN-38, IC₅₀Was 4.3 nM.

In summary, SN-38 conjugates of two anti-Trop-2 antibodies hRS7 and MAB650 showed the same efficacy against several tumor cell lines in these in vitro assays, similar to free SN-38. Since the targeting function of anti-Trop-2 antibodies will be a much more significant factor in vivo than in vitro, the data supports that anti-Trop-2-SN-38 ADCs as a class will be very effective in vivo, as demonstrated for the sablizumab gaulthikan in the above examples.

Example 7 cytotoxicity of anti-Trop-2 ADC (162-46.2-SN-38)

New anti-Trop-2 ADCs were prepared with SN-38 and 162-46.2, resulting in a drug to antibody substitution ratio of 6.14. Cytotoxicity assays were performed in BxPC-3 human pancreatic cancer and MDA-MB-468 human triple negative breast cancer using two different Trop-2-positive cell lines as targets to compare 162-46.2-SN-38 and hRS7-SN-38 ADCs.

One day prior to addition of ADC, cells were harvested from tissue culture and plated at 2000 cells/well into 96-well plates. The following day cells were exposed to the drug in the range of 3.84X 10^-12To 2.5X 10^-7M of Saxizumab govitegradn, 162-46.2-SN-38 or free SN-38. Unconjugated 162-46.2 and hRS7 were used at the same protein equivalent dose as controls for 162-46.2-SN-38 and saxilizumab govitegradine, respectively. The plates were incubated at 37 ℃ for 96 hours. After this incubation period, MTS substrate was added to all plates and color was read at half hour intervals until untreated control cells reached an OD of about 1.0_492nmAnd (6) reading. Growth inhibition was measured as percent growth relative to untreated cells using Microsoft Excel and Prism software (non-linear regression to generate sigmoidal dose response curves that yield IC₅₀-value).

162-46.2-SN-38 ADCs have similar IC compared to Saxizumab gavitin₅₀-value (not shown). IC of Saxizumab gavelvetikon when tested against BxPC-3 human pancreatic cancer cell line (not shown)₅₀5.8nM, in contrast, for 162-46.2-SN-38, IC₅₀10.6nM and for free SN-38, IC₅₀It was 1.6 nM. IC of Saxizumab gavietin when tested against MDA-MB-468 human breast cancer cell line (not shown)₅₀The concentration of the active carbon was 3.9nM,in contrast, for 162-46.2-SN-38, IC₅₀6.1nM and for free SN-38, IC₅₀It was 0.8 nM. Free antibody alone showed little cytotoxicity against Trop-2 positive cancer cell lines.

In summary, all three ADCs exhibited equivalent cytotoxic effects against multiple Trop-2 positive cancer cell lines, comparing the in vitro efficacy of three different anti-Trop-2 antibodies conjugated to the same cytotoxic drug. These data support that one class of anti-Trop-2 antibodies incorporated into drug-conjugated ADCs are potent anti-cancer therapeutics for Trop-2 expressing solid tumors.

Example 8 clinical trials with IMMU-132 (Saxizumab-gaulthikang) anti-Trop-2 ADC comprising an hRS7 antibody conjugated with SN-38

SUMMARY

This example reports the results of a phase I clinical trial with IMMU-132 and an ongoing phase II extension, where IMMU-132 is an ADC of an internalized humanized hRS7 anti-Trop-2 antibody conjugated to SN-38 via a pH sensitive linker (average drug-antibody ratio 7.6). Trop-2 is a type I transmembrane calcium transducin produced by many human carcinomas at high density (approximately 1X 10)⁵) Frequency and specificity of expression, with limited normal tissue expression. Preclinical studies in nude mice bearing a Capan-1 human pancreatic tumor xenograft revealed that IMMU-132 was able to deliver up to 120-fold more SN-38 to tumors than SN-38 derived from maximally tolerated irinotecan therapy.

This example reports an initial phase I trial of 25 patients who failed multiple previous treatments (some including topoisomerase-I/II inhibitory drugs), and an ongoing phase II extension now reports 69 patients including colorectal cancer (CRC), small cell and non-small cell lung cancer (SCLC, NSCLC, respectively), Triple Negative Breast Cancer (TNBC), pancreatic cancer (PDC), esophageal cancer, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but was found to be largeStrong expression in most archived tumors (. gtoreq.2)⁺Immunohistochemical staining). In the 3+3 trial design, IMMU-132 was administered on days 1 and 8 of a repeated 21-day cycle, starting at 8 mg/kg/dose and then 12mg/kg and 18mg/kg prior to dose-limiting neutropenia. To optimize cumulative treatment with minimal delay, phase II centered on 8mg/kg and 10mg/kg (n ═ 30 and 14, respectively). Of 49 patients reporting related AEs at this time, moderate granulocytopenia ≧ 3 occurred in 28% (4% grade 4). The most common non-hematologic toxicities initially seen in these patients were fatigue (55%; > G3 ═ 9%), nausea (53%; > G3 ═ 0%), diarrhea (47%; > G3 ═ 9%), alopecia (40%) and vomiting (32%; > G3 ═ 2%); alopecia also occurs frequently. Homozygote UGT1a1 x 28/' 28 was found in 6 patients, 2 of which had more severe hematological and GI toxicity.

In phase I and extended, 48 patients (not including PDC) are now evaluated by RECIST/CT for optimal response. 7 (15%) patients had Partial Response (PR) including CRC patient (N ═ 1), TNBC patient (N ═ 2), SCLC patient (N ═ 2), NSCLC patient (N ═ 1) and esophageal cancer patient (N ═ 1), and 27 patients (56%) had Stable Disease (SD), with a total of 38 patients (79%) having disease response; 8 of the 13 CT evaluable PDC patients (62%) had SD with a median Time To Progression (TTP) of 12.7 weeks, compared to 8.0 weeks in the last treatment. The remaining 48 patients had a TTP of 12.6+ weeks (range 6.0 to 51.4 weeks). Plasma CEA and CA19-9 were associated with increased titers of these antigens in blood. Despite months of administration, no anti-hRS 7 or anti-SN-38 antibodies were detected.

The conjugate cleared from serum within 3 days, consistent with an in vivo animal study in which 50% of SN-38 was released daily, > 95% of SN-38 in serum bound to the non-glucuronidated form of IgG, and at a concentration 100-fold higher than that reported for patients administered irinotecan. These results show that saxizumab govitikang is therapeutically active in metastatic solid cancers with controlled diarrhea and neutropenia.

Pharmacokinetics

Both ELISA methods were used to measure clearance of IgG (captured with anti-hRS 7 idiotype antibody) and clearance of intact conjugate (captured with anti-SN-38 IgG/probed with anti-hRS 7 idiotype antibody). SN-38 was measured by HPLC. The total IMMU-132 fraction (intact conjugate) cleared faster than IgG (not shown), reflecting the known gradual release of SN-38 from the conjugate. HPLC determination of SN-38 (unbound SN-38 and total SN-38) showed > 95% SN-38 in serum bound to IgG. Low concentrations of SN-38G suggest that SN-38 bound to IgG is protected from glucuronidation. Comparison of the ELISA for the conjugate and SN-38 HPLC revealed that the two overlap, suggesting that ELISA is an alternative to monitoring SN-38 clearance.

A summary of dosing regimens and patient pools is provided in table 3.

TABLE 3 clinical trial parameters

Clinical trial status

A total of 69 patients (including 25 patients in stage I) were reported to have multiple metastatic cancers, with a number of 3 prior treatments. 8 patients had clinical progression and were withdrawn prior to CT assessment. 13 CT evaluable pancreatic cancer patients were reported separately. The median TTP (time to progression) for PDC patients was 11.9 weeks (ranging from 2 to 21.4 weeks) compared to the median of 8-week TTP from the previous treatment.

A total of 48 patients with different cancers were evaluated for at least 1 CT from which the best response (not shown) and time to progression (TTP; not shown) were determined. To summarize the best response data for 8 evaluable TNBC (triple negative breast cancer) patients, 6 of 8 had a total response [ PR + SD ] (75%), 2 PR (partial response), 4 SD (stable disease) and 2 PD (progressive disease). For SCLC (small cell lung cancer), among the 4 evaluable patients, 2 out of 4 had a total response (50%), 2 PR, 0 SD and 2 PD. For CRC (colorectal lung cancer), among the 18 evaluable patients, 12 of the 18 had a total response (67%), 1 PR, 11 SD and 6 PD. For esophageal cancer, among 4 evaluable patients, 3 out of 4 had a total response (75%), 1 PR, 2 SD and 1 PD. For NSCLC (non-small cell lung cancer), among 5 evaluable patients, 4 out of 5 had a total response (80%), 1 PR, 3 SD and 1 PD. Of all patients treated, among 48 evaluable patients, there were total responses (71%), 7 PR, 27 SD and 14 PD for 34 of 48. These results demonstrate that anti-TROP-2 ADC (hRS7-SN-38) shows significant clinical efficacy against a wide range of solid tumors in human patients.

The reported side effects of treatment (adverse events) are summarized in table 4. As is apparent from the data in table 4, therapeutic efficacy of saxizumab govitikang was achieved at the dose of ADC, showing acceptably low levels of adverse side effects.

Table 4.

The study reported in table 4 was continued and 261 patients were enrolled to date. Results (not shown) were generally performed along the lines shown in table 4, with only neutropenia showing that more than 10% of the patients tested developed grade 3 or higher adverse events. The incidence of grade 3 or higher responses was below 10% for all other adverse events. This distinguishes the ADCs of the present invention from the vast majority of ADCs, and in certain embodiments, the claimed methods and compositions relate to anti-Trop-2 ADCs that exhibit efficacy in a variety of solid tumors, with less than 10% of patients experiencing grade 3 or greater adverse events for all adverse events except neutropenia. In a subsequent study, no anti-hRS 7 or anti-SN-38 antibody response was detected in a total of 421 samples from 121 baseline patients and at least one available follow-up sample, despite repeated multiple treatment cycles.

The CT data confirms an exemplary partial response (not shown) against the Trop-2 ADC. As an exemplary PR in CRC, a 62 year old female first diagnosed with CRC underwent primary hemisection. Four months later, she underwent hepatectomy for liver metastasis and received 7 months of treatment with FOLFOX and 1 month of 5FU treatment. She presented multiple lesions mainly in the liver (3 + Trop-2 according to immunohistology) and entered the saxizumab govitegradine test at an initial dose of 8mg/kg for about 1 year after initial diagnosis. PR was achieved with a 37% reduction in target lesions in her first CT assessment (not shown). The patient continued treatment with a maximum 65% reduction in CEA after 10 months of treatment (not shown) from 781ng/mL to 26.5ng/mL, then progressed after 3 months.

As an exemplary PR in NSCLC, a 65 year old male is diagnosed with stage IIIB NSCLC (squamous cell). Initial treatment of carboplatin/etoposide with 7000cGy XRT (3 months) resulted in a response lasting 10 months. He then started to receive a Tarceva (Tarceva) maintenance treatment, and he had considered performing the IMMU-132 trial except for receiving a lumbar laminectomy. He received the first dose of IMMU-132 after 5 months of Tarceva treatment, when the right lung presented 5.6cm lesions and abundant pleural effusion. Two months later, when the first CT showed a reduction in the primary target lesion to 3.2cm, he had just completed the 6 th dose (not shown).

As an exemplary PR in SCLC, a 65 year old female was diagnosed with poorly differentiated SCLC. After receiving carboplatin/etoposide (topoisomerase-II inhibitor) ending with no response after 2 months, followed by topotecan (topoisomerase-I inhibitor), which likewise ends with no response after 2 months, she received topical XRT (3000cGy), ending after 1 month. However, by the next month, progress continues. The patient began using IMMU-132(12 mg/kg; decreased to 6.8 mg/kg; Trop-2 expression 3+) the next month and after 2 months of IMMU-132 treatment, target lesions were reduced by 38%, including a significant reduction in the occurrence of major lung lesions (not shown). Patients progressed after 3 months after receiving 12 doses.

These results are significant because they demonstrate that anti-Trop-2 ADCs are effective even in patients who fail or progress after multiple previous treatments. In summary, the primary toxicity at the doses used was controlled neutropenia, with few grade 3 toxicities. IMMU-132 shows evidence of activity (PR and durable SD) in relapsed/refractory patients with triple negative breast, small cell lung, non-small cell lung, colorectal and esophageal cancer, including patients who had a previous history of relapse with topoisomerase-I inhibitors. These results show the efficacy of anti-Trop-2 ADCs in a wide range of cancers that are resistant to existing therapies.

Example 9 comparative efficacy of different anti-Trop-2 ADCs

Comparison of the therapeutic efficacy of a mouse anti-Trop-2 monoclonal antibody (162-46.2) conjugated to SN-38 with that of a saxizumab gavatican-drug conjugate (ADC) was performed in mice bearing a human gastric cancer xenograft (NCI-N87). NCI-N87 cells were expanded in tissue culture and harvested with trypsin/EDTA. Female athymic nude mice were administered 1X 10 cells per mouse by subcutaneous injection of 200. mu.l of NCI-N87 cell suspension mixed with matrigel 1:1⁷And (4) cells. Once the tumor size reached about 0.25cm³(after 6 days) the animals were divided into 7 different treatment groups of 9 mice each. For SN-38ADC, mice received 500 μ g intravenous injections once a week for two weeks. Control mice received non-tumor targeting hA20-SN-38ADC at the same dose/schedule. The last group of mice received saline only and served as untreated controls. Tumors were measured and mice were weighed twice weekly. If the tumor volume of the mice exceeds 1.0cm³Of mice were then euthanized for disease progression.

The average tumor volume of SN-38-ADC treated mice was determined (not shown). Both the saxizumab govitegam and 162-46.2-SN-38 significantly inhibited tumor growth when compared to saline and hA20-SN-38 control mice (P <0.001), as determined by area under the curve (AUC). Disease stabilization was achieved with saxizumab govitikang treatment in 7 out of 9 mice with a mean time To Tumor Progression (TTP) of 18.4 ± 3.3 days. Mice treated with 162-46.2-SN-38 achieved positive responses in 6 of 9 mice, with the remaining 3 achieving disease stabilization. The mean TTP was 24.2 ± 6.0 days, significantly longer than the saxizumab gowittign treated animals (P ═ 0.0382).

These results confirm the in vivo efficacy of different anti-Trop-2 ADCs for the treatment of human gastric cancer.

Example 10 overview of treatment of patients with advanced metastatic pancreatic cancer with anti-Trop-2 ADC

IMMU-132 (sapizumab goverikang) is an anti-Trop-2 ADC comprising a cancer cell internalizing humanized anti-Trop-2 hRS7 antibody conjugated to irinotecan's active metabolite SN-38 through a pH-sensitive linker with an average drug-antibody ratio of 7.6. Trop-2 is a type I transmembrane calcium transduction protein expressed at high density, frequency and specificity in many epithelial cancers including pancreatic ductal adenocarcinoma, with limited normal tissue expression. All 29 pancreatic tumor microarray samples tested by immunohistochemistry were Trop-2 positive and human pancreatic cancer cell lines were found to express 115k-891kTrop-2 copies on the cell membrane.

We report above the results of an IMMU-132 phase I study that recruited 13 patients with different tumor types using a 3+3 design. Phase I dose-limiting toxicity is neutropenia. Over 80% of 24 patients evaluated in this study had long-term stable disease, with partial Response (RECIST) observed in patients with colorectal cancer (CRC), Triple Negative Breast Cancer (TNBC), small and non-small cell lung cancer (SCLC, NSCLC) and Esophageal (EAC) cancer. This example reports the results of a phase IMMU-132I/II study cohort of patients with metastatic PDC. A median of 2 PDC patients who failed previous treatments (ranging from 1 to 5) were given IMMU-132 on days 1 and 8 of a repeated 21-day cycle.

Of the PDC patient subgroup (N ═ 15), 14 received previous gemcitabine-containing treatment regimens. Initial toxicity data from 9 patients neutropenia was found [ 3 of 9 ≧ G3, 33%; and 1 with G4 febrile neutropenia ], resulting in dose delay or dose reduction. 2 patients had grade 3 diarrhea; no patients experienced grade 3 to 4 nausea or vomiting. Of 9 patients, 5 developed hair loss (grade 1 to 2). The best response was evaluated in 13 of 14 patients, 8 of which were stable for 8 to 21.4 weeks (median 12.7 weeks; 11.9 weeks for all 14 patients). One patient who is continuing treatment has not yet been evaluated for first CT. Wherein five people have progressive disease according to RECIST; due to clinical progression, 1 dose was followed by 1 withdrawal and was not evaluated. The serum CA19-9 titer of 3 stable disease patients decreased by 23% to 72%. No patient produced an antibody response to IMMU-132 or SN-38 despite multiple administrations. Peak and trough serum samples showed faster clearance of IMMU-132 than IgG, which was expected based on the known local release of SN-38 within tumor cells. The SN-38 concentration bound to IgG in the peak sample from one patient administered IMMU-132 at 12mg/kg showed a level of about 4000ng/mL, which is 40-fold higher than the SN-38 titer reported for patients treated with irinotecan.

We conclude that IMMU-132 is active in 62% (8/13) PDC patients who failed multiple previous treatments (long-term stable disease), with controlled neutropenia and little GI toxicity. On days 1 and 8 of the 21-day cycle, patients with advanced PDC may be given a repeat treatment cycle (>6) of 8 to 10mg/kg IMMU-132 with some dose modulation or growth factor support for neutropenia in subsequent treatment cycles. The results of these studies are consistent with the results of patients with advanced CRC, TNBC, SCLC, NSCLC, EAC, who showed partial response and long-term stable disease upon IMMU-132 administration. In summary, monotherapy IMMU-132 is a novel and effective treatment regimen for PDC patients, including patients with tumors that have previously been resistant to other treatment regimens for PDC.

Method and results

Trop-2 expressionUse by flow cytometryPE beads were assayed for Trop-2 expression on the surface of various cancer cell lines. The number of Trop-2 molecules detected in different cell lines results: BxPC-3 pancreatic cancer (891,000); NCI-N87 gastric cancer (383,000); MDA-MB-468 breast cancer (341,000); SK-MES-1 squamous cell lung carcinoma (27,000); capan-1 pancreatic cancer (115,000); AGS gastric cancer (78,000) COLO 205 colon cancer (52,000). Trop-2 expression was also observed in 29 of 29 pancreatic cancer tissue microarrays (100%) (not shown).

SN-38 accumulationSN-38 accumulation in nude mice bearing Capan-1 human pancreatic cancer xenografts (about 0.06 to 0.27 g). Mice were injected intravenously with irinotecan 40mg/kg (773 μ g; total SN-38 equivalents 448 μ g). This dose is the MTD of the mice. Human dose equivalent is 3.25mg/kg or about 126mg/m². Alternatively, mice were injected intravenously with IMMU-1321.0mg (SN-38: antibody ratio 7.6; Sn-38 equivalent 20. mu.g). This dose is much lower than the MTD of the mice. The human equivalent dose is about 4mg/kg IMMU-132 (about 80. mu.g/kg SN-38 equivalents). Necropsies were performed on 3 animals at each of the following time intervals, at 5 minutes, 1 hour, 2 hours, 6 hours, and 24 hours in irinotecan-injected mice or at 1 hour, 6 hours, 24 hours, 48 hours, and 72 hours in IMMU-132-injected mice. Tissues were extracted and analyzed by reverse phase HPLC analysis of SN-38, SN-38G and irinotecan. Extracts from IMMU-132 treated animals were also acid hydrolyzed to release SN-38 (i.e., SN-38 (Total) from the conjugate]). The results demonstrate that IMMU-132ADC is likely to deliver more than 120-fold more SN-38 to tumors compared to irinotecan, although less than 22-fold equivalent SN-38 was administered with ADC (not shown).

