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NZ749410B2 - Conjugates for targeted cell surface editing - Google Patents
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NZ749410B2 - Conjugates for targeted cell surface editing - Google Patents

Conjugates for targeted cell surface editing Download PDF

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Publication number
NZ749410B2
NZ749410B2 NZ749410A NZ74941017A NZ749410B2 NZ 749410 B2 NZ749410 B2 NZ 749410B2 NZ 749410 A NZ749410 A NZ 749410A NZ 74941017 A NZ74941017 A NZ 74941017A NZ 749410 B2 NZ749410 B2 NZ 749410B2
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NZ
New Zealand
Prior art keywords
sialidase
her2
cells
cell
conjugate
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NZ749410A
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NZ749410A (en
Inventor
Carolyn R Bertozzi
Melissa Gray
Elliot C Woods
Han Xiao
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The Board Of Trustees Of The Leland Stanford Junior University
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Application filed by The Board Of Trustees Of The Leland Stanford Junior University filed Critical The Board Of Trustees Of The Leland Stanford Junior University
Priority claimed from PCT/US2017/040411 external-priority patent/WO2018006034A1/en
Publication of NZ749410A publication Critical patent/NZ749410A/en
Publication of NZ749410B2 publication Critical patent/NZ749410B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase

Abstract

Provided are conjugates comprises an antibody that binds to a cell surface molecule of a target cell, which is conjugated to a prokaryotic sialidase, a eukaryotic sialidase or a derivative thereof. Also provided are compositions and kits that include the conjugates, as well as methods of using the conjugates. Methods of making conjugates are also provided. onjugates. Methods of making conjugates are also provided.

Description

CONJUGATES FOR TARGETED CELL SURFACE EDITING REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US. Provisional Patent Application No. 62/357,645 filed July 1, 2016, which application is incorporated herein by reference in its entirety.
ENT REGARDING FEDERALLY SPONSORED RESEARCH This ion was made with Government support under contracts 07 and CA108781 awarded by the al Institutes of Health. The Government has certain rights in the invention.
INTRODUCTION ies that enhance the immune response to cancer are proving revolutionary in the fight against intractable tumors. Immune cells ate s from activating and inhibitory receptors to ine their response to a challenging target—activating signals alert them to the presence of pathology while inhibitory signals tell the cell that it has confronted a healthy “self”. Successful tumors evolve mechanisms to thwart immune cell ition, often by overexpressing ligands for inhibitory receptors. This discovery has led to new therapeutic strategies aimed at blocking inhibitory immune cell signaling, as embodied in clinically approved T cell checkpoint inhibitors targeting PD-t and CTLA-4.
Ongoing pre-clinical studies have focused on combining therapies targeting multiple immunologic pathways. For example, antibodies against PD-1/PD-L1 in combination with those targeting other T cell checkpoint inhibitors demonstrate improved umor activity in syngeneic tumor models. A complement to these interventions are therapies targeting innate immune cells, particularly natural killer (NK) cells, macrophages and dendritic cells.
Provided are conjugates including a targeting moiety that binds to a cell e molecule of a target cell and a target cell surface-editing enzyme. Also provided are compositions and kits that include the conjugates, as well as methods of using the conjugates. Methods of making conjugates are also provided.
BRIEF DESCRIPTION OF THE FIGURES schematically illustrates an immune evasion gy and a method of reducing such immune evasion according to one embodiment of the present disclosure. depicts the preparation of antibody-sialidase conjugates and their electrophoretic and ESl-MS analysis. depicts electrophoretic analysis of sialidases, ysis activities, and flow cytometry and imaging analysis of their ties depicts cell-surface sialylation levels and ligand levels of various receptors with or without sialidase treatment in different breast cancer cell lines. depicts the cytotoxicity of isolated peripheral blood NK cells in the absence or presence of sialidase. depicts s assays used for the characterization of wild-type and heat- inactivated V. cholerae sialidase. depicts the ESI-MS spectra for Vibrio ae sialidase, Salmonella urium sialidase, anti-Her2—IgG and its conjugates. depicts the hydrolytic activities of V. cholerae sialidase and anti-Her2—lgG-Sia.
Also depicted is hydrolytic ties of S. typhlmurium sialidase and anti-Her2—lgG-StSia. depicts images showing the level of urface sialic acid in various cell lines, with or without trastuzumab-sialidase conjugate treatment. Also shown is flow cytometry data comparing removal of cell-surface sialic acid by two trastuzumab-sialidase conjugates in s cell lines. depicts images showing Sambucus nigra ligands on various cell lines in the absence or ce of anti-Her2—lgG-Sia ate. depicts the cytotoxicity of isolated eral blood NK cells in the presence of anti-Her2—lgG or anti-Her2—lgG-Sia. depicts the activity of trastuzumab and trastuzumab-sialidase conjugate against HER-2 expressing cancer cells. s the siglec expression levels and cytotoxicity of isolated monocyte populations and differentiated hages in the ce of sialidase, trastuzumab, and trastuzumab-sialidase conjugates against HER2+ expressing cancer cells. depicts the cytotoxicity of isolated yo T cells in the presence of sialidase, trastuzumab, or zumab-sialidase conjugate against HER2+ expressing cancer cells. depicts the cytotoxicity of peripheral blood NK cells with anti-Her2—lgG or a mixture of anti-Her2-IgG and sialidase in the e or presence of blocking antibodies as specified