IMMU-132 clinical protocolThe protocol used for the phase I/II study is indicated in table 5 below.

IMMU-132 was administered to the patient according to the protocol outlined above. An exemplary case study is as follows. A 34 year old white man male initially diagnosed with metastatic pancreatic cancer (liver) progressed on multiple chemotherapy regimens including gemcitabine/erlotinib/FG-3019, FOLFIRINOX and GTX prior to the introduction of IMMU-132(8mg/kg dose administered on days 1 and 8 of a 21 day cycle). Patients received drug treatment for 4 months with good symptom tolerance, improved pain, a maximum 72% reduction in CA19-9 (from 15885U/mL to 4418U/mL), and stable disease according to CT RECIST criteria, with evidence of tumor necrosis. Treatment must be suspended due to liver abscesses; the patient then stopped for about 6 weeks, followed by 6 months of treatment.

Conclusion

Preclinical studies indicate that IMMU-132 delivers 120-fold amounts of SN-38 to human pancreatic tumor xenografts compared to when irinotecan is administered. As part of a large study in which patients with multiple metastatic solid cancers were recruited, the phase 2 dose of IMMU-132 was determined to be 8mg/kg to 10mg/kg based on controlled neutropenia and diarrhea as major side effects. To date no anti-antibody or anti-SN-38 antibody has been detected, even in repeated cycles of treatment.

One study of 14 patients with advanced PDC who relapsed after a median of 2 prior treatments showed that the anti-tumor activity confirmed by CT consisted of 8/13 (62%) patients with stable disease. The median duration of TTP for the 13 CT evaluable patients was 12.7 weeks, compared to 8.0 weeks estimated for the last previous treatment. Known drug ADCs with nanomolar toxicity conjugated to antibody-targeted Trop-2, which is ubiquitous on many epithelial cancers, by providing cleaved linkers at the tumor site represent a new effective strategy for pancreatic cancer treatment with ADCs. The prolongation of progression time of pancreatic cancer patients, particularly patients resistant to various prior treatments, compared to the current standard of care for pancreatic cancer patients is surprising and unpredictable.

Example 11 combining antibody-targeted radiation (radioimmunotherapy) with anti-Trop-2-SN-38 ADC improves pancreatic cancer treatment

We have previously reported⁹⁰Y-humanized PAM4IgG (hPAM 4;⁹⁰Y-Securlizumab tetraxetan) in nude mice bearing human pancreatic tumors, enhanced in combination with Gemcitabine (GEM) (Gold et al, Int J. cancer 109:618-26, 2004; clin Cancer Res 9:3929S-37S, 2003). These studies have led to ranking in combination with GEM⁹⁰Clinical testing of Y-hPAM4IgG showed encouraging objective response. While GEM is known for its radiosensitizing ability, it is not a very effective pancreatic cancer therapeutic by itself and its dose is limited by hematologic toxicity, which is also true for⁹⁰Y-hPAM4IgG was also limiting.

As discussed in the examples above, anti-Trop-2 ADCs composed of hRS7IgG linked to SN-38 show anti-tumor activity in various solid tumors. The ADC was very well tolerated in mice (e.g., ≧ 60mg), but only 4.0mg (0.5mg, twice weekly × 4) was significantly therapeutic. Trop-2 is also expressed in most pancreatic cancers.

This study examined the presence of a human pancreatic cancer cell line, Capan-1, at 0.35cm³In nude mice with subcutaneous xenografts⁹⁰A combination of Y-hPAM4IgG and Saxizumab gavoritan. Mice (n-10) alone with a single dose⁹⁰Y-hPAM4IgG (130. mu. Ci, i.e. Maximum Tolerated Dose (MTD) or 75. mu. Ci), treatment with Saxizumab gavatikang alone (as described above), or with both⁹⁰Y-hPAM4 agentAt a quantitative level of 2 doses with⁹⁰The first ADC injection given the same day as Y-hPAM4 for combination therapy. All treatments were tolerated with weight loss ≦ 15%. Objective responses occurred in most animals, but they were more robust in both combination groups than each agent administered alone. 0.13-mCi⁹⁰All animals in the Y-hPAM4IgG + saxizumab gavatica group reached a tumor-free state within 4 weeks, while other animals continued to have evidence of persistent disease. These studies provide the first evidence that combined radioimmunotherapy and ADC can enhance the efficacy at safe doses.

In the PAM4 clinical trial being performed, a four week clinical treatment cycle was performed. At week 1, the subject is administered¹¹¹A dose of In-hPAM4, followed by a dose of gemcitabine administered at least 2 days later. At weeks 2,3 and 4, subjects were administered⁹⁰Y-hPAM4 dose, followed by gemcitabine (200 mg/m) at least 2 days later²). Upgrade at 3X 6.5mCi/m²The following begins. The maximum tolerated dose of the baseline pancreatic cancer patient was 3X 15mCi/m²(hematologic toxicity is dose limiting). Of the 22 patients evaluable for CT, the disease control rate (CR + PR + SD) was 68%, with 5 (23%) patients having partial response and 10 (45%) patients having stable optimal response according to RECIST criteria.

Preparation of antibody-drug conjugates (ADCs)

SN-38 conjugated hRS7 antibodies were prepared as described above and according to previously described protocols (Moon et al, J MedChem 2008,51: 6916-. A reactive bifunctional derivative of SN-38 (CL2A-SN-38) was prepared. CL2A-SN-38 has the formula (maleimido- [ x ] -Lys-PABOCO-20-O-SN-38, where PAB is p-aminobenzyl and 'x' contains a short PEG). After reduction of the disulfide bond in the antibody with TCEP, CL2A-SN-38 was reacted with the reduced antibody to generate SN-38 conjugated RS 7.

Prepared as previously described⁹⁰Y-hPAM4(Gold et al, Clin Cancer Res 2003,9: 3929S-37S; Gold et al, Int J Cancer 2004,109:618-26)。

Combined RAIT + ADC

Trop-2 antigen is expressed in most epithelial cancers (lung, breast, prostate, ovarian, colorectal, pancreatic), and the saxizumab govitikang conjugates are examined in various human cancer-mouse xenograft models. By using⁹⁰Preliminary clinical trials of Y-hPAM4IgG plus a radiosensitizing amount of GEM were encouraging with evidence of tumor shrinkage or disease stabilization. However, treatment of pancreatic cancer is very challenging. Thus, the combination therapy was examined to determine if it would induce a better response. In particular, the administration of Saxizumab govitegam in an effective but non-toxic dose with⁹⁰RAIT combination of Y-hPAM4 IgG.

The results demonstrate that the Saxizumab govitikang and⁹⁰the combination of Y-hPAM4 was more effective than either therapy alone or the sum of the individual therapies (not shown). At 75. mu. Ci⁹⁰At the dose of Y-hPAM4, only 1 of 10 mice was tumor-free after 20 weeks of treatment (not shown), as observed with saxizumab gavatica alone (not shown). However, the combination of Saxizumab gavitikang and⁹⁰the combination of Y-hPAM4 resulted in 4 out of 10 mice without tumors after 20 weeks (not shown), and the remaining subjects showed significant reduction in tumor volume compared to either treatment alone (not shown). At 130. mu. Ci⁹⁰The difference was even more pronounced at Y-hPAM4, with 9 out of 10 animals in the combination treatment group being tumor-free compared to 5 out of 10 in the RAIT group alone (not shown). These data demonstrate that the Saxizumab gavelutin and⁹⁰synergistic effect of the combination of Y-hPAM 4. RAIT + ADC significantly improved the time to progression and increased the frequency of tumor-free treatment. Is added to the bag with⁹⁰The combination of ADC of the MTD of RAIT of Y-hPAM4 with saxizumab gagoniteachings had minimal additional toxicity, as indicated by% weight loss in animals responding to treatment (not shown).

The effect of different sequences of treatment on tumor survival indicates that the best effect was obtained when RAIT was administered first, followed by administration of ADC (not shown). In contrast, when ADC was administered first, followed by RAIT, the incidence of tumor-free animals decreased (not shown). Neither unconjugated hPAM4 nor hRS7 antibodies had antitumor activity when administered alone (not shown).

Example 12 treatment of metastatic Breast cancer refractory to therapy with Saxizumab gavoritacin (IMMU-132)

The patient was a 57 year old female with stage IV triple negative breast cancer (ER/PR negative, HER-neu negative), initially diagnosed in 2005. She received left lumpectomy in 2005 and then dose-intensive ACT adjuvant therapy in 9 months in 2005. She then received radiation therapy, which was completed in 11 months. When the patient palpated to determine a contralateral (right) breast lump in early 2012, local recurrence of the disease was identified and then treated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil) chemotherapy. Her disease recurred in the same year with metastatic lesions on the chest wall skin. Then she accepts carboplatin +A chemotherapy regimen during which thrombocytopenia develops. Her disease progressed and began to use doxorubicin weekly for 6 doses. Skin diseases are also progressing. The FDG-PET scan at 09/26/12 showed disease progression on the chest wall and enlarged solid axillary lymph nodes. Oxycodone is administered to patients for pain management.

When the chest wall lesion opened and bleeded, she was given from 10 months 2012 until 2 months 2013(once every 2 weeks for 4 months). Then give her a doseIt is not well tolerated due to neuropathy in the hands and feet and constipation. Skin lesions are progressive, howeverAnd then enrolled in the IMMU-132 trial after giving informed consent. The patient also had a history of hyperthyroidism and visual impairment, with a high risk of CNS disease (however, brain MRI is negative for CNS disease). When enrolled in the trial, the skin lesions (targets) of the right breast were measured to be 4.4 cm and 2.0 cm in maximum diameter. She had another non-target lesion in her right breast and had one enlarged lymph node in each of the right and left axilla.

The first IMMU-132 infusion (12mg/kg) started on 12/3/2013, well tolerated. The second infusion was delayed until one week later due to a reduction in Absolute Neutrophil Count (ANC) of grade 3 (0.9) on the scheduled day of infusion. After a delay of one week and after acceptanceThereafter, she was given a second dose of IMMU-132, which was reduced by 25% to 9 mg/kg. Thereafter, she received IMMU-132 on a schedule, once a week for 2 weeks, and then rested for a week, according to the protocol. After 3 treatment cycles, her first response assessment on day 17, 5 months, 2013 showed a 43% reduction in the sum of the long diameters of the target lesions, constituting a partial response according to RECIST criteria. She is continuing treatment at the 9mg/kg dose level. Since she began treatment with IMMU-132, her overall health and clinical symptoms were significantly improved.

Example 13 treatment of refractory metastatic small cell lung cancer with Saxizumab gavelutinkang (IMMU-132)

This is an MRI evidence of metastasis involving the left lung, mediastinal lymph node and left parietal brain in a 65 year old female diagnosed with small cell lung cancer. Previous chemotherapy included carboplatin, etoposide, and topotecan, but no response was noted. Radiation therapy also failed to control her disease. IMMU-132 was then given at a dose of 18mg/kg once every three weeks for a total of 5 infusions. After the second dose, she developed hypotension and grade 2 neutropenia, which improved prior to the next infusion. After the fifth infusion, the CT study showed that her target left lung mass contracted 13%. Brain MRI also showed 10% reduction in this metastasis. She continued her administration of IMMU-132 every 3 weeks for an additional 3 months and continued to show objective and subjective improvement in her condition with a 25% reduction in left lung mass and a 21% reduction in brain metastases.

Example 14 treatment of gastric cancer patients with stage IV metastatic disease with Saxizumab govitikang (IMMU-132)

This patient was a 60 year old male with a history of smoking and excessive alcohol intake for over 40 years. He experienced weight loss, diet discomfort and pain that could not be relieved with antacids, frequent abdominal pain, lower back pain and recently accessible lymph nodes in both axilla. He sought medical advice based on a biopsy through a gastroscope showing adenocarcinoma, including some squamous features, at the gastroesophageal junction after examination. Radiologic studies (CT and FDG-PET) also revealed metastatic disease in the left and right axilla, mediastinal region, lumbar spine and liver (2 tumors in the right lobe, 1 tumor in the left lobe, all measured diameters from 2cm to 4 cm). His gastric tumor was excised and then a course of chemotherapy was performed with epirubicin, cisplatin and 5-fluorouracil. After a rest period of 4 months and 6 weeks, he switched to docetaxel chemotherapy, which also failed to control his disease based on progression confirmed by metastatic tumors and some CT measurements of general exacerbations.

The patients were then given treatment with IMMU-132(hRS7-SN-38), infused with 10mg/kg doses every other week, for a total of 6 doses, before being subjected to CT studies to assess their disease status. These infusions were well tolerated with mild nausea and diarrhea and were controlled with symptomatic medication. The CT study revealed a 28% reduction in the sum of his exponential metastatic lesions, so he continued to receive the therapy for an additional 5 courses. The follow-up CT study showed that the disease remained reduced by about 35% from baseline measurements prior to IMMU-132 treatment according to RECIST criteria, and his general condition appeared to improve as well, with the patient regaining his optimistic attitude under control of his disease.

Example 15 clinical trials of IMMU-132 in various Trop-2-positive cancers

Abstract

Saxizumab govitegam (IMMU-132, also known as hRS7-CL2A-SN-38) is an antibody-drug conjugate (ADC) that targets the surface glycoprotein Trop-2 expressed on many epithelial tumors for the delivery of the active metabolite SN-38 of irinotecan. Unlike most ADCs that use a super toxic drug and a stabilizing linker, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate linkage between SN-38 and the linker. Flow cytometry and immunohistochemistry disclose that Trop-2 is expressed in a variety of tumor types including gastric, pancreatic, Triple Negative Breast (TNBC), colon, prostate, and lung cancers. Although cell binding experiments revealed no significant difference between IMMU-132 and the parental hRS7 antibody, surface plasmon resonance analysis using Trop-2CM5 chips showed that IMMU-132 had a significant binding advantage over hRS 7. The conjugate retained binding to neonatal receptors, but lost greater than 60% of antibody-dependent cell-mediated cytotoxic activity compared to hRS 7.

Exposure of tumor cells to free SN-38 or IMMU-132 demonstrates the same signaling pathway: pJNK1/2 and p21WAF1/Cip1 are upregulated, followed by cleavage of caspases 9, 7 and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA fragmentation. Pharmacokinetics of intact ADCs in mice revealed a Mean Residence Time (MRT) of 15.4 hours, while carrier hRS7 antibody cleared at a similar rate to the unconjugated antibody (MRT about 300 hours). IMMU-132 treatment of mice with human gastric cancer xenografts (17.5 mg/kg; twice weekly for 4 weeks) resulted in significant anti-tumor effects compared to mice treated with non-specific controls. Clinically relevant dosing regimens of IMMU-132 were administered once every other week, once a week, or twice a week in mice with human pancreatic or gastric cancer.

Current phase I trials evaluate this ADC as a potential therapeutic agent in pre-treated patients with multiple metastatic solid tumors. In particular embodiments, the therapy is used to treat patients who have previously been found to be resistant to or have relapsed from standard anticancer therapy, including but not limited to treatment with irinotecan (the parent compound of SN-38). These results were surprising and unexpected and could not be predicted.

Saxizumab govitegam was administered on days 1 and 8 of a 21-day cycle, repeating the cycle until dose-limiting toxicity or progression. Dose escalation follows a standard 3+3 regimen, allowing 4 planned dose levels and dose delays or decreases. 25 patients (52-60 years old, median 3 in previous chemotherapy regimens) were treated with dose levels of 8mg/kg (N-7), 10mg/kg (N-6), 12mg/kg (N-9) and 18mg/kg (N-3). Neutropenia is dose-limiting with a maximum tolerated dose of 12mg/kg at cycle 1, but too toxic for repeated cycles. Lower doses were accepted for extension therapy, where there was no treatment-related grade 4 toxicity, and grade 3 toxicity was limited to fatigue (N ═ 3), neutropenia (N ═ 2), diarrhea (N ═ 1), and leukopenia (N ═ 1). Using the CT-based RECIST1.1 criteria, 3 patients achieved partial responses (triple negative breast cancer, small cell lung cancer, colon cancer), and 15 additional patients had disease stabilization as the best response; with continued treatment for 16 to 36 weeks, 12 patients maintained disease control. Patients were not pre-selected based on tumor Trop-2 expression.

It was concluded that, saxizumab gavelutinkang is a promising ADC conjugate with acceptable toxicity and encouraging therapeutic activity in patients with refractory cancer. The 8mg/kg and 10mg/kg doses were selected for phase II studies.

Introduction to

Two new antibody-Drug conjugates (ADCs) incorporating different ultra-toxic (picomolar potency) drugs have been approved, leading to further development of other ADCs based on similar principles, including the use of ultra-toxic drugs (Younes et al, 2011, NatRev Drug Discov 11: 19-20; Sievers and Senter, 2013, Ann Rev Med 64: 15-29; Krop and wine, 2014, Clin Cancer Res 20: 15-20). Alternatively, Moon et al, (2008, J Med Chem51:6916-26) and Govindan et al, (2009, Clin Cancer Res 15:6052-61) selected SN-38, which is a topoisomerase I inhibitor as an active metabolite of irinotecan, a well-known but pharmacologically complex approved drug (Mathijssen et al, 2001, Clin Cancer Res 7: 2182-94). Several linkers used to conjugate SN-38 were evaluated for release from IgG at different rates over a period of hours to days (Moon et al, 2008, J Med Chem51: 6916-26; Govindan et al, 2009, Clin Cancer Res 15: 6052-61; Cardillo et al, 2011, Clin Cancer Res17: 3157-69). The best linker named CL2A, which exhibits intermediate conjugate stability in serum, was chosen to attach to the hydroxyl group on the SN-38 lactone ring, thereby protecting this ring from exposure to the less toxic carboxylate form when bound to the linker and containing a short polyethylene glycol moiety to enhance solubility (cardiollo et al, 2011, ClinCancer Res17: 3157-69). When the carbonate linkage between the linker and SN-38 is cleaved, the active form of SN-38 is released, which occurs at low pH such as found in lysosomes and in the tumor microenvironment or possibly through enzymatic degradation.

The antibody selected for this ADC targets the tumor-associated antigen Trop-2 (trophoblast cell surface antigen) (cardiolo et al, 2011, Clin Cancer Res17:3157-69), which uses a humanized RS7 monoclonal antibody that shows prior internalization (Stein et al, 1993, Int J Cancer55: 938-46. Trop-2 is an important tumor target for ADCs) because it is overexpressed on many epithelial tumors, particularly of the more aggressive type (Ambrogi et al, 2014, PLoSOne9: e 96993; Cubas et al, 2009, biom biophysis Act 1796: 309-14; Trerotola et al, 2013, Oncogene32: 222-33. Trop-2 is also present on many normal tissues, but preclinical studies of monkeys expressing this antigen only observed this new ADC dose-limiting neutropenia and diarrhea, there is no evidence of toxicity of the expression of Trop-2 on normal tissues (cardio et al, 2011, Clin Cancer Res17: 3157-69). Thus, preclinical data indicate activity in several human tumor xenograft models and show a high therapeutic window (cardiolo et al, 2011, Clin Cancer Res17:3157-69), a phase I clinical trial was initiated to determine the maximum tolerance and optimal dose of this novel ADC in heavily pretreated patients with multiple relapsed/refractory metastatic epithelial tumors. The assay was registered on clinical trials. gov (NCT 01631552).

Materials and methods

Inclusion criteriaThe main objective was to determine the safety and tolerability of the saxizumab govitikang (IMMU-132) as a single agent. The trial was designed as a standard 3+3 phase I design, starting with a dose of 8mg/kg per injection, administered once per week for 2 weeks in a 3 week treatment cycle.

Both males > 18 years of age and non-pregnant non-lactating females are eligible if they are diagnosed with one of thirteen different types of epithelial tumors. Although pre-selection based on Trop-2 expression is not required, these tumors are expected to have Trop-2 expression in > 75% of cases based on immunohistological studies on archived samples. Patients are required to have measurable metastatic disease (no single lesion ≧ 5 cm) and have relapsed or at least one approved standard chemotherapeutic regimen for this indication is refractory. Other key criteria include adequate (≦ 1 grade) hematological, hepatic and renal functions, and no known history of irinotecan anaphylaxis, or grade 3 gastrointestinal toxicity to previous irinotecan or other topoisomerase-I treatments. Since patients with this variety of diseases are allowed, prior irinotecan treatment is not a prerequisite. Patients with gilbert's disease or patients who do not tolerate previously administered irinotecan or who have known CNS metastatic disease are excluded.

Design of researchBaseline assessment was performed within 4 weeks after treatment initiation, with periodic monitoring of blood cell counts, serum chemistry, vital signs and any adverse events. Anti-antibody and anti-SN-38 antibody responses were measured by ELISA, with samples taken at baseline and then before the start of each even-numbered treatment cycle. The first CT examination was obtained from 6 to 8 weeks after the start of treatment and then continued at intervals of 8 to 12 weeks until progression. Need to make sure thatAdditional follow-up was performed to monitor any ongoing treatment-related toxicity. Toxicity was graded using NCI CTCAE version 4.0 and efficacy was assessed by RECIST 1.1.

An ELISA was developed to detect Trop-2 in serum with a sensitivity of 2ng/mL, but no further screening was performed after testing 12 patients and no evidence of circulating Trop-2 was found. Although not a qualifying criterion, since the epitope recognized by the antibody hRS7 of ADC is not preserved in formalin-fixed paraffin-embedded sections (Stein et al, 1993, Int J Cancer55: 938-46), previously archived tumor samples are required for Trop-2 assays by immunohistology using goat polyclonal antibody anti-human Trop-2(R & D Systems, Minneapolis, MN). Staining was performed as follows.

Treatment regimensReconstitution of lyophilized saxizumab gavelutikang in saline and infusion for 2 to 3 hours (100mg antibody containing about 1.6mg SN-38, average drug: antibody ratio [ DAR)]7.6: 1). Most patients received acetaminophen, antihistamines (H1 and H2 blockers), and dexamethasone prior to the initiation of each infusion. The prophylactic use of antiemetics or antidiarrheals is prohibited. Treatment consisted of 2 consecutive doses given on days 1 and 8 of a 3 week treatment cycle, which was intended to allow the patient to continue treatment for up to 8 cycles (i.e., 16 treatments) unless there was unacceptable toxicity or progression. Patients who show stable or responsive disease after 8 cycles can continue treatment.

Dose-limiting toxicity (DLT) is considered to be grade 3 febrile neutropenia of any duration; grade 3 thrombocytopenia is accompanied by significant bleeding or grade 4 thrombocytopenia is greater than or equal to 5 days; any grade 3 nausea, vomiting or diarrhea that persists for >48 hours despite optimal medical management; or grade 4 (life threatening) nausea, vomiting or diarrhea of any duration; or at least any other grade 3 non-hematologic toxicity that may be caused by the study drug; and the occurrence of any grade 3 infusion-related reactions.

The Maximum Tolerated Dose (MTD) is judged by the patient's tolerance to the first treatment cycle. Treatment of any patient with grade 2 treatment-related toxicities, except alopecia, was delayed in weekly increments for up to 2 weeks on the scheduled treatment day. Once toxicity is eliminated to grade 1 or less, treatment is resumed. The regimen also required initially a reduction in all subsequent therapeutic doses (to 25% if recovered within 1 week and 50% if recovered within 2 weeks), but this was relaxed later in the trial when the regimen was modified to allow for supportive treatment after the first week. However, if toxicity does not recover or worsen within 3 weeks, treatment is terminated. Importantly, the reduced dose delay does not constitute a DLT, thus allowing treatment to continue, but at lower doses. Thus, patients requiring a dose delay/reduction who are able to continue treatment are not considered as evaluable for DLT, and then replaced.

Since DLT events result in the termination of all further treatments, a secondary objective is to assess the dose level that can be tolerated with minimal dose delay or reduction over multiple treatment cycles. The dose level is designated as the maximum acceptable dose and the patient is asked to tolerate the given dose level in the first cycle without delay or decrease during the cycle and resulting in the start of the second cycle.

Pharmacokinetics and immunogenicityBlood samples were taken within about 30 minutes after the end of the infusion (e.g., peak) and before each subsequent injection (e.g., trough). Samples were isolated and sera were frozen by ELISA to determine total IgG and saxizumab govitikang concentrations. Serum samples from 7 patients were also assayed for SN-38 content, total SN-38 (representing SN-38 bound to IgG and free SN-38) and free SN-38 (i.e., unbound SN-38).

Results

Patient characteristics25 patients were recruited (table 6). The median age ranged from 52 to 60 years with 76% having ECOG1 fitness status and the remaining ECOGs being 0. Most patients suffer from metastatic pancreatic cancer (PDC) (N ═ 7), followed by Triple Negative Breast Cancer (TNBC) (N ═ 4), colorectal cancer (CRC) (N ═ 3), Small Cell Lung Cancer (SCLC) (N ═ 2), Gastric Cancer (GC) (N ═ 2)) And single case Esophageal Adenocarcinoma (EAC), Hormone Refractory Prostate Cancer (HRPC), non-small cell lung cancer (NSCLC), Epithelial Ovarian Cancer (EOC), renal cancer, tonsil cancer, and bladder cancer (UBC).

Performing an immunohistological analysis of archived tissues from 17 patients, of which 13 (76.4%) had 2+ to 3+ membrane and cytoplasmic staining on > 10% of tumor cells in the samples; 3 samples (17.6%) were negative. Several representative cases are disclosed below.

All patients with metastatic disease at the typical site of primary cancer entered the trial. CT determines a median sum of the maximum tumor diameters of 9.7cm (ranging from 2.9cm to 29.8cm) for all patients, and 14 patients identified 3 or more target lesions (median 4 for all patients, ranging from 1 to 10 lesions) and 2 non-target lesions (ranging from 0 to 7 lesions) in their baseline study. The median of the previous systemic treatments was 3, with 7 patients (2 for PDC and GC and 1 for CRC, TNBC, tonsil each) receiving one previous treatment and 7 patients receiving 5 or more previous treatments; 11 patients received radiation therapy. 9 patients were given prior topoisomerase I treatment with 2/3CRC, 4/7PDC, 1 EAC with irinotecan, and 2/2 SCLC patients with topotecan, where 3 patients (2 with SCLC and 1 with CRC) did not respond to anti-topoisomerase 1 therapy. In addition, 7 of 23 patients (2 undetermined) responded to their last treatment with a median duration of 3 months (ranging from 1 to 11 months).

Almost all patients received multiple treatments of saxizumab govitikang (median, 10 doses) until there was evidence of confirmed disease progression by CT using RECIST 1.1; one patient was withdrawn due to systemic deterioration and 1 patient had no measured target lesion at the time of observation of a new lesion in the first follow-up.

Dose assessmentNo dose delay or reduction, nor DLT events in 3 patients (1 CRC, 2 PDC) selected at a starting dose level of 8.0 mg/kg. At the next dose level of 12mg/kg, 9 patients were enrolled because of the delay required for the regimen encountered in administering the second dose. 5 patients experienced a delay in the first cycle (4 patients were delayed by 1 week, with 2 patients given bone marrow growth factor support and 1 patient delayed by 2 weeks before the second dose was given). All patients, except 1, received 12mg/kg as their second dose. Of the 9 patients at the 12mg/kg dose level, 4 patients had the 3 rd dose, they started the second cycle, decreasing to 9mg/kg, and the second cycle was delayed for another 1 week in 3 patients. Although these regimens require a delay/reduction, none of the 9 patients had a dose limiting event during the first cycle (e.g., 1 patient had disease-associated grade 3 hemoglobin after the first dose, 2 patients had grade 3 neutropenia after the first dose were administered myeloid growth factor, 1 patient had grade 3 neutropenia after the first dose without support for recovery, 2 patients had grade 3 neutropenia after the second dose, 2 patients had grade 2 neutropenia after the first dose or the second dose, and 1 patient had no adverse events), thus allowing an increase to 18mg/kg dose levels. Here, all three patients had a dose delay after the first treatment, and only 1 patient received the second treatment at a dose of 18 mg/kg. Two patients had dose-limited grade 4 neutropenia, 1 patient was after the first dose, another patient was after the second 18mg/kg dose, the latter patient also experienced grade 2 diarrhea after this dose. Thus, 0/9 patient was in the first cycle at 12mg/kgWith DLT, this level is declared as MTD.

Additional dose finding studies continue to improve dose levels, which will allow multiple cycles to be administered with minimal delay between treatments/cycles. Thus, an additional 4 patients were enrolled at the 8mg/kg dose level and a new intermediate level of 10mg/kg was turned on. Of the first 3 patients enrolled at 8mg/kg, two CRC patients continued to be treated at 8mg/kg, 31 and 11 total treatments, while PDC patients received a dose of 8mg/kg 3 times before lowering the dose to 6mg/kg due to grade 2 neutropenia at the fourth dose, then completed more than 3 treatments at that level, and then exited as a result of disease progression. Another 4 patients received 3 to 9 doses of 8mg/kg, followed by withdrawal as the disease progressed. Two of these patients received only 1 dose, and the subsequent regimen required a reduction to 6mg/kg due to grade 2 rash and grade 2 neutropenia.

5 of 6 patients enrolled at 10mg/kg received 6 to 30 doses without a decrease, followed by withdrawal due to disease progression. One GC patient (#9) developed grade 3 febrile neutropenia and grade 4 hemoglobin after receiving 1 dose. While febrile neutropenia is considered likely to be associated with treatment, it was found that perforation of the inside stomach wall may promote grade 4 hemoglobin and is considered irrelevant since it occurs shortly after the first dose. Eventually, the patient worsens rapidly and dies 4 weeks after the first dose.

Thus, while the overall results support 12mg/kg as the MTD, phase II clinical studies are ongoing to evaluate these 2 dose levels as 8 to 10mg/kg are better tolerated in the first cycle and allow cycles to be repeated with minimal toxicity.

Adverse events297 saxizumab gavatica infusions over 2 to 3 hours, with most investigators choosing a predose before each infusion. There were no infusion related adverse events. Although more than half of patients experience fatigue, nausea, hair loss, diarrhea, and neutropenia, these are believed to be at least likely to be related to saxib(ii) gavietinate therapy-related bead; these are mainly level 1 and level 2 (not shown). The most reported grade 3 or 4 toxicities were neutropenia (N ═ 8), but 6 of these patients were initially treated at 12mg/kg and 18 mg/kg. Febrile neutropenia occurred in 2 patients, one was GC patient #9, which had been mentioned to receive only one 10mg/kg dose, and the other was PDC patient (#19), which received 4 12mg/kg doses. Most patients have mild diarrhea, with only 3 cases (12%) experiencing grade 3 diarrhea. Two cases occurred at the 12mg/kg dose level, 1 after receiving 4 doses and another after the first dose, but the patient received more than 6 more 12mg/kg doses, reporting only grade 2 diarrhea. Subsequently, both patients were prescribed over-the-counter antidiarrheal drugs and continued treatment. There were no other significant toxicities associated with the saxizumab gavitin, but two patients reported a grade 2 rash and 3 patients had a grade 1 pruritus.

Efficacy ofThe optimal response is measured by the change in the target lesion and time-progression data from the patient with at least one post-treatment CT measurement of the target lesion (not shown). The 4 patients with disease progression are not shown in the graph because they either did not follow-up CT assessment (N ═ 1) or they had new lesions and therefore progressed regardless of their target lesion status (N ═ 3). Overall, the target lesions were reduced by more than 30% in 3 patients (partial response, PR). Two of these patients (#3 and #15) had confirmed follow-up CT, while the third patient (#22) progressed on the next CT examination after 12 weeks. Disease Stability (SD) in 15 patients and disease Progression (PD) in 7 patients was taken as the best response for RECIST 1.1. The median time from initiation of treatment to progression was 3.6 months for 24 patients (excluding 1 patient who received only 1 treatment and was withdrawn) [ range 1 to 12.8 months](ii) a The median time from initiation of treatment to progression for all patients with SD or PR (N ═ 18) was 4.1 months (ranging from 2.6 to 12.8 months). Of 9 patients receiving prior treatment with topoisomerase-I inhibitor, 2 patients had significantly reduced target lesions (28% and 38%), 5 patients had stable disease, of which 2 were of sustained duration (4.1 months and 6.9 months, respectively), and 2 patients had stable disease at his dateProgress was made in the first evaluation.

Comparison of TTP to survival of these patients indicates that 16 patients survived 15 to 20 months after treatment initiation, two of which had PR (15 patients (TNBC) and 3 patients (CRC), and 4 additional patients had SD (2 CRC, 1 HRPC, 1 TNBC) (not shown).

In addition to 3 patients with PR as the best response, there are several obvious cases with prolonged stable disease. One 50 year old TNBC patient (patient 18; immunohistology Trop-2 expression 3+) experienced a 13% reduction after only 3 doses, accumulated a 19% reduction in target lesions (SLD reduced from 7.5 cm to 6.1 cm) after 16 doses, and then progressed for 45 weeks after starting treatment and receiving 26 doses. A63-year-old CRC female (patient 10; immunohistology 2+), who had 7 prior treatments, including 3 separate courses of irinotecan-containing regimens, had an overall 23% reduction in target lesions at 5 after receiving 5 doses of 10mg/kg of saxilizumab gaulthikang, and a cumulative maximum 28% reduction after 18 doses. Her plasma CEA dropped from a baseline level of 38.5ng/mL to 1.6 ng/mL. After receiving 25 doses (27 weeks), she had a PD increase of 20% compared to the SLD nadir. Interestingly, plasma CEA at the end of treatment was only 4.5 ng/mL. A68 year old HRPC patient (patient 20; no immunohistology) presented 5 target lesions (13.3cm) and 5 non-target lesions (3 bone metastases). He received 34 treatments over a 12.7 month period until progression, during which time PSA levels gradually increased. Another notable case is a 52 year old male with esophageal cancer (patient 25; immunohistology 3+), who received 6 prior treatments, including 6 months of FOLFIRI as his 3 rd course of treatment. Treatment was started with 18mg/kg of saxizumab goviitacon, which dropped to 13.5mg/kg due to neutropenia. He had SD within 30 weeks receiving 15 doses and then progressed. A60 year old PDC female (#11) with liver metastases was treated at 10 mg/kg. After 8 doses, her baseline CA19-9 serum titer dropped from 5880 units/mL to 2840 units/mL, and the disease stabilized (12% contraction as the best response) some time or 15 weeks (11 doses) before new lesions were found. However, since CA19-9 was still reduced (2814 units/mL), the patient received 8 additional treatments at 10mg/kg (3 months) and subsequently left the study as their target lesion progressed.

At this time, the potential utility of testing Trop-2 expression in archived samples of small samples from 16 patients with different cancers was not sufficient for definitive assessment, mainly because most showed elevated expression.

PK and immunogenicityTable 7 provides the concentrations of saxizumab govitikang and IgG in the 30 min serum samples, showing a general trend of increasing values with increasing dose. In representative cases, TNBC patients (#15) received multiple doses, starting at 12mg/kg, followed by a decrease over the course of treatment. The concentrations of IgG and of saxizumab govitegafur were similar over time over multiple doses in 30 min serum according to ELISA (not shown), and were adjusted lower as the dose was reduced. Although residual IgG could be found in serum drawn immediately before the next dose (valley samples), no saxizumab govitegam was detected (not shown).

The total SN-38 concentration in the 30 min serum sample of patient 15 after the first dose (C1D1) of cycle 1 was 3,930ng/mL, but when the second dose (C1D2) of the first cycle of saxizumab govacizumab therapy was reduced to 9.0mg/kg, the level was reduced to 2,947ng/mL (not shown). When the dose was further reduced to 6.0mg/kg, a further reduction to 2,381ng/mL was observed in cycle 6. The amount of free SN-38 in these samples ranged from 88ng/mL to 102ng/mL (2.4% to 3.6% of total SN-38), indicating that > 96% of the SN-38 in serum in these peak samples bound to IgG. 28 30 min serum samples from 7 patients were analyzed by HPLC and the free SN-38 averaged 2.91. + -. 0.91% of the total SN-38 in these samples. The concentration of free SN-38G measured in 4 patients never exceeded the SN-38 level and was typically several times lower. For example, patient #25 had an assay evaluated in a 30 minute sample over 12 injections of 8 treatment cycles. At an initial dose of 18mg/kg, he had 5,089ng/mL SN-38 in the acid hydrolyzed sample (total SN-38) and only 155.2ng/mL in the non-hydrolyzed sample (free SN-38; 3.0%). The free SN-38G (glucuronidated form) in this sample was 26.2ng/mL, or only 14.4% of the total unbound SN-38+ SN-38G in the sample. The patient continued to be treated at 13.5mg/kg, with SN-38 averaging 3309.8 + -601.8 ng/mL in the remaining 11 acidic hydrolysis samples, while free SN-38 averaging 105.4 + -47.7 ng/mL (i.e., 96.8% bound to IgG), and free SN-38G averaging 13.9 + -4.1 ng/mL (11.6% of the total SN-38+ SN-38G). Importantly, the concentration of SN-38G was similar in the acid hydrolyzed and non-hydrolyzed samples in almost all patients, indicating that the SN-38 bound to the conjugate was not glucuronidated.

TABLE 7 intact Saxizumab gavitilikang (ADC) and hRS7 according to ELISA

Serum concentration of IgG (. mu.g/mL). The determination was performed in a sample taken 0.5 hours after the first dose.

None of these patients had a positive baseline level (i.e., >50ng/mL) or a positive antibody response to IgG or SN-38 during their treatment.

Discussion of the related Art

Trop-2 is abundantly expressed in many epithelial tumors, making it an antigen of interest for targeted therapy (Cubas et al, 2009, Biochim biophysis Acta1796: 309-14), particularly because it is considered a prognostic marker and Oncogene for several Cancer types (cardiolo et al, 2011, Clin Cancer Res17: 3157-69; ambrogri et al, 2014, PLoS One9: e 96993; Cubas et al, 2009, Biochim biophysis Acta1796: 309-14; Trerotola et al, 2013, Oncogene32: 222-33). Despite its expression in normal tissues and its relationship to another well-studied tumor-associated antigen, EpCam, some preliminary warnings on the safety of Trop-2 for the development of immunotherapeutics were raised (Trerotola et al, 2009, Biochim biophysis Acta 1805:119-20), and our studies on cynomolgus monkeys expressing Trop-2 in human-like tissues indicated that the tolerance of sachimazezumab goviatib was very good at a human equivalent dose of about 40mg/kg (cardiolo et al, 2011, Clin cancer res17: 3157-69). At higher doses, animals experience neutropenia and diarrhea, which are side effects known to be associated with SN-38 derived from irinotecan treatment, but lack evidence of significant histopathological changes in normal tissues expressing Trop-2 (cardiolo et al, 2011, Clin Cancer Res17: 3157-69). Thus, it was found in other preclinical studies that saxizumab govitegam is effective at low nanomolar concentrations and at non-toxic doses in various human epithelial tumor xenografts, with phase I trials conducted on patients who failed to treat their various metastatic epithelial tumors with one or more standard therapies.

One of the main findings of this study is that, despite the use of a more conventional drug (a drug active in the picomolar range, whereas SN-38 has a potency in the low nanomolar range) that is not considered to be ultra-toxic, the saxizumab gavatica anti-Trop-2-SN-38 conjugate demonstrated clinical activity in a variety of solid cancers at doses with moderate and controlled toxicity, thus exhibiting a high therapeutic index. 25 patients who had no accidents were co-administered 297 doses of saxizumab gavatinib; 4 patients received >25 injections. Importantly, no antibody response to hRS7IgG or SN-38 was detected even in patients with multiple treatment cycles up to 12 months. Although Trop-2 is expressed in low amounts in various normal tissues (Cardillo et al, 2011, Clin cancer Res17:3157-69), neutropenia is the only dose-limiting toxicity, with the use of myeloid growth factors in 2 patients given ≧ 12mg/kg of Saxizumab govitegradine to support accelerated recovery and allow continued treatment of patients who have exhausted the choice of other therapies. Although an MTD of 12mg/kg is declared, the selection of 8.0mg/kg and 10.0mg/kg dose levels is further expanded as patients are more likely to tolerate additional cycles at these levels with minimal supportive care, and responses are observed at these levels. Only 2 of 13 patients (15.4%) experienced grade 3 neutropenia at these dose levels. Grade 3 and 4 neutropenia incidence for irinotecan monotherapy administered once weekly or once every three weeks in the anterior or second line setting is 14% to 26% (Camptosar-irinotecan hydrochloride injection (prescription information, package insert) Pfizer, 2012). Only 1 patient with a dose level of 10mg/kg had grade 3 diarrhea when using saxizumab govitegam. This incidence was less than 31% in patients with 4 doses of irinotecan administered weekly experiencing grade 3 and grade 4 advanced diarrhea (Camptosar-irinotecan hydrochloride injection (prescription information, package insert) Pfizer, 2012). Other common toxicities attributed to saxizumab goverikang include fatigue, nausea and vomiting, mostly grade 1 and 2, and alopecia. At the 10mg/kg and 12mg/kg dose levels, two cases of febrile neutropenia and one case of grade 3 deep vein thrombosis also occurred. UGT1a1 monitoring does not begin until after dose detection is complete, and therefore, currently, no assessment of its contribution to toxicity can be reported.

Patients enrolled in the trial were not pre-selected for Trop-2 expression, primarily because immunohistological evaluation of tissue microarrays for various cancers (such as prostate, breast, pancreatic, colorectal, and lung cancers) indicated that antigen was present in > 90% of the samples (not shown). In addition, Trop-2 was not found in the sera of 12 patients with various metastatic cancers, further suggesting that serum assays cannot be used for patient selection. Although we attempted to collect archival samples of tumors from patients enrolled in the trial, there is currently insufficient evidence to suggest that selection of patients based on immunohistological staining will be associated with anti-tumor activity and therefore patient enrichment based on Trop-2 expression.

As a monotherapy, saxizumab goviitakang has good anti-tumor activity in patients with multiple metastatic, relapsed/refractory epithelial tumors, showing significant reduction of target lesions including sustained disease stabilization by CT using RECIST1.1 criteria. 3 of 25 patients (12%) (SCLC [ after progression with topotecan ], 1 each of TNBC and colon cancer) had > 30% reduction in target lesions, followed by 2.9 months, 4.3 months and 7.1 months of progression from the start of treatment, respectively. 15 patients (60%) had SD, with 9 patients progressing after more than 4 months from the start of treatment. Of 9 patients on prior therapy with drugs or regimens containing topoisomerase I inhibitors, 7 had responded or disease stabilized. Three of these patients failed to respond to their previous topoisomerase I inhibitor treatment (irinotecan or topotecan), but the desizumab gavitabine was able to induce tumor shrinkage in both of them: 13% in colon cancer patients and 38% in SCLC patients. Thus, the therapeutic activity of saxizumab govitegam was likely in those patients who failed or relapsed from the previous topoisomerase I regimen, which was further examined in a phase II extended study.

Although the largest number of patients enrolled in this trial had advanced pancreatic ductal carcinoma (N ═ 7; median time progression was 2.9 months); ranging from 1.0 to 4.0 months), even in this refractory disease, there is a target lesion and encouraging reduction in serum concentration of CA19-9 suggesting activity (Picozzi et al, 2014, set forth in the AACR Special Conference "functional Cancer: Innovations in Research and Treatment, New orans, LA USA, p. B99). However, the response of TNBC and SCLC patients is of particular interest in view of the need for targeted therapy in these indications. Indeed, other partial responses observed in additional partial responses in patients with TNBC (golden nberg et al, 2014, proposed in AACR San Antonio Breast cancer Symposium, San Antonio, TX) and SCLC (golden nberg et al, 2014, Sci transmed) observed during the ongoing extension of this trial suggest further emphasis on these cancers, but encouraging responses in NSCLC, EAC, UBC and CRC are also being addressed. In fact, in the recent update of the ongoing Saclizumab gaulthikang trial, 29% overall response rate (PR) and 46% clinical benefit rate (PR + SD ≧ 6 months) were observed in 17 TNBC patients studied so far. Long-term survival (15 to 20 months) was observed for patients in the near 25% (6/25) study, and included 2 PR and 4 SD, including TNBC patients (N ═ 2), CRC patients (N ═ 3), and HRPC patients (N ═ 1).

Analysis of serum samples 30 minutes after the end of infusion showed > 96% SN-38 binding to IgG. When the phase II portion of the assay is complete, more detailed pharmacokinetics will be provided. HPLC analysis also detected only trace amounts of free SN-38G in serum, whereas treatment with irinotecan resulted in a > 4.5-fold higher AUC for the less active SN-38G than SN-38 (Xie et al, 2002, J ClinOncol 20: 3293-. Comparison of SN-38 delivery in tumor-bearing animals given to Saxizumab gavatica and irinotecan indicates that SN-38 bound to IgG is not glucuronidated, whereas in animals given to irinotecan > 50% of the total SN-38 in serum is glucuronidated (Goldenberg et al, 2014, J Clin Oncol 32: Abstract 3107). More importantly, the analysis of SN-38 concentration in the Capan-1 human pancreatic cancer xenograft administered saxizumab gavitikang was about 135 times higher than that administered irinotecan (golden enberg et al, 2014, Sci trans Med). Thus, there are several distinct advantages of saxizumab govitikang over a non-targeted form of topoisomerase-I inhibitor: (i) the mechanism of selectively retaining the conjugate in the tumor (anti-Trop-2 binding), and (ii) the targeted SN-38 also appears to be fully protected (i.e., not glucuronidated and in lactone form), so any SN-38 that is commensally released into the tumor microenvironment by direct internalization of the conjugate or by its release from the tumor-bound conjugate into the tumor microenvironment, would be its most effective form. These results suggest that a moderately toxic but well understood cytotoxic agent SN-38 may be effective as part of a tumor targeting ADC such as saxizumab govitegam. However, by administering the ADC with a moderately toxic drug conjugated at a high drug to antibody ratio (7.6:1), higher concentrations of SN-38 can be delivered to the targeted cancer, as improved SN-38 concentrations are achieved with the saxizumab govitegam compared to SN-38 released from irinotecan.

In conclusion, this phase I experience shows that the Saxizumab govitegam is resistant to moderate and controllable toxicity, which is related to the activity of SN-38, with no evidence of the known content ofThe normal tissue of Trop-2 is damaged. Importantly, the saxizumab goviekin is active in patients with multiple metastatic solid tumors even after prior treatment failure with topoisomerase-I inhibitors. Thus, from initial experience, it can be seen that the saxizumab goviitan has a high therapeutic index, even in patients with tumors, such as SCLC and TNBC, for which the tumor is not known to be responsive to topoisomerase I inhibitors. The clinical trial is continuing, focusing on TNBC, SCLC and other Trop-2⁺The initial dose in patients with cancer was 8mg/kg and 10 mg/kg.

Example 16 use of IMMU-132 in Triple Negative Breast Cancer (TNBC)

The Trop-2/TACTD 2 gene has been cloned (Fornaro et al, 1995, Int J Cancer62: 610-18) and found to encode a transmembrane Ca functionally linked to cell migration and anchorage-independent growth⁺⁺Signal transmitters (Basu et al, 1995, Int J Cancer62: 472-72; Ripani et al, 1998, Int J Cancer 76:671-76), which have higher expression than normal tissues, including in a variety of human epithelial cancers of breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck and ovarian cancers (Cardillo et al, 2011, Clin Cancer Res17: 3157-69; Stein et al, 1994; Int J Cancer Suppl 8: 98-102; Cubas et al, 2009, BiochimBiophys Acta 196: 309-14; Trerotola et al, 2013, Oncogene32: 222-33). It has been reported that increased expression of Trop-2 is necessary and sufficient for stimulating cancer growth (Trerotola et al, 2013, Oncogene32: 222-33), while the bicistronic cyclin D1-Trop-2mRNA chimera is an Oncogene (Guerra et al, 2008, cancer Res 68: 8113-21). Importantly, elevated expression is associated with a more aggressive disease and poor prognosis for several Cancer types (Cubas et al, 2009, Biochim Biophys Acta 196: 309-14; Guerra et al, 2008, Cancer Res 68: 8113-21; Bignoti et al, 2010, Eur J Cancer46: 944-53; Fang et al, 2009, Int J Coloracal Dis 24: 875-84; Muhlmann et al, 2009, J Clin Pathol 62:152-58)The disease types include breast cancer (Ambrogi et al 2014, PLoS One9: e 96993; Lin et al 2013, Exp MolPathol 94: 73-8). The increase in Trop-2mRNA is a strong predictor of low survival and lymph node metastasis in invasive ductal breast cancer patients, and the Kaplan-Meier survival curve shows significantly shorter survival in breast cancer patients with high Trop-2 expression (Lin et al, 2013, Exp Mol Pathol 94: 73-8).

Method of producing a composite material

DAR determination by HIC-analysis of clinical batches of IMMU-132 by Hydrophobic Interaction Chromatography (HIC) using a butyl-NPR HPLC column (TosohBioscience, King of Prussia, PA). IMMU-132 injections (100 μ g) were resolved using a 15 minute linear gradient of 2.25-1.5M NaCl in 25mM sodium phosphate (pH 7.4) at 1mL/min and room temperature.

DAR determination by LC-MSAs the interchain disulfide is reduced and the resulting sulfhydryl group is used for drug conjugation (or blocking), the heavy and light chains are resolved during LC-MS analysis without addition of a reducing agent and analyzed independently. Different batches of IMMU-132 were injected on an Agilent 1200 series HPLC using an Aeris Widepore C4 reverse phase HPLC column (3.6. mu.M, 50X 2.1mm) and resolved by reverse phase HPLC with a 14 minute linear gradient of 30% to 80% acetonitrile in 0.1% formic acid. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was performed with an online Agilent 6210ESI-TOF mass spectrometer with Vcap, fragmentation voltage (fragmentor), and ion picker set at 5000V, 300V, and 80V, respectively. The entire RP-HPLC peak representing all kappa or heavy chain species was used to generate the deconvolution mass spectrum.

Cell linesAll human cancer cell lines used in this study were purchased from the american type culture collection (Manassas, VA) and all cell lines were identified by Short Tandem Repeat (STR) assay of the ATCC, unless otherwise indicated.

Trop-2 surface expression on various human breast cancer cell linesThe expression of Trop-2 on the cell surface is based on flow cytometry. Briefly, solutions were separated with Accutase cells (Becton Dickinson (BD); Fran)klin Lakes, NJ; catalog No. 561527) and Trop-2 expression was determined using QuantiBRITE PE beads (BD catalog No. 340495) and PE-conjugated anti-Trop-2 antibodies (eBiosciences, catalog No. 12-6024) following the manufacturer's instructions. Data were acquired on a FACSCalibur flow cytometer (BD) using CellQuestPro software and analyzed using Flowjo software (TreeStar; Ashland OR).

In vitro cytotoxicity assaySensitivity to SN-38 was determined using the 3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium dye reduction assay (MTS dye reduction assay; Promega, Madison, Wis.). Briefly, cells were plated into 96-well clear flat-bottom plates as described above. SN-38 dissolved in DMSO was diluted with medium to a final concentration of 0.004nM to 250 nM. Plates were placed in a humid chamber at 37 deg.C/5% CO₂Incubation was continued for 96 hours, then MTS dye was added and placed back in the incubator until untreated control cells had an absorbance greater than 1.0. Growth inhibition was measured as percent growth relative to untreated cells. Dose-response curves were generated from the mean of triplicate determinations and IC was calculated using Prism GraphPad software₅₀The value is obtained.

In vitro specificity test by flow cytometry using cells stained with rH2AXFor the pharmaceutical activity test, HCC1806 and HCC1395TNBC cell lines cells were plated in 6-well plates at 5 × 10⁵Individual cells/well were seeded and maintained at 37 ℃ overnight. After cooling the cells on ice for 10 minutes, the cells were incubated with about 20. mu.g/ml IMMU-132 or hA20 anti-CD 20-SN38 (both reagents have equal SN 38/well) on ice for 30 minutes, washed three times with fresh medium, and then returned to 37 ℃ overnight. Cells were briefly trypsinized, pelleted by centrifugation, fixed in 4% formalin for 15 minutes, then washed and permeabilized in 0.15% Triton-X100 in PBS for an additional 15 minutes. After washing twice with 1% bovine serum albumin-PBS, cells were incubated with mouse anti-rH 2AX-AF488(EMD Millipore Corporation, Temecula, Calif.) for 45 minutes at 4 ℃. The signal intensity of rH2AX was measured by flow cytometry using BD FACSCalibur (BD Biosciences, San Jose, CA).

IHC of Trop-2 in tumor microarrays and patient samplesThis involves standard IHC methods on tissue and microarray sections. Scoring is based on within-sample>Staining intensity in 10% of tumor cells, including negative, 1+ (weak), 2+ (moderate), and 3+ (strong).

In vivo therapeutic study in xenograft modelAdministration of 250. mu.g of ADC to 20g of mice produced an SN-38 equivalent (12.5mg/kg) equal to 0.2mg of SN-38/kg. For irinotecan (irinotecan-HCl injection; AREVAPharmaceuticals, Inc., Elizabethtown, KY), 10mg irinotecan/kg was converted on a mass basis to 5.8mg SN-38/kg.

Immunoblotting-cells (2X 10)⁶) The plates were plated in 6-well plates overnight. The following day, they were treated with SN-38 or IMMU-132 at an SN-38 concentration equivalent of 0.4 μ g/mL (1 μ M) for 24 hours and 48 hours. The parent hRS7 was used as a control for the ADC.

Quantification of SN-38 in mice with human tumor xenografts-administering irinotecan or IMMU-132 to two groups, each group having 15 animals bearing subcutaneous implants of human pancreatic cancer cell lines. At 5 different time intervals, 3 animals per group were euthanized. Capan-1 tumors (0.131 + -0.054 g; N ═ 30) were removed and homogenized in deionized water (DI) (1 part tissue +10 parts DI water); the serum was diluted with an aliquot of DI water. Serum and tissue homogenates were extracted and analyzed by reverse phase HPLC (RP-HPLC). Although the extracted sample was sufficient to detect the product from irinotecan-treated animals, the sample from animals given IMMU-132 was divided into 2 portions, one of which was subjected to an acid hydrolysis step to release all SN-38 bound to IgG that would otherwise not be detectable in the extracted sample.

Statistics of-performing statistical analysis using GraphPad Prism version 5.00 for Windows, GraphPad Software, LaJolla California USA. Each study identified the specific test performed.

Results

SN-18 Structure and characteristicsIMMU-132 utilization of topoisomerase IInhibitor SN-38, water-soluble metabolite of anticancer camptothecin, irinotecan (7-ethyl-10- [4- (1-piperidino) -1-piperidino)]Carbonyloxycamptothecin having therapeutic activity in colorectal, lung, cervical and ovarian cancers (Garcia-Carbonero et al, 2002, Clin cancer Res8: 641061). An important advantage of choosing SN-38 is that the in vivo pharmacology of drugs is well known. Irinotecan must be cleaved by esterases to form SN-38, which is 2-3 orders of magnitude more potent than irinotecan, with activity in the low nanomolar range (Kawato et al, 1991, Cancer Res 51: 4187-91). At physiological pH, camptothecin exists in a balance comprising a more active lactone form and a less active (10% potency) open carboxylic acid form (Burke and Mi, 1994, J Med Chem37: 40-46).

The design of the SN-38 derivative CL2A-SN-38 used in IMMU-132 addresses the multiple challenges of using the drug in ADC format and involves the following features: (i) placing short polyethylene glycol (PEG) moieties in a cross-linking agent to render the highly insoluble drug water soluble; (ii) incorporation of maleimide groups for rapid thiol-maleimide conjugation with mildly reduced antibodies, with a specially designed synthetic procedure capable of high yield incorporation of maleimide with assembly of carbonate linkages; (iii) the benzyl carbonate site provides a pH mediated cleavage site to release the drug from the linker; and (iv) importantly, the crosslinker is attached to the 20-hydroxy position of SN-38, which allows the lactone ring of the drug to not open to the less active carboxylic acid form under physiological conditions (Giovanella et al, 2000, Ann NY Acad Sci 922: 27-35). The synthesis of SN-38 derivatives and conjugation of CL2A-SN-38 to lightly reduced hRS7IgG has been described above. Limited reduction procedures only disrupt the interchain disulfide bridges between heavy-heavy and heavy-light chains, but not within domains, producing 8 site-specific thiols per antibody molecule. It was then conjugated with CL2A-SN-38, purified by diafiltration, and lyophilized for storage. During manufacture, conditions were adjusted to minimize any loss of SN-38 from IMMU-132, and the final lyophilized product consistently had < 1% free SN-38 upon reconstitution. However, when placed in serum and maintained at 37 ℃, SN-38 is released from the conjugate with a half-life of about 1 day (not shown).

The release of SN-38 appears to be an important feature of IMMU-132, this type of linker was selected based on efficacy studies that tested SN-38 for optimal therapeutic activity with conjugates with intermediate release rates in serum from a 10 hour release half-life to a highly stable with different SN-38 release rates (Moon et al, 2008(30, 31). we subsequently improved the manufacturing process for this type of linker (named CL2A) (cardiolo et al, 2011, Clin Cancer Res17: 3157-64) by removing phenylalanine residues, and then again comparing the efficacy with that of another stably linked anti-Trop-2 conjugate (CL2) designed to release SN-38 only under lysosomal conditions (i.e., in the presence of cathepsin B and pH 5.0) in animal models, anti-Trop-2 conjugates prepared with CL2A linker gave better therapeutic response than when SN-38 was stably linked, indicating that even rapidly internalized antibodies would benefit by allowing SN-38 to be released in serum with a half-life of about 1 day (Govidan et al, 2013, Mol cancer ther 12: 968-78). Since clinical studies with radiolabeled antibodies found that the antibodies localized to the tumor within a few hours, reaching peak concentrations within 1 day (Sharkey et al, 1995, Cancer Res 55:5935s-45s), selectively enhanced SN-38 concentrations were delivered locally in the tumor by internalization of the intact conjugate, extracellular release of free drug, or both mechanisms co-action.

Drug-antibody ratio (DAR) determination. Five clinical batches of IMMU-132 were evaluated by hydrophobic interaction HPLC (HIC-HPLC) which resolved three peaks representing species with DAR of 6, 7 and 8, the maximum score containing DAR ═ 8 (not shown). IMMU-132 was consistently produced by this manufacturing method, total DAR (DAR) in these five clinical batches_AVE) 7.60. + -. 0.03 (not shown). HIC-HPLC results were confirmed by liquid chromatography-mass spectrometry (LC-MS) (not shown). Analysis showed that of the 8 available thiol groups>99% coupled to a CL2A linker with or without SN-38. No unsubstituted (or N-ethylmaleimide-capped) heavy or light chains were detected. Thus, between speciesThe difference in DAR results from the release of SN-38 from the splice during manufacturing, rather than from a lower initial substitution ratio. Once prepared and lyophilized, IMMU-132 was stable for several years.

Effect of DAR on pharmacokinetics and antitumor efficacy of mice. With Trop-2⁺Mice with human gastric cancer xenografts (NCI-N87) were treated 2 times at 7-day intervals with an equivalent protein (0.5mg) dose of IMMU-132 with a DAR of 6.89, 3.28 or 1.64 per treatment (not shown). Animals treated with ADC with DAR 6.89 had significantly improved Median Survival Time (MST) compared to mice given ADC with DAR 3.38 or 1.64 (MST ═ 39 days vs 25 and 21 days; P, respectively; P)<0.0014). There was no difference between the group treated with 3.28 or 1.64DAR conjugate and the saline control group.

To further elucidate the importance of higher DAR, mice bearing NCI-N87 gastric tumor were administered 0.5mg of IMMU-132 with DAR of 6.89 twice a week for two weeks (not shown). Another group received two protein (1mg) doses of IMMU-132 conjugate with a DAR of 3.28. Although both groups received the same total amount of SN-38(36 μ g) in each dosing regimen, the group treated with 6.89DAR conjugate inhibited tumor growth to a significantly greater extent than tumor-bearing animals treated with 3.28DAR conjugate (P ═ 0.0227; AUC). In addition, treatment with lower DAR was not significantly different from untreated controls. Overall, these studies indicate that lower DAR decreases efficacy.

An examination of the pharmacokinetic behavior of the conjugates prepared at these different ratios was performed in non-tumor bearing mice given 0.2mg of each conjugate, either unconjugated hRS7IgG or hRS7IgG reduced and then capped with N-ethylmaleimide. Sera were taken at 5 time intervals from 0.5 h to 168 h and assayed for hRS7IgG by ELISA. There was no significant difference in the clearance of these conjugates compared to the unconjugated IgG (not shown). Thus, the level of substitution does not affect the pharmacokinetics of the conjugate, and, equally importantly, the reduction of interchain disulfide bonds does not appear to destabilize the antibody.

Trop-2 expression in TNBC and SN-38 sensitivity. Trop-2 expression was determined by Immunohistochemistry (IHC) in several tissue microarrays of human tumor specimens. In a microarray containing 31 TNBC samples and 15 hormone receptor or HER-2 positive breast cancers, more than 95% of tumors stained positively, and 65% of cases indicated 3+ staining.

Table 8 lists 6 human breast cancer cell lines, including 4 TNBC, whose indications

Trop-2 surface expression and sensitivity to SN-38. Trop-2 surface expression in 5 of 6 cell lines exceeded 90,000 copies per cell. SN-38 potency ranged from 2nM to 6nM in 5 of 6 cell lines, MCF-7 had the lowest sensitivity of 33 nM. The in vitro potency of IMMU-132 was not provided, as almost all SN-38 associated with IMMU-132 was released into the culture medium over the 4 day incubation period, and thus its potency would be similar to that of SN-38. Thus, a different strategy is needed to illustrate the importance of antibody targeting as a mechanism for delivering SN-38.

Antigen positive (HCC1806) or negative (HCC1395) TNBC cell lines incubated with IMMU-132 or non-binding anti-CD 20SN-38 conjugate for 30 minutes at 4 ℃. The cells were then washed to remove unbound conjugate and then incubated overnight at 37 ℃. Cells were fixed and permeabilized, then stained with fluorescent anti-phospho histone h2a.x antibody to detect dsDNA breaks by flow cytometry (Bonner et al, 2008, Nat Rev Cancer8:957-67) (table 9). Trop-2 when incubated with IMMU-132⁺Median Fluorescence Intensity (MFI) of breast cancer cell line HCC1806 increased from 168 (untreated baseline) to 546, indicating an increase in the presence of dsDNA fragmentation, while MFI of cells incubated with non-binding conjugate remained at baseline levels. In contrast, MFI of Trop-2 antigen negative cell line HCC1395 remained at baseline levels after treatment with IMMU-132 or non-binding control conjugate. Thus, by only binding the conjugate with anti-Trop-2Evidence of dsDNA fragmentation in incubated Trop-2 expressing cells eventually revealed the specificity of IMMU-132 relative to unrelated ADCs.

In vivo efficacy of saxizumab gavitinecan in TNBC xenografts.Efficacy of IMMU-132 was assessed in nude mice bearing MDA-MB-468TNBC tumors (not shown). IMMU-132 at doses of 0.12 or 0.20mg/kg SN-38 equivalents (0.15mg and 0.25mg IMMU-132/dose) induced significant tumor regression (P) compared to saline, irinotecan (10 mg/kg; about 5.8mg/kg SN-38 equivalent weight), or control anti-CD 20 ADC, hA20-CL2A-SN-38 administered at the same 2 dose levels<0.0017, area under the curve, AUC). Since mice convert irinotecan to SN-38(38) more efficiently than humans (on average about 25% in our studies, see below), doses of irinotecan will yield SN-38 of about 145 μ g to 174 μ g, whereas IMMU-132 is administered at doses containing only 9.6 μ g of SN-38. However, because IMMU-132 selectively targets SN-38 to tumors, it is more effective. These results confirm the results of studies in other solid tumor models (Cardillo et al, 2011, Clin Cancer Res17:3157-69), showing that specifically targeting tumors with small amounts of SN-38 with IMMU-132 is more effective than targeting tumors with larger doses of irinotecan or, for that matter, a mixture of hRS7IgG and an equivalent amount of free SN-38 (Cardillo et al, 2011, Clin Cancer Res17: 3157-69). The unconjugated RS7 antibody did not show any anti-tumor effect even at 1mg repeat doses per animal (Cardillo et al, 2011, Clin cancer Res17: 3157-69). However, in vitro studies of Trop-2 expressing gynaecological cancers have indicated cell killing by antibody dependent cytotoxicity with RS7mAb (Bignoti et al, 2010, Eur J Cancer46: 944-53; Raji et al, 2011, J Exp Clin Cancer Res 30: 106; Varughese et al, 2011, Gynecol Oncol 122: 171-7; Varughese et al, 2011, Am JObstet Gyneol 205: 567). Furthermore, another monovalent Fab of anti-Trop-2 antibodies was reported to be therapeutically active in vitro and in animal studies.

On day 56 of treatment, four of seven tumors in mice given 0.12mg/kg hA20-CL2A-SN-38 control ADC had progressed to 1.0cm³End point (not shown). At this point, the animals were treated with IMMU-132, with a higher dose of 0.2mg/kg being selected in an attempt to affect the progression of these much larger tumors. Despite the large tumor size of several animals, all mice demonstrated a therapeutic response with significant shrinkage of tumor volume after five weeks (total volume [ TV, respectively)]＝0.14±0.14cm³Alignment 0.74 + -0.41 cm³(ii) a P ═ 0.0031, two-tailed t test). Similarly, we selected two tumors progressing to about 0.7cm in the irinotecan treatment group³And one was treated again with irinotecan and the other was treated with IMMU-132 (not shown). Within 2 weeks of the end of treatment, the tumor in irinotecan-treated animals decreased by 23% and then began to progress, while the tumor treated with IMMU-132 stabilized and the tumor size decreased by 60%. These results demonstrate that even in tumors that continue to grow after exposure to SN-38 via non-specific ADCs, significantly enhanced therapeutic responses can be achieved when treated with Trop-2-specific IMMU-132. However, no specific therapeutic effect of IMMU-132 was achieved in MDA-MB-231 (not shown). This cell line had the lowest Trop-2 levels, but was also least sensitive to SN-38.

Mechanism of action of IMMU-132 in TNBCIn TNBC cell lines MDA-MB-468 and HER2⁺Apoptotic pathways used by IMMU-132 were detected in the SK-BR-3 cell line to confirm that ADC functions based on its incorporated SN-38 (not shown). SN-38 and IMMU-132 alone mediate p21 in MDA-MB-468 within 24 hours^WAF1/Cip1Is more than 2-fold up-regulated, and by 48 hours, p21 in these cells^WAF1/Cip1The amount of (A) starts to decrease (31% and 43% in the case of SN-38 or IMMU-132, respectively). Interestingly, in HER2⁺In SK-BR-3 tumor lines, neither SN-38 nor IMMU-132 mediated p21 in the first 24 hours^WAF1/Cip1Up-regulated above the constitutive level, but as seen in MDA-MB-468 cells, p21 was found after 48 hours of exposure to SN-38 or IMMU-13248^WAF1/Cip1Is reduced in amount>57 percent. Both SN-38 and IMMU-132 resulted in 24 hoursThe pro-caspase-3 cleaves into its active fragment, but a greater degree of active fragment is observed after 48 hours of exposure. Notably, in both cell lines, IMMU-132 mediated greater degrees of procaspase-3 cleavage, with the highest levels observed after 48 hours compared to SN-38 exposed cells. Finally, both SN-38 and IMMU-132 mediated Poly ADP Ribose Polymerase (PARP) cleavage starting at 24 hours and almost completely cleaved after 48 hours. Taken together, these results confirm that IMMU-132 has a similar mechanism of action to free SN-38 when administered in vitro.

SN-38 delivered by IMMU-132 vs irinotecan in a human tumor xenograft model-determination of the constitutive products derived from irinotecan or IMMU-132 in the sera and tumors of mice subcutaneously implanted with human pancreatic cancer xenografts (Capan-1) administered with irinotecan (773 μ g; SN-38 eq. 448 μ g) and IMMU-132(1.0 mg; SN-38 eq. 16 μ g).

Irinotecan is rapidly cleared from the serum and converts to SN-38 and SN-38G within 5 minutes. No product was detected within 24 hours. For irinotecan, SN-38 and SN-38G, the AUCs over 6 hours were 21.0. mu.g/mL. multidot.h, 2.5. mu.g/mL. multidot.h and 2.8. mu.g/mL. multidot.h, respectively (SN-38 conversion in mice, [2.5+2.8)/21 ═ 25.2% ]). Animals given IMMU-132 had much lower concentrations of free SN-38 in serum, but free SN-38 was detected within 48 hours (not shown). Free SN-38G was detected only at 1 hour and 6 hours and was 3 to 7 times lower than free SN-38.

In the Capan-1 tumors excised from irinotecan-treated animals, irinotecan levels were high within 6 hours, but were not detectable within 24 hours (AUC)_{5 minutes to 6 hours}48.4 μ g/g · h). SN-38 is much lower and is only detected within 2 hours (i.e., AUC)_{5 minutes to 2 hours}0.4 μ G/G · h), SN-38G values were almost 3 times higher (AUC 1.1 μ G/G · h) (not shown). Tumors taken from animals administered IMMU-132 do not have any detectable free SN-38 or SN-38G, but rather all SN-38 in the tumor binds to IMMU-132. Importantly, since SN-38G was not detected in tumors, this suggested that SN-38 bound to IMMU-132 was not glucuronidated. In thatThe AUC of SN-38 binding to IMMU-132 in these tumors was 54.3 μ g/g.h, 135 times higher than the amount of SN-38 in tumors of animals treated with irinotecan for a2 hour period where SN-38 could be detected, even though mice given irinotecan gave 28-fold higher SN-38 equivalents compared to IMMU-132 administration (i.e., 448 μ g SN-38 equivalents versus 16 μ g SN-38 equivalents, respectively)

Discussion of the related Art

We describe a novel ADC targeting Trop-2 and early clinical results suggest it is in TNBC as well as other Trop-2⁺Has good tolerance and efficacy in cancer patients (Bardia et al, 2014, San Antonio Breastcancer Symposium, P5-19-27). IMMU-132 represents a second generation ADC due to its unique properties. In general, ADCs require 4 best available broad attributes: (i) selective targeting/activity; (ii) binding, affinity, internalization, and immunogenicity of the antibodies used in the ADC; (iii) (iii) the drug, its potency, metabolism and pharmacological disposition, and (iv) how the drug binds to the antibody. Target selectivity is the most common requirement for all ADCs, as this will play a major role in defining the therapeutic index (the ratio of toxicity to tumor versus normal cells). Trop-2 appears to have a high prevalence in many epithelial cancers, but it is also expressed by several normal tissues (Cubas et al, 2009, Biochim biophysis Acta1796: 309-14; Trerotola et al, 2013, Oncogene32: 222-33; Stepan et al, 2011,59:701-10), which may affect specificity. However, expression in normal tissues appears to be lower than in Cancer (Bignotti et al, 2010, Eur J Cancer46: 944-53) and Trop-2 appears to be masked by normal tissue structures that limit antibody accessibility, whereas in Cancer these tissue barriers are involved by invasive tumors. Evidence for this can be seen from preliminary toxicology studies in monkeys, where histopathological damage to normal tissues expressing Trop-2 did not occur despite elevated doses of IMMU-132 to levels that lead to irinotecan-like neutropenia and diarrhea (cardiolo et al, 2011, Clin Cancer Res17: 3157-69). These results appear to have been clinically confirmed, and specific organ toxicity has not been noted in patients to date, except for the parent compound irinotecanIn addition to known toxicities (Bardia et al, 2014, San Antonio Breast Cancer Symposium, P5-19-27), it is easier to control these toxicities with IMMU-132.

A generally accepted and important criterion for ADC therapy is that the antibody should be internalized, delivering its chemotherapeutic agent intracellularly, which is usually metabolized in lysosomes. Despite IMMU-132 internalization, we believe that the linker in the ADC that provides for local release of SN-38 that may induce bystander effects on cancer cells is another feature that distinguishes this platform from platforms using superantivens. Indeed, a super-toxic agent with a stable linkage to IgG is the only construct that is able to maintain a useful therapeutic window for these types of compounds. However, the use of more moderately toxic drugs does not give the freedom of linkers to prematurely release the drug once in circulation. Our panel explored release of SN-38 linker from conjugates with different half-lives from about 10 hours to highly stable linkers from serum, but it was a linker with intermediate stability that provided the best therapeutic response in mouse-human tumor xenograft models (Moon et al, 2008, J Med Chem51: 6916-26; Govindan et al, 2009, Clin Chem Res 15: 6052-61). Since this preliminary work, we found that the highly stable linkage of SN-38 was significantly less effective than the CL2A linker with more moderate stability in serum (Govindan et al, 2013, Mol Cancer Ther 12: 968-78).

Another current principle of ADC design is the use of ultra-fine cytotoxic drugs to compensate for low levels of antibody enhancement in tumors, typically at 0.003% to 0.08% injected dose per gram (Sharkey et al, 1995, Cancer Res 55:5935s-45 s). It has been found that the drug-antibody substitution ≦ 4:1 for this current generation of ultra-toxic drug conjugates is optimal because higher ratios can adversely affect their pharmacokinetics and reduce the therapeutic index by incidental toxicity (Hamblett et al, 2004, Clin cancer Res 10: 7063-70). In this second generation ADC platform, we chose to use an IgG conjugation method that site-specifically links the drug to interchain disulfides by slightly reducing the IgG, thereby exposing 8 binding sites. With CL2A-SN-38 linkers, we achieved a DAR of 7.6:1, LC-MS data showing that each of the 8 coupling sites carries a CL2A linker, but it is clear that some SN-38 was lost during the manufacturing procedure. However, 95% of the CL2A linkers have 7-8 SN-38 molecules. We subsequently found that (a) coupling to these sites did not destabilize the antibody, and (b) conjugates prepared with these sites with higher levels of substitution did not impair antibody binding nor affect pharmacokinetic properties. In fact, we demonstrate that the conjugates prepared at the maximum substitution level have the best therapeutic response in a mouse-human tumor xenograft model.

From a tolerability point of view, one of the more prominent features of IMMU-132 is that SN-38 bound to IgG is not glucuronidated, which is a critical step in irinotecan detoxification, most SN-38 produced is readily converted to the inactive SN-38G form in the liver in the case of treatment with irinotecan estimation of AUC for SN-38G shows that it is typically 4.5 to 32 times higher than SN-38 (Gupta et al, 1994, Cancer Res 54: 3723-25; Xie et al, 2002, J ClinOncol 20: 3293-.

Prevention of glucuronidation of SN-38 bound to an antibody can also help to improve the therapeutic effect of SN-38 delivered to a tumor. Tumor extracts from animals administered irinotecan found high levels of irinotecan with SN-38 and SN-38G concentrations 10-fold lower. In contrast, the movement given to IMMU-132The only SN-38 found in a tumor is that bound to IgG. We hypothesize that conjugates that remain in the tumor will eventually be internalized, releasing their SN-38 payload, or SN-38 can be released outside the tumor cell; however, it is released in a fully active form with a low probability of conversion to SN-38G, which occurs mainly in the liver. It is also important to emphasize that SN-38 is maintained in the active lactone form by coupling a linker to the 20-hydroxy position of SN-38 (Zhao et al, 2000, J Org Chem 65: 4601-6). Overall, these results suggest that IMMU-132 is able to deliver and concentrate SN-38 to Trop-2 in a selective manner compared to SN-38 derived from non-targeted irinotecan⁺Tumors in which SN-38 delivered by IMMU-132 may be released in the tumor as a fully active, non-glucuronidated lactone.

Irinotecan is not generally used to treat breast cancer patients. However, the experiments shown here with the TNBC cell line indicate that concentrating higher amounts of SN-38 into the tumor can enhance its activity. In MDA-MB-468TNBC and HER2⁺In the SK-BR-3 tumor line, IMMU-132 mediates the activation of an intrinsic apoptotic pathway in which the pre-caspases are cleaved into their active fragments and PARP is cleaved. Demonstration of double-stranded DNA breaks in cancer cells treated with IMMU-132(Bardia et al, 2014, San Antonio Breast cancer symposium, P5-19-27) compared to unrelated SN-38 ADCs confirms the selective delivery of SN-38 to target cells. Most importantly, these laboratory study results were confirmed by treatment of patients with metastatic TNBC who had undergone extensive pretreatment, where a persistent objective response has been observed (Bardia et al, 2014, San Antonio Breast cancer symposium, P5-19-27). It also appears that IMMU-132 is active in patients with other cancers and who have failed previous treatment regimens involving topoisomerase I inhibitors (Starodub et al, 2015, Clin cancer Res21: 3870-78).

In summary, SN-38 conjugated with a moderately stable linker at very high drug to antibody ratios was also effective in animal models and also clinically constitutes a second generation ADC platform. Our findings indicate that Trop-2 is a clinically relevant and novel target for Trop-2+ solid tumors, particularly TNBC.

Example 17 investigation of the mechanism of action of IMMU-132

Saxizumab govitegam (IMMU-132, also known as hRS7-CL2A-SN-38) is an antibody-drug conjugate (ADC) that targets the surface glycoprotein Trop-2 expressed on many epithelial tumors for the delivery of the active metabolite SN-38 of irinotecan. Unlike most ADCs that use a super toxic drug and a stabilizing linker, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate linkage between SN-38 and the linker. Flow cytometry and immunohistochemistry disclose that Trop-2 is expressed in a variety of tumor types including gastric, pancreatic, Triple Negative Breast (TNBC), colon, prostate, and lung cancers. Although cell binding experiments revealed no significant difference between IMMU-132 and the parental hRS7 antibody, surface plasmon resonance analysis using Trop-2CM5 chips showed that IMMU-132 had a significant binding advantage over hRS 7. The conjugate retained binding to neonatal receptors, but lost greater than 60% of antibody-dependent cell-mediated cytotoxic activity compared to hRS 7.

Exposure of tumor cells to free SN-38 or IMMU-132 demonstrates the same signaling pathway: pJNK1/2 and p21WAF1/Cip1 are upregulated, followed by cleavage of caspases 9, 7 and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA fragmentation.

Pharmacokinetics of intact ADCs in mice revealed a Mean Residence Time (MRT) of 15.4 hours, while carrier hRS7 antibody cleared at a similar rate to the unconjugated antibody (MRT about 300 hours). IMMU-132 treatment of mice with human gastric cancer xenografts (17.5 mg/kg; twice weekly for 4 weeks) resulted in significant anti-tumor effects compared to mice treated with non-specific controls. Clinically relevant dosing regimens with IMMU-132 administered once every other week, once a week, or twice a week in mice bearing human pancreatic or gastric cancer xenografts demonstrated similar significant anti-tumor effects in both models. Current phase I/II clinical trials (clinical trials. gov, NCT01631552) confirm anti-cancer activity of IMMU-132 in Trop-2 expressing cancers including patients with gastric and pancreatic cancer.

Introduction to

There will be an estimated 22,220 new cases of diagnosed stomach Cancer in the united states this year, with an additional 10,990 deaths from the disease (Siegel et al, 2014, CA Cancer J Clin 64: 9-29). Although the 5-year survival rate is on the rise (currently 29%), it is still quite low compared to most other patients including colon, breast and prostate cancer (65%, 90% and 100%, respectively). In fact, among human cancers, only esophageal cancer, liver cancer, lung cancer and pancreatic cancer have a worse 5-year survival rate. Pancreatic Cancer remains the fourth leading cause of death for all cancers in the united states, with a 5-year survival rate of only 6% (Siegel et al, 2014, CA Cancer J Clin 64: 9-29). As is clear from this rigorous statistical data for gastric and pancreatic cancer, new therapeutic approaches are needed.

Trop-2 is a 45-kDa glycoprotein belonging to the family of TACTD genes, in particular TACTD 22. Overexpression of this transmembrane protein in many different epithelial cancers is associated with a poor overall prognosis. Trop-2 is essential for anchorage-independent cell growth and tumorigenesis (Wang et al, 2008, Mol Cancer Ther 7: 280-85; Trerotola et al, 2013, Oncogene32: 222-33). It acts as a calcium signaling agent for the intact cytoplasmic tail that needs to be phosphorylated by the protein kinase C12-14. Growth-promoting signaling associated with Trop-2 includes NF-. kappa.B, cyclin D1 and ERK (Guerra et al, 2013, Oncogene32: 1594-1600; Cubas et al, 2010, Mol Cancer 9: 253).

In pancreatic Cancer, Trop-2 overexpression is observed in 55% of the patients studied, positively correlated with metastasis, tumor grade and poor progression-free survival in patients undergoing surgery with intent to treat (Fong et al, 2008, Br J Cancer 99: 1290-95). Also, in gastric cancer, 56% of patients show Trop-2 overexpression in their tumors, which in turn is associated with shorter disease-free survival and poorer prognosis in patients with lymph node involvement of Trop-2-positive tumor cells (Muhlmann et al, 2009, J Clin Pathol 63: 152-58). In view of these characteristics, and the fact that Trop-2 is associated with many refractory cancers, Trop-2 is an attractive target for therapeutic intervention with antibody-drug conjugates (ADCs).

The general paradigm of using antibodies to target drugs to tumors includes several key features, including: (a) an antigen target that is preferentially expressed on tumors relative to normal tissue, (b) an antibody that has good affinity and is internalized by tumor cells, and (c) a super-toxic drug stably coupled to the antibody (Panowski et al, 2014, mAbs 6: 34-45). Along these lines, we developed an antibody named RS7-3G11(RS7) that binds to Trop-2 in many solid tumors (Stein et al, 1993, Int J Cancer55: 938-46; Basu et al, 1995, Int J Cancer62: 472-79), has nanomolar affinity (Cardillo et al, 2011k, Clin Cancer Res17:3157-69), and is internalized by cells once bound to Trop-2 (Shih et al, 1995, Cancer Res 55:5857s-63 s).

By immunohistochemistry, Trop-2 is expressed in some normal tissues, but is generally much less intense than tumor tissues and is often present in tissue regions with restricted vascular access (Trerotola et al, 2013, Oncogene32: 222-33). Based on these characteristics, RS7 was humanized and conjugated to 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan. In vitro cytotoxicity of many cell lines the IC of SN-38 has been found₅₀Values in the unit nanomolar range, in contrast to the IC of many of the super-toxic drugs currently used in ADCs₅₀Values are in the picomolar range (Cardillo et al, 2011, Clin Cancer Res17: 3157-69). Although the prevailing view is to use ultra-toxic agents such as auristatins or maytansinoids to stably link an ADC to an antibody with only 2 to 4 drugs per antibody, the therapeutic window for these agents is narrow, resulting in re-engineering ADCs to expand their therapeutic index (Junutula et al, 2010, Clin cancer res16: 4769-78).

As a departure from this approach, we used linkers that release SN-38 in human serum with a half-life of about 1 day, conjugated to 7 to 8 SN-38 molecules per antibody. It is hypothesized that the use of a less stable linker allows SN-38 to be released at the tumor site after the ADC targets the cell, so that the drug can reach the surrounding tumor cells, rather than just the cells directly targeted by the ADC. The resulting ADC, hRS7-CL2A-SN-38 (Saxizumab govitegam or IMMU-132), has shown anti-tumor activity against a variety of tumor types (Cardillo et al, 2011, Clin Cancer Res17: 3157-69). Recently, IMMU-132 has demonstrated significant anti-tumor activity against a preclinical model of Triple Negative Breast Cancer (TNBC) (golden nberg et al, 2014, poster published at st. Most importantly, in the current phase I/II clinical trial, IMMU-132 showed activity in TNBC patients (Bardia et al, 2014, poster published on st ontoneo breast cancer seminar, 12 months 9 to 13 days, abstract P5-19-2), thus this paradigm shift in ADC chemistry was validated using less toxic drugs and linkers that release SN-38 over time rather than relying entirely on internalization of the ADC to achieve activity.

SN-38 is a known topoisomerase-I inhibitor that induces significant damage to cellular DNA. It mediates upregulation of the early pro-apoptotic proteins p53 and p21WAF1/Cip1, leading to caspase activation and poly-ADP-ribose polymerase (PARP) cleavage. Expression of p21WAF1/Cip1 is associated with G1 arrest in the cell cycle and is therefore a marker of the intrinsic apoptotic pathway. We previously demonstrated that IMMU-132 can also mediate the upregulation of early pro-apoptotic signaling events (p53 and p21WAF1/Cip1), leading to PARP cleavage in NSCLC cell lines (Calu-3) and pancreatic cell lines (BxPC-3) consistent with an intrinsic pro-apoptotic signaling pathway (Cardillo et al, 2011, Clin Cancer Res17: 3157-69).

Here we further characterized IMMU-132, with particular attention to the treatment of solid cancers, especially human gastric and pancreatic cancers. Trop-2 surface expression in a range of solid tumor types was examined and correlated with in vivo expression in tumor xenografts. Mechanistic studies further elucidate evidence of intrinsic pro-apoptotic signaling events mediated by IMMU-132, including increased double-stranded dna (dsdna) breaks and subsequent caspase activation. Finally, clinically relevant and non-toxic dosing regimens were compared in gastric and pancreatic cancer disease models, tested on a twice weekly, once weekly and once every other week schedule to determine which treatment cycles could be optimally applied in the clinical setting without loss of efficacy.

Experimental procedures

Cell lines and chemotherapeutic agentsAll human cancer cell lines used were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). Each human cancer cell line was stored and routinely tested for mycoplasma according to ATCC recommendations, all of which were validated by Short Tandem Repeat (STR) assays of ATCC. IMMU-132(hRS7-SN-38) and control ADC (anti-CD 20 hA20-SN-38 and anti-CD 22 hLL2-SN-38) were prepared as described previously and stored at-20 deg.C (Cardillo et al, 2011, Clin Cancer Res17: 3157-69). SN-38(Biddle Sawyer Pharma, LLC, New York, NY) was purchased and stored in 1mM aliquots in DMSO at-20 ℃.

Trop-2ELISARecombinant human Trop-2 with His-tag (nano Biological, inc., Bejing, China, cat # 10428-H09H) and recombinant mouse Trop-2 with His-tag (nano Biological, inc., cat # 50922-M08H) were plated at 1 μ g onto Ni-NTA Hissorb strips (Qiagen GmbH, cat # 35023) at room temperature for 1 hour. The plates were washed four times with PBS-Tween (0.05%) wash buffer. Serial dilutions of hRS7 were prepared in 1% BSA-PBS dilution buffer to give a test range of 0.1ng/mL to 10. mu.g/mL. The plates were then incubated at room temperature for 2 hours and then washed 4 times, followed by the addition of peroxidase-conjugated secondary antibody (goat anti-human Fc fragment specific; Jackson Immunoresearch, Cat. No. 109-. After 45 min incubation, plates were washed and substrate solution (o-phenylenediamine dihydrochloride (OPD); Sigma, Cat. No. P828) was added to all wells. The plates were incubated in the dark for 15 minutes and then stopped with 4N sulfuric acid. Plates were read at 450nm on a Biotek ELX808 plate reader. Data were analyzed and plotted using Prism Graphics pad Software (v4.03) (Advanced Graphics Software, Inc.; Encinitas, Calif.).

In vitro cell bindingLumigLO chemiluminescent substrate system (KPL, Gaithersberg, MD) for the detection of cell-bound antibodies. Briefly, cells were plated into 96-well black flat-bottom transparent plates overnight. Antibodies were serially diluted 1:2 and added in triplicate to give concentrations ranging from 0.03nM to 66.7 nM. After 1 hour incubation at 4 ℃, the media was removed, the cells were washed with fresh cold media, and then 1:20,000 dilution of goat-anti-human horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) was added for 1 hour at 4 ℃. The plate was washed again before adding the LumiGLO reagent. The luminescence of the plates was read using an Envision plate reader (Perkin Elmer, Boston MA). The data were analyzed by non-linear regression to determine the equilibrium dissociation constant (KD). Statistical comparisons of KD values were made using F-test on best-fit curves of data using Prism GraphPad Software (v4.03) (advanced graphics Software, inc.; Encinitas, CA). Significance is set as P<0.05。

Antibody-dependent cell-mediated cytotoxicity (ADCC)-performing a 4 hour LDH release assay to evaluate ADCC activity elicited by IMMU-132, hRS7IgG, hLL2-SN-38 and hLL2 IgG (hLL2 is a non-binding anti-CD 22 conjugate for solid tumor cell lines). Briefly, target cells (MDA-MB-468, NIH: OVCAR-3 or BxPC-3) were incubated at 1X 10⁴Individual cells/well were plated in 96-well black flat-bottom plates and incubated overnight. The following day, peripheral blood mononuclear effector cells (PBMCs) were freshly isolated from donors and added to designated wells on the reaction plate at an E: T ratio of 50: 1. Human PBMC collection was performed under approval by the new england institutional review board (Newton, MA). The test agent was added to its designated wells at a final concentration of 33.3 nM. One set of wells received ADCC assay medium only for background control and the other set of wells received cells plus triton x100 only for maximum cell lysis control. The plates were incubated at 37 ℃ for 4 hours. After 4 hours, target cell lysis was assessed by a homogeneous fluorescent LDH release Assay (cytoTox-One Homogenous Membrane Integrity Assay; Promega, Cat. G7891).

Plates (544nm to 590nm) were read using an Envision plate reader (PerkinElmer LAS, inc.; Shelton, CT). Data were analyzed by Microsoft Excel. The percent specific lysis was calculated as follows:

wherein:

experiment: effector + target cell + antibody

Effector + target control: effector + target cell

Maximum lysis: target cells + Triton-X100

Target control: target cells only

Surface plasmon resonance Bonding (BIACORE)Briefly, rhTrop-2/TACTD 2 (Nano Biological, Inc.) or recombinant human neonatal receptor (FcRn) generated as described (Wang et al, 2011, drug disks 39:1469-77) was immobilized on a CM5 sensor chip (GEHealthcare; catalog No. BR-1000-12) using an amine coupling kit (GE Healthcare; catalog No. BR-1000-50) following the manufacturer's instructions for low density chips. Three independent dilutions of hRS7IgG and IMMU-132 were prepared in running buffer (400nM, 200nM, 100nM, 50nM and 25 nM). Each set will be run individually on a BIACORE (BIACORE-X; Biacore Inc., Piscataway, NJ) and the data analyzed using BIAevaluation software (Biacore Inc., v 4.1). Analysis was performed using a 1:1(Langmuir) binding model and fit, using all five concentration points for each sample run to determine the best fit (lowest χ)²Value). KD values were calculated using the formula KD ═ KD1/ka1, where KD1 is the dissociation rate constant and ka1 is the association rate constant.

Immunohistological evaluation of Trop-2 distribution in formalin-fixed paraffin-embedded tissuesTumor xenografts were removed from mice, fixed in 10% buffered formalin and paraffin embedded. After deparaffinization, 5 μm sections were incubated with Tris/EDTA buffer (DaKo Target recovery Solution, pH 9.0; Dako, Denmark) in an NxGen visualization Chamber (Biocare Medical, Concord, CA) at 95 deg.CIncubate for 30 minutes. Goat polyclonal anti-human Trop-2 antibody was used at 10. mu.g/mL (R)&D Systems, Minneapolis, MN) and stained with the Vector vectasalinr ABC kit (Vector Laboratories, inc., Burlingame, CA). Normal goat antibody was used as a negative control (R)&DSystems, Minneapolis, MN). The tissue was counterstained with hematoxylin for 6 seconds.

Trop-2 surface expression on human cancer cell linesThe expression of Trop-2 on the cell surface is based on flow cytometry. Briefly, cells were harvested with an Accutase cell isolation solution (Becton Dickinson (BD), Franklin Lakes, NJ; catalog No. 561527) and assayed for Trop-2 expression using QuantiBRITE PE beads (BD catalog No. 340495) and PE-conjugated anti-Trop-2 antibodies (eBiosciences, catalog No. 12-6024) following the manufacturer's instructions. Data were acquired on a FACSCalibur flow cytometer (BD) using CellQuestPro software. Staining was analyzed by Flowjo software (Tree Star, Ashland OR).

PharmacokineticsNaive female NCr nude (nu/nu) mice 8 to 10 weeks old were purchased from Tastic Farms (Germantown, NY). Mice (N ═ 5) were injected intravenously with 200 μ g of IMMU-132 (parental hRS7) or modified hRS7-NEM (hRS7 treated with TCEP and conjugated with N-ethylmaleimide). Animals were bled through the retroorbital plexus at 30 minutes, 4 hours, 24 hours, 72 hours, and 168 hours post-injection. Serum concentrations of total hRS7IgG due to competition for anti-hRS 7IgG idiotypic antibody binding with horseradish peroxidase conjugate of hRS7 were determined by ELISA. Serum concentrations of intact IMMU-132 were determined using anti-SN-38 antibody capture and horseradish peroxidase conjugated anti-hRS 7IgG antibody for detection. Pharmacokinetic (PK) parameters were calculated by non-compartmental analysis using Phoenix WinNonlin software (version 6.3; Pharsight Corp., mountain View, Calif.).

In vitro assessment of double stranded DNA breaksFor drug activity assays BxPC-3 cells were seeded at 5 × 105 cells/well in 6-well plates and kept at 37 ℃ overnight. After cooling on ice for 10 minutes, cells were incubated with IMMU-132, hA20-SN-38, or hRS7-IgG at a final concentration of 20. mu.g/ml on ice for 30 minutes, washed three times with fresh medium,the temperature was then returned to 37 ℃ to continue the culture overnight. The next morning, cells were briefly trypsinized, centrifuged, stained with FixableVisability Stain 450(BD Biosciences, San Jose, Calif.), washed with 1% BSA-PBS, then fixed in 4% formalin for 15 minutes, washed again, and permeabilized in 0.15% Triton-X100 in PBS for an additional 15 minutes. After washing twice with 1% BSA-PBS, cells were incubated with mouse anti-gamma H2AX-AF488(EMD Millipore corporation, Temecula, Calif.) for 45 minutes at 4 ℃. Signal intensity of γ H2AX was measured by flow cytometry using BD facscan (BD Biosciences, San Jose, CA).

In vivo therapeutic studyNCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, were purchased from Taconnic Farms (Germantown, NY). NCI-N87 gastric tumor xenografts were established by harvesting cells from tissue culture and making final cell suspensions in matrigel (BD Bioscience; San Jose, Calif.) at 1:1, receiving a total of 1X 10 subcutaneous injections per mouse in the right flank⁷And (4) cells. For BxPC-3, 1g of xenografts was harvested and made into tumor suspension in HBSS at a concentration of 40% tumor w/v. This suspension was mixed with matrigel 1:1 to give a final tumor suspension of 20% w/v. Mice were then injected subcutaneously with 300 μ L. Tumor Volume (TV) was determined by two-dimensional measurement using calipers, the volume being defined as: l x w²And/2, wherein L is the longest dimension of the tumor and w is the shortest dimension of the tumor. For IHC, tumors were allowed to grow to about 0.5cm³Mice were then euthanized and tumors removed, formalin fixed and paraffin embedded. For the treatment study, mice were randomized into treatment groups when tumor volume was about 0.25cm³Treatment is initiated. Treatment regimens, doses, and number of animals in each experiment are described in the results and legend. The lyophilized IMMU-132 and control ADC (hA20-SN-38) were reconstituted and diluted in sterile saline as needed.

Mice were euthanized and tumors once grown to greater than 1.0cm³The size of (d) is considered to have died from the disease. The optimal response to treatment is defined as: partially responsive to shrinkage from the starting size>30 percent; the stability of the disease is ensured,tumor volume contracted to 29% or increased by no more than 20% of the original size; progression was by 20% increase in tumor size from the initial size or nadir. Time To Progression (TTP) was determined as the time after treatment initiation when the tumor grew more than 20% from its nadir size.

Statistical analysis of tumor growth was based on area under the curve (AUC). A profile of individual tumor growth was obtained by linear curve modeling. Prior to statistical analysis of growth curves, an f-test was used to determine the equality of variance between groups. The two-tailed t-test was used to assess statistical significance between various treatment groups and controls other than the saline control, using the single-tailed t-test (significance, P.ltoreq.0.05). Survival studies were analyzed using a Kaplan-Meier plot (log rank analysis) using the Prism GraphPad Software (v4.03) Software package (Advanced Graphics Software, inc., Encinitas, CA).

Immunoblotting-cells (2X 10)⁶) The plates were plated overnight in 6-well plates the next day they were treated with free SN-38 (dissolved in DMSO) or IMMU-132 with SN-38 concentrations equivalent to 0.4. mu.g/mL (1. mu.M.) parent hRS7 was used as a control for ADC cells were lysed in buffer containing 10mM Tris (pH 7.4), 150mM NaCl, protease inhibitors and phosphatase inhibitors (2mM Na2PO4, 10mM NamaF.) A total of 20. mu.g of protein was resolved in 4-20% SDS polyacrylamide gel, transferred to nitrocellulose membrane and blocked with 5% skimmed milk in 1 × TBS-T (Tris buffered saline, 0.1% Tween-20) for 1 hour at room temperature the membrane was probed overnight under 4oC catalog, then incubated with anti-rabbit secondary antibody (1:2500) for 1 hour at room temperature using chemiluminescence kit (super, Dura, eromo Scientific, Rofornt, IL catalog for detecting signals in rabbit secondary catalog No. 29959, No. 7-7, No. 7-2000, No. 7-III-7, No. 7-III-7, No. 7-III-20-III-V-III.

Results

Trop-2 expression levels in multiple solid tumor cell linesSurface expression of Trop-2 is evident in a number of human solid tumor lines including gastric, pancreatic, breast, colon and lung cancers (table 10). None of the tumor types had higher expression than any other type, and variability was observed within a given tumor cell type. For example, in gastric adenocarcinoma, Trop-2 levels range from very low 494 ± 19(Hs 746T) to high 246,857 ± 64,651(NCI-N87) surface molecules per cell.

Gastrointestinal tumor xenografts stained for Trop-2 expression showed cytoplasmic and membrane staining (not shown). Staining intensity correlated well with the results of surface Trop-2 expression as determined by FACS analysis. For pancreatic cancer, all three had homogeneous staining, and BxPC-3 represented 2+ to 3+ staining. NCI-N87 gastric adenocarcinoma has a more heterogeneous staining pattern, with 3+ staining of the apical lining of the gland and less pronounced staining of surrounding tumor cells. COLO 205 demonstrated only very focal 1+ to 2+ staining, whereas HT-29 showed very rare 1+ staining of a few cells.

TABLE 10 analysis of Trop-2 surface expression levels in various solid tumor lines by FACS. a is

a three separate determinations were performed, providing the mean and standard deviation.

IMMU-132 binding characteristicsTo further demonstrate that hRS7 does not cross-react with murine Trop-2, ELISA was performed on plates coated with recombinant murine Trop-2 or human Trop-2 (not shown). Humanized RS7 specifically binds only to human Trop-2(KD ═ 0.3 nM); has no cross-reactivity with mouse Trop-2. Control polyclonal Rabbit anti-mouse Trop-2 and anti-human Trop-2 antibodiesIndeed cross-reacted with and bound to both forms of Trop-2 (data not shown).

IMMU-132 was examined for binding to various cell lines, compared to the parent hRS7 and modified hRS7, hRS7-NEM (hRS7 treated with TCEP and conjugated with N-ethylmaleimide) (not shown). In all cases, the calculated KD values were in the sub-nanomolar range, with no significant differences between hRS7, IMMU-132, and hRS7-NEM within a given cell line.

The comparison of the binding of IMMU-132 and hRS7 was further investigated using surface plasmon resonance (BIACORE) analysis (not shown). A low-density Trop-2 biosensor chip (density 1110RU) was used with recombinant human Trop-2. Three independent binding runs demonstrated not only that IMMU-132 was not adversely affected by the SN-38-conjugation process, but also that the binding affinity for Trop-2 was higher than that of hRS7 for Trop-2 (0.26 ± 0.14nM vs. 0.51 ± 0.04 nM; P ═ 0.0398, respectively).

The action mechanism is as follows: ADCC and intrinsic apoptosis signaling pathways-comparing the ADCC activity of IMMU-132 with that of hRS7 in three different cell lines TNBC (MDAMB-468), ovary (NIH: OVCAR-3) and pancreas (BxPC-3) (not shown). In all three, hRS7 mediated cell lysis significantly (P) compared to all other treatments including IMMU-132 (P)<0.0054). ADCC was reduced by more than 60% when IMMU-132 was used to target cells, compared to hRS 7. For example, in MDA-MB-468, hRS 7-mediated specific cleavage was 29.8 + -2.6%, in contrast to IMMU-132-mediated specific cleavage of 8.6 + -2.6% (not shown; P)<0.0001). Similar ADCC activity loss was also observed in NIH, OVCAR-3 and BxPC-3 (not shown; P, respectively)<0.0001 and P<0.0054). This reduced ADCC activity appears to be the result of antibody changes during the conjugation process, as this same loss of specific cell lysis is evident with hRS7-NEM, which lacks the CL2A-SN-38 linker, with cysteine blocked instead by N-ethylmaleimide (not shown). There was no CDC activity associated with hRS7 or IMMU-132 (data not shown).

IMMU-132 has previously been shown to mediate the up-regulation of early pro-apoptotic signaling events (p53 and p21WAF1/Cip1), ultimately leading to cleavage of PARP 20. To better define the apoptotic pathway used by IMMU-132, NCI-N87 human gastric carcinoma and BxPC-3 pancreatic carcinoma cell line were exposed to 1 μ M free SN-38 or equivalent amounts of IMMU-132 (not shown)). Both free SN-38 and IMMU-132 mediated upregulation of p21WAF1/Cip1, but up to 48 hours upregulation was the same between NCI-N87 cells exposed to free SN-38 versus NCI-N87 cells exposed to IMMU-132 (not shown), whereas in BxPC-3, maximal upregulation was apparent within 24 hours (not shown). Both free SN-38 and IMMU-132 demonstrated cleavage of the first caspase-9 and-7 within 48 hours of exposure. The pro-caspase-3 was cleaved in both cell lines, with the highest degree of cleavage observed after 48 hours. Finally, both free SN-38 and IMMU-132 mediate PARP cleavage. This first became apparent at 24 hours with increased lysis at 48 hours. Taken together, these data confirm that SN-38 contained in IMMU-132 has the same activity as free SN-38.

In addition to these later apoptotic signaling events, an early event associated with this pathway, namely phosphorylation of JNK (pJNK), was also evident in BxPC-3 cells exposed for short periods to free SN-38 or IMMU-132 but not naked hRS7 (not shown). The increase in pJNK amount was evident at 4 hours and did not change significantly at 6 hours. Compared to IMMU-132, the phosphorylation intensity was higher in cells exposed to free SN-38, but both were significantly higher than the control. As an end point of IMMU-132 mechanism of action, dsDNA fragmentation measurements were performed in BxPC-3 cells. Exposure of BxPC-3 to IMMU-132 for only 30 minutes resulted in greater than 2-fold induction of γ H2AX compared to non-targeted control ADCs (table 11). About 70% of the cells were positive for γ H2AX staining, in contrast < 20% for naked hRS7, ADC-independent hA20-SN-38 and untreated controls (P < 0.0002).

TABLE 11 IMMU-132 mediated dsDNA fragmentation in BxPC-3: gamma H2AX induction.^a

a IMMU-132 vs all 3 control treatments, P <0.0002 (one-tailed t-test; N ═ 3).

Pharmacokinetics of IMMU-132Binding to human neonatal receptor (FcRn) was determined by BIACORE analysis (not shown), three independent binding was performed at three different concentrations (400nM to 25nM) for each agent using a low density FcRn biosensor chip (density 1302RU), overall, hRS7 and IMMU-132 both demonstrated KD values in the nanomolar range (92.4 ± 5.7nM and 191.9 ± 47.6nM, respectively), with no significant difference between the two, mice were injected with IMMU-132, and the clearance of IMMU-132 versus hRS7IgG was compared to the parent hRS7 using two ELISAs (not shown), mice injected with hRS7 demonstrated a similar biphasic clearance pattern (not shown) observed with the hRS7 targeting portion (not shown) of imm-132, where α and β were about 3 hours and about 200 hours, respectively, in contrast, a rapid clearance half-life of intact mu-132 was observed, a rapid clearance half-life was observed for MRT 11 hours and a 15 hours (not shown).

To further confirm that disruption of interchain disulfide bonds does not alter the PK of the targeted antibody, the PK of the parent hRS7 was compared to the modified hRS7(hRS 7-NEM). There was no significant difference between the two agents in half-life, Cmax, AUC, clearance or MRT (not shown).

Efficacy of IMMU-132 in human gastric cancer xenograftsThe efficacy of IMMU-132 has previously been demonstrated in non-small cell lung, colon, TNBC and pancreatic cancer xenograft models (Cardillo et al, 2011, Clin cancer Res17: 3157-69; Goldenberg et al, poster published on the St.Andonio Breast cancer workshop, 12 months 9 to 13 days, Abstract P5-19-08). To further extend the results of these studies to other gastrointestinal cancers, IMMU-132 (not shown) was tested in mice bearing the human gastric cancer xenograft NCI-N87. Treatment with IMMU-132 achieved significant tumor regression compared to saline and non-targeted hA20 (anti-CD 20) -SN-38ADC controls (not shown; P)<0.001). Of the 7 mice in the IMMU-132 group, 6 were partial responders, with the last administration to the animalsThe duration of the treatment dose is more than 18 days after one treatment dose. This resulted in a mean Time To Progression (TTP) of 41.7. + -. 4.2 days, compared to 4.1. + -. 2.0 days (P) for non-responders in the control ADC group<0.0001). Overall, Median Survival Time (MST) for IMMU-132 treated mice was 66 days, compared to 24 days for control ADC and 14 days for saline control animals (not shown; P)<0.0001)。

Clinically relevant dosing regimensThe highest repeat doses of IMMU-132 tolerance currently being tested clinically are 8mg/kg and 10mg/kg given on days 1 and 8 of the 21-day cycle. A human dose of 8mg/kg is converted to a murine dose of 98.4mg/kg, or about 2mg for 20g mice. Three different dose regimens of fractionated 2mg IMMU-132 were examined in the human pancreatic cancer xenograft model (BxPC-3). The total dose was graded using one of three different dosing regimens: one group received two doses of 1mg of IMMU-132 (treatment days 1 and 15), one group received 4 doses of 0.5mg (treatment days 1, 8, 22 and 29), and a final group received 8 doses of 0.25mg (treatment days 1, 4, 8, 11, 22, 25, 29 and 32). All three dosing regimens provided significant anti-tumor effects in both tumor growth inhibition and overall survival compared to untreated control animals (not shown; P, respectively)<0.0009 and P<0.0001). There was no significant difference in TTP between the three different treatment groups, with the TTP of the 1-mg administered group being 22.4 ± 10.1 days and the TTP of the 0.25-mg administered group being 31.7 ± 14.5 days (TTP of the untreated control group being 5.0 ± 2.3 days).

Similar dose-schedule experiments were performed in mice bearing NCI-N87 human gastric tumor xenografts (not shown). All three dose regimens had significant anti-tumor effects when compared to untreated control mice, but were not different from each other (AUC; P < 0.0001). Also, all three dose regimens provided significant survival benefit (P <0.0001) compared to untreated controls in terms of overall survival, with no difference in any of the three different regimens.

To further differentiate possible dosing regimens, chronic IMMU-132 dosing was performed on mice bearing NCI-N87 tumors, wherein the mice received a 0.5mg IMMU-132 injection once a week for two weeks, then stopped for one week, and then started another cycle (not shown), as in current clinical trial protocols. In total, four treatment cycles were administered to the animals.

This dosing regimen slowed tumor growth with 15.7 ± 11.1 days TTP, in contrast to 4.7 ± 2.2 days TTP (P ═ 0.0122) for control ADC-treated mice. Overall, chronic dosing increased median 19 survival from 21 days for control ADC-treated mice to 63 days for those animals administered IMMU-132 by a factor of 3 (P ═ 0.0001). Importantly, in all of these different dosing regimen evaluations, no treatment-related toxicity was observed in mice as evidenced by no significant loss in body weight (data not shown).

Discussion of the related Art

In a current phase I/II clinical trial (clinical trials. gov, NCT01631552), IMMU-132 (saxilizumab gaulthikang) demonstrated objective responses in patients with multiple solid tumors (Starodub et al 2015, Clin Cancer Res21: 3870-78). As this phase I/II clinical trial continues, the efficacy of IMMU-132 needs to be further explored in an expanding list of Trop-2-positive cancers. In addition, the uniqueness of IMMU-132 needs to be further elucidated as we advance clinical development compared to other clinically relevant ADCs using superantitoxin drugs.

The work provided herein further characterizes IMMU-132 and demonstrates its efficacy in clinically relevant dosing regimens for gastric and pancreatic cancer. The prevailing view of a successful ADC is that it should use antibodies that recognize antigens with high tumor expression levels relative to normal tissues, as well as antibodies that preferentially internalize when bound to tumor cells (Panowski et al, 2014, mAbs 6: 34-45). All currently approved ADCs use a super toxic drug (pMIC50) conjugated to an antibody at a high substitution ratio (2 to 4 drugs per antibody) through a highly stable linker. IMMU-132 inThree main aspects deviate from this paradigm: (i) SN-38, a moderately cytotoxic drug (nMIC)₅₀) Used as a chemotherapeutic agent, (ii) SN-38 is site-specifically conjugated to 8 interchain thiols of an antibody, each antibody yielding a 7.6 drug substitution, and (iii) using a carbonate linker that is cleavable at low pH but will also release a drug with a serum half-life of about 24 hours (cardiollo et al, 2011, Clin Cancer Res17: 3157-69). As we show, IMMU-132 consists of antibodies that internalize upon binding to an epitope, are specific for human Trop-2, are highly expressed in many different types of epithelial tumors, and are present at low concentrations in their corresponding normal tissues (Shih et al, 1995, Cancer Res 55:5857s-63 s). Despite its presence in normal tissues, previous studies in monkeys (which also express Trop-2 in similar tissues) indicated that there were relatively mild and reversible histopathological changes even at very high doses where dose-limiting neutropenia and diarrhea occurred, suggesting that antigens in normal tissues were sequestered in some way, or that these normal tissues were protected from severe damage using less toxic drugs (Cardillo et al, 2011, Clin Cancer Res17: 3157-69).

Herein, we extended the assessment of Trop-2 expression on a variety of human solid tumor lines, examining in vitro expression in xenografts in a more quantitative manner than previously reported, but importantly, suggesting that Trop-2 expression ranges from homogeneous (e.g., NCIN87) to very focal (e.g., COLO 205). In summary, the in vitro measured surface expression level of Trop-2 correlates with staining intensity upon IHC analysis of xenografts. Of particular interest, even in tumors like COLO 205, IMMU-132 was able to trigger specific tumor regression by immunohistology revealing the presence of only a focal pocket of Trop-2 expressing cells, suggesting that bystander effects may occur as a result of the release of SN-38 from conjugates bound to antigen presenting cells (Cardillo et al, 2011, Clin Cancer Res17: 3157-69). In fact, SN-38 readily penetrates the cell membrane, so its local release in the tumor microenvironment provides another mechanism for its entry into the cell, without the need to internalize the intact conjugate. Importantly, SN-38 bound to the conjugate remains in a fully active state; that is, it is not glucuronidated and will be in the form of a lactone ring when released (Sharkey et al, 2015, Clin Cancer Res,21: 5131-8). This property is unique, as distinguished from the ability of IMMU-132 to localize the fully active form of SN-38 in a more selective manner than any other sustained release SN-38 or irinotecan agents studied to date.

Phase I clinical trial identification with IMMU-132 was given 8mg/kg to 10mg/kg weekly over a 21 day period for two weeks for further study at phase II (Starodub et al, 2015, Clin Cancer Res21: 3870-78). Patients with a variety of metastatic solid tumors, including pancreatic cancer and gastric cancer, show an extended period of disease stability after relapse from various prior therapies (Starodub et al, 2015, Clin cancer res21: 3870-78; Starodub et al, 2014, J ClinOncol 32:5s (supplement abstract 3032)). Additional studies were conducted in the xenograft model to determine whether different dosing regimens could be more effective. For this purpose, a human dose corresponding to 8mg/kg (mouse dose of 98.4 mg/kg) was graded in three different dosing regimens, including every other week, once a week or twice a week, over a 21-day cycle. In the pancreatic and gastric tumor models, no significant difference in treatment response was observed for all three regimens, with tumors progressing only after cessation of treatment. Thus, these data support continued use of the once-weekly dosing regimen that is currently clinically ongoing.

With clinical trials recommending therapeutic doses of IMMU-132 of 8mg/kg to 10mg/kg per treatment (Starodub et al 2015 Clin cancer Res21:3870-78), it was important to examine whether antibodies alone might contribute to IMMU-132 activity. Previous studies in a nude mouse-human xenograft model included unconjugated hRS7IgG alone (e.g., repeat doses of 25mg/kg to 50mg/kg), with no evidence of therapeutic activity (cardiollo et al, 2011, Clin cancer res17: 3157-69); however, studies in mice do not always predict immune function. The in vitro ADCC activity of hRS7 has been reported in Trop-2 positive ovarian and uterine cancers (Raji et al, 2011, J Exp Clin Cancer Res 30: 106; Bignoti et al, 2011, Int J Gynecol Cfacer 21: 1613-21; Varughese et al, 2011, Am JObstet Gynecol 205: 567; Varughese et al, 2011, Cancer 117: 3163-72). We confirmed unconjugated hRS7ADCC activity in three different cell lines, but found that IMMU-132 lost 60% to 70% of its effector function. Since reduced/NEM blocked IgG has a similar loss of ADCC activity, it appears that the attachment of the CL2A-SN-38 component is not itself responsible.

Antibodies can also trigger cell death by acting on various apoptotic signaling pathways. However, we did not observe any role of the unconjugated antibody in many apoptotic signaling pathways, but rather noted that IMMU-132 elicited intrinsic apoptotic events similar to SN-38. Early events include phosphorylation of JNK1/2 and upregulation of p21WAF1/Cip1, leading to activation of caspase-9, caspase-7 and caspase-3, with the end result of PARP cleavage and significant levels of dsDNA fragmentation as measured by increased amounts of phosphorylated histone H2AX (γ H2AX) 41. These data suggest that the primary mechanism of action of IMMU-132 is related to SN-38.

Surface plasmon resonance (BIACORE) analysis did not detect a significant difference in binding of IMMU-132 to human neonatal receptor (FcRn), although the mean level of binding of IMMU-132 was about 2-fold lower. FcRn binding is associated with prolonged IgG half-life in serum (Junghans and Anderson, 1996, Proc Natl Acad Sci USA 93:5512-16), but the overall importance of the results of this study is unclear as the in vitro affinity of the antibody for FcRn may not be related to in vivo clearance (Datta-Mannan et al, 2007, Jbiol Chem 282: 1709-17). Use in tumor-bearing mice¹¹¹Previous experiments with In-DTPA-IMMU-132 revealed that¹¹¹In-DTPA-hRS7 cleared the conjugate slightly faster from serum than it did, although both had similar tumor uptake (Cardillo et al, 2011, Clin cancer Res17: 3157-69). In the current study, an ELISA assay that additionally measures clearance of IgG components, it was found that IMMU-132 and reduced and NEM blocked IgG cleared at a similar rate to the unconjugated hRS7, suggesting that coupling to interchain disulfides does not destabilize the antibody. As expected, when using monitoring complete affixesThe clearance of the compound in the ELISA (captured using anti-SN-38 antibody and a probe with anti-idiotype antibody) was faster than when only the IgG fraction was monitored. This difference only reflects the release of SN-38 from the conjugate with a half-life of about 1 day. We also examined the clearance rates of the saxizumab govitegradine conjugates prepared at different substitution levels by ELISA and again found no significant difference in their clearance rates (Goldenberg et al 2015, Oncotarget 8: 22496-. Overall, these data suggest that mild reduction of the antibody and subsequent site-specific modification of some or all of the interchain disulfides has minimal, if any, effect on serum clearance of IgG, but that the overall clearance of IMMU-132 will be primarily defined by the rate of SN-38 release from the linker.

In addition, extensive cell binding experiments demonstrated no significant differences in binding of IMMU-132, unconjugated antibody or NEM modified antibody, suggesting that site-specific attachment to interchain disulfides protects the antigen binding properties of the antibody. Interestingly, when analyzed by BIACORE, it was able to measure association and dissociation rates more accurately in addition to overall affinity, with IMMU-132 significantly improved by a factor of 2 in the calculated KD value for Trop-2 binding when compared to naked hRS 7.

We speculate that this improvement may be due to increased hydrophobicity when SN-38 is conjugated to an antibody. Hydrophobic residues, as well as the hydrophobicity of the blocking regions of the protein binding site, have been shown to confer greater affinity to epitopes (Park et al, 2000, Nat Biotechnol 18: 194-98; Berezov et al, 2001, J Med Chem 44: 2565-74; Young et al, 2007, Proc Natl Acad Sci USA 104: 808013). These regions need not be located at the protein-protein interface, but may be located in surrounding, less energetic contact residues (Li et al, 2005, Structure 13: 297-. Although there is no SN-38 conjugation site in the Complement Determining Region (CDR) of hRS7, SN-38 on the antibody can replace some water molecules around the epitope, resulting in no compromise of the prospect of improved binding affinity observed for IMMU-132 relative to naked hRS 7.

Most of the work in ADC development has involved the use of stable linkers and super-toxic drugs, with pre-clinical studies indicating specific optimal requirements for these binders (Panowski et al, 2014, mAbs 6: 34-45; Phillips et al, 2008, Cancer Res 68: 9280-90). For example, comparison of T-DM1 with another less stable derivative, T-SSPDM1, revealed that intact T-SSP-DM1 cleared at a rate of about 2-fold faster than TDM-1 in non-tumor-bearing mice (Phillips et al, 2008, Cancer Res 68: 9280-90; Erickson et al, 2012, Mol Cancer Ther 11:1133-42), with 1.5-fold higher levels of T-DM1 compared to T-SSPDM1 in tumors. Unexpectedly and most interestingly, the amount of free active maytansinoid catabolites in the targeted tumors was very similar between the two ADCs (Erickson et al, 2012, Mol cancer ther 11: 1133-42).

In other words, T-SSP-DM1 was able to overcome its drawbacks in linker stability, as the fact of lower stability leads to more efficient drug release in tumors compared to more stable T-DM 1. Not surprisingly, the active drug-metabolite equivalence between these two ADCs in tumors resulted in similar anti-tumor effects in tumor-bearing animals. Finally, T-DM1 was selected based on toxicity issues arising when using super-toxic drugs and less stable linkers (Phillips et al, 2008, cancer Res 68: 9280-90). Since SN-38 is at least one log-fold less toxic than these maytansinoids, it is expected to release less toxicity from ADCs. However, even if it was released in serum, the amount of SN-38 localized in human gastric or pancreatic tumor xenografts was 136-fold higher than tumor-bearing mice injected with irinotecan at doses > 20-fold higher than SN-38 equivalents (Sharkey et al, 2015, Clin Cancer Res,21: 5131-8). Although we tested more stable linkers in the development of IMMU-132, they were significantly less effective than IMMU-132 in xenograft tumor models (Govindan et al, 2013, Mol Cancer Ther 12: 968-78).

Similarly, linkers that release SN-38 more rapidly (e.g., serum half-life of about 10 hours) are also less effective in xenograft models (Moon et al, 2008, J Med Chem51: 6916-26; Govindan et al, 2009, Clin ChemRes 15:6052-61), suggesting that there is an optimal window for SN-38 release leading to improved efficacy. Thus, current data demonstrates that IMMU-132 is a more efficient method of targeting and releasing drugs at tumors than irinotecan.

Early clinical studies have shown encouraging objective responses in various solid tumors and importantly indicate a better safety profile than irinotecan treatment with lower incidence of diarrhea (Starodub et al, 2015, ClincCancerRes 21: 3870-78).

In summary, IMMU-132 (Saxizumab govitegam) is a paradigm shift in ADC development. It conjugates 7-8 molecules of the more tolerated active metabolite SN-38 of incantan with an anti-Trop-2 antibody using a moderately stable linker. Although these appear to have counterintuitive characteristics compared to the hypercoxic ADC, non-clinical studies have demonstrated that IMMU-132 targets Trop-2 expressing tumors very efficiently, with significant efficacy and without significant toxicity. IMMU-132 also exhibits anti-tumor effects in early I/II clinical trials against a variety of solid tumors including pancreatic, gastric, TNBC, small cell and non-small cell lung cancers, with controlled toxicity in these patients, and no immune response to IgG or SN-38 was detected even after several months of dosing (Starodub et al, 2015, Clincearr Res21: 3870-78). Given the elevated expression of Trop-2 in such a wide variety of solid tumors, IMMU-132 continues to be studied clinically, particularly in advanced cancers that are refractory to most current treatment strategies.

Example 18 further results of phase I/II clinical Studies

Triple Negative Breast Cancer (TNBC)

Continuing with the phase I/II clinical trial (NCT01631552) discussed in the above example, a cumulative number of 56 TNBC patients received 10mg/kg of treatment. Prior to initiating IMMU-132 treatment, the patient population has previously been extensively treated with at least 2 prior treatments including taxane treatment. Previous treatments include cyclophosphamide, doxorubicin, carboplatin, gemcitabine, capecitabine, eribulin, cisplatin, anastrozole, vinorelbine, bevacizumab, and tamoxifen. Despite this extensive treatment history, TNBC patients responded well to IMMU-132, 2 confirmed Complete Responses (CR), 13 Partial Responses (PR) and 25 Stable Disease (SD), with an objective response rate of 29% (15/52) (not shown). By adding the incidence of CR to the incidence of PR and the incidence of SD, TNBC treatment resulted in a favorable response rate of 71% for IMMU-132 treated patients (not shown). The median time to progression in this heavily pretreated TNBC patient population was 9.4 months, and to date 2.9 to 14.2 months. However, 72% of patients in this study were still undergoing treatment. Metastatic NSCLC

Clinical trials are also being conducted on patients with metastatic non-small cell lung cancer (NSCLC), and to date 29 evaluable patients who received 8mg/kg or 10mg/kg IMMU-132 treatment have been accumulated. The optimal response was determined according to RECIST1.1 criteria (not shown). Among 29 patients, there were 8 PR and 13 SD. The time to progression of NSCLC patients showed that 21/33 (64%) of NSCLC patients exhibited PR or SD (not shown). The median time to progression is 9/4 months, ranging from 1.8 to 15.5+ months, and 47% of patients are still receiving treatment. Progression-free survival of NSCLC patients treated with 8mg/kg or 10mg/kg IMMU-132 was determined (not shown). Median PFS at 8mg/kg was 3.4 months at 10mg/kg for 3.8 months. However, studies are still ongoing and the median progression-free survival number may be improved.

Metastatic SCLC

Comparable results were observed for metastatic SCLC patients (not shown). The time to progression (not shown) showed a median of 4.9 months, ranging from 1.8 to 15.7+ months, and 7 patients were still receiving IMMU-132 treatment. Progression-free survival (not shown) showed a median PFS at 8mg/kg of 2.0 months and a median PFS at 10mg/kg of 3.6 months. The median OS was 8.1 months at 8mg/kg, and a median OS of 10mg/kg could not be determined.

Urothelial cancer

Similar results were obtained for urothelial cancer patients treated with either 8mg/kg or 10mg/kg IMMU-132. The best response data for 11 evaluable patients showed 6 PR and 2 SD (not shown). The time to progression (not shown) showed a median of 8.1 months, ranging from 3.6 months to 9.7+ months.

In summary, a sustained phase I/II clinical trial showed that IMMU-132 had superior efficacy in at least TNBC, NSCLC, SCLC and urothelial cancers when administered at the doses of ADC. Excellent therapeutic effects appear in these severely pretreated and resistant metastatic cancers without inducing severe toxicity that may hamper clinical use. IMMU-132 showed an acceptable safety profile in severe pre-treated patients with multiple solid cancers, with a median of 2 to 5 prior treatments. For grade 3 or higher adverse events, only the incidence of neutropenia exceeds 20% of the patient population. The study further demonstrates that repeated doses of IMMU-132 can be administered to human patients at therapeutic doses without causing interference with host anti-IMMU-132 antibodies. These results demonstrate the safety and utility of IMMU-132 for the treatment of various Trop-2 positive cancers in human patients.

***

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without undue experimentation, can make various changes and modifications to the invention to adapt it to various usages and conditions without departing from the spirit and scope of the invention. All patents, patent applications, and publications cited herein are incorporated by reference.

Claims (28)

1. A method of treating a Trop-2-positive cancer comprising administering to a human patient having a Trop-2-positive cancer an ADC comprising SN-38 conjugated to a linker moiety that is linked to an anti-Trop-2 antibody or antigen-binding fragment thereof, wherein the patient has relapsed from treatment with a checkpoint inhibitor or is refractory to treatment with a checkpoint inhibitor.

2. The method of claim 1, wherein the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4.

3. The method of claim 1, wherein the checkpoint inhibitor is selected from the group consisting of MPDL3280A, pembrolizumab, nivolumab, ipilimumab, pidilizumab, MDX-1105, covolizumab, BMS-936559, and tremelimumab.

4. The method of claim 1, wherein the checkpoint inhibitor is MPDL 3280A.

5. The method of claim 1, wherein the anti-Trop-2 antibody is an hRS7 antibody comprising the light chain CDR sequences CDR1(KASQDVSIAVA, SEQ ID NO:1), CDR2(SASYRYT, SEQ ID NO:2) and CDR3(QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO: 6).

6. The method of claim 1, wherein the anti-Trop-2 antibody competes for binding to Trop-2 with an antibody comprising the following sequences: light chain CDR sequences CDR1(KASQDVSIAVA, SEQ ID NO:1), CDR2(SASYRYT, SEQ ID NO:2) and CDR3(QQHYITPLT, SEQ ID NO:3) and heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO: 6).

7. The method of claim 1, wherein the linker is CL2A and the structure of the ADC is MAb-CL2A-SN-38

MAb-CL2A-SN-38。

8. The method of claim 7, wherein the anti-Trop-2 antibody is an hRS7 antibody comprising the light chain CDR sequences CDR1(KASQDVSIAVA, SEQ ID NO:1), CDR2(SASYRYT, SEQ ID NO:2) and CDR3(QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO: 6).

9. The method of claim 1, wherein the cancer is selected from the group consisting of colorectal cancer, lung cancer, gastric cancer, bladder cancer, renal cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, esophageal cancer, and prostate cancer.

10. The method of claim 1, wherein the cancer is triple negative breast cancer.

11. The method of claim 1, wherein the cancer is selected from the group consisting of triple negative breast cancer, HER +, ER +, progesterone + breast cancer, metastatic non-small cell lung cancer, metastatic endometrial cancer, metastatic urothelial cancer, and metastatic pancreatic cancer.

12. The method of claim 1, wherein the ADC is administered at a dose of between 3mg/kg and 18 mg/kg.

13. The method of claim 12, wherein the dose is selected from the group consisting of 3mg/kg, 4mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 16mg/kg, and 18 mg/kg.

14. The method of claim 1, wherein the ADC is administered at a dose of between 8mg/kg and 12 mg/kg.

15. The method of claim 1, wherein the ADC is administered at a dose of between 8mg/kg and 10 mg/kg.

16. The method of claim 1, wherein the ADC is administered at a dose of 10 mg/kg.

17. The method of claim 1, wherein the treatment results in a reduction in tumor size of at least 15%, at least 20%, at least 30%, or at least 40%.

18. The method of claim 1, wherein the ADC comprises 4 or more SN-38 molecules conjugated to the antibody or antigen-binding fragment thereof.

19. The method of claim 1, wherein the ADC comprises 6 to 8 SN-38 molecules conjugated to the antibody or antigen-binding fragment thereof.

20. The method of claim 1, wherein the cancer is metastatic.

21. The method of claim 20, further comprising reducing the size or eliminating metastasis.

22. The method of claim 7 wherein the 10-hydroxyl position of SN-38 in MAb-CL2A-SN-38 is 10 using the 'COR' moietyOEster or 10-O-carbonate derivatives wherein "CO" is a carbonyl group and the "R" group is selected from (i) N, N-disubstituted aminoalkyl group "N (CH)₃)₂-(CH₂)_n- ", wherein n is 1 to 10, and wherein the terminal amino group is optionally in quaternary salt form; (ii) alkyl residue "CH₃-(CH₂)_n- ", where n is 0 to 10; (iii) alkoxy moiety "CH₃-(CH₂) n-O- ", wherein n is 0-10; (iv) "N (CH)₃)₂-(CH₂)_n-O- ", wherein n is 2-10; or (v) "R₁O-(CH₂-CH₂-O)_n-CH₂-CH₂-O- ", wherein R₁Is ethyl or methylAnd n is an integer having a value of 0-10.

23. The method of claim 1, further comprising administering to the patient at least one additional anti-cancer therapy selected from the group consisting of surgery, external radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy, interfering RNA therapy, treatment with a therapeutic agent, and gene therapy.

24. The method of claim 23, wherein the therapeutic agent is a drug, toxin, immunomodulator, secondary antibody, antigen-binding fragment of a secondary antibody, pro-apoptotic agent, toxin, rnase, hormone, radionuclide, anti-angiogenic agent, siRNA, RNAi, chemotherapeutic agent, cytokine, chemokine, prodrug, or enzyme.

25. The method of claim 24, wherein the drug is selected from the group consisting of 5-fluorouracil, afatinib, aplidine, azalipine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryodin-1, busulfan, calicheamicin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecin, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinesib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrroline doxorubicin (2P-DOX), and, Cyano-morpholinodoxorubicin, doxorubicin glucuronide, epidoxorubicin glucuronide, erlotinib, estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogen receptor binding agent, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5' -O-dioleoyl-FudR (FUdR-dR), fludarabine, flutamide, farnesyl-protein transferase inhibitors, fraxidil, fotattinib, ginetidine, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, iderivil, ifosfamide, imatinib, L-asparaginase, lapatinib, lenalidomide, iminoctadine, LFM-A13, lomustine, erlotinib-A3578, etoposide, 3',5' -O-dioleonide, fludarabicin, farnes, Nitrogen mustard, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, noviban, neratinib, nilotinib, nitrosourea, olaparib, plicamycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozotocin, SU11248, sunitinib, tamoxifen, temozolomide (an aqueous form of DTIC), carboplatin, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uramustine, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloid, and ZD 1839.

26. The method of claim 24, wherein the immunomodulator is selected from the group consisting of cytokines, lymphokines, monokines, stem cell growth factor, lymphotoxins, hematopoietic factors, Colony Stimulating Factor (CSF), Interferons (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH), liver growth factor, prostaglandins, fibroblast growth factor, prolactin, placental prolactin, OB protein, Transforming Growth Factor (TGF), TGF- α, TGF- β, insulin-like growth factor (IGF), erythropoietin, thrombopoietin, Tumor Necrosis Factor (TNF), TNF- α, TNF- β, mullerian tube inhibitory substances, mouse gonadotropin-related peptides, inhibin, activin, vascular endothelial growth factor, Interleukin (IL), granulocyte colony stimulating factor (G-CSF), granulocyte colony stimulating factor (GM-CSF), interferon-56, interferon-4656, interferon-5, interleukin-16, IL-5, IL-16, IL-ligand, IL-16, IL-ligand, IL-5, IL-16, IL-16, IL-5, IL-16, IL-16, IL-16, IL-16, IL-IL.

27. The method of claim 24, wherein the radionuclide is selected from the group consisting of¹¹C、¹³N、¹⁵O、³²P、³³P、⁴⁷Sc、⁵¹Cr、⁵⁷Co、⁵⁸Co、⁵⁹Fe、⁶²Cu、⁶⁷Cu、⁶⁷Ga、⁶⁷Ga、⁷⁵Br、⁷⁵Se、⁷⁵Se、⁷⁶Br、⁷⁷As、⁷⁷Br、^80mBr、⁸⁹Sr、⁹⁰Y、⁹⁵Ru、⁹⁷Ru、⁹⁹Mo、^99mTc、^103mRh、¹⁰³Ru、¹⁰⁵Rh、¹⁰⁵Ru、¹⁰⁷Hg、¹⁰⁹Pd、¹⁰⁹Pt、¹¹¹Ag、¹¹¹In、^113mIn、¹¹⁹Sb、^121mTe、^122mTe、¹²⁵I、^125mTe、¹²⁶I、¹³¹I、¹³³I、¹⁴²Pr、¹⁴³Pr、¹⁴⁹Pm、¹⁵²Dy、¹⁵³Sm、¹⁶¹Ho、¹⁶¹Tb、¹⁶⁵Tm、¹⁶⁶Dy、¹⁶⁶Ho、¹⁶⁷Tm、¹⁶⁸Tm、¹⁶⁹Er、¹⁶⁹Yb、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、^189mOs、¹⁸⁹Re、¹⁹²Ir、¹⁹⁴Ir、¹⁹⁷Pt、¹⁹⁸Au、¹⁹⁹Au、¹⁹⁹Au、²⁰¹Tl、²⁰³Hg、²¹¹At、²¹¹Bi、²¹¹Pb、²¹²Bi、²¹²Pb、²¹³Bi、²¹⁵Po、²¹⁷At、²¹⁹Rn、²²¹Fr、²²³Ra、²²⁵Ac、²²⁷Th and²⁵⁵fm.

28. The method of claim 24, wherein the toxin is selected from the group consisting of ricin, abrin, ribonuclease (rnase), dnase I, staphylococcal enterotoxin-a, pokeweed antiviral protein, gelonin, diphtheria toxin, pseudomonas exotoxin and pseudomonas endotoxin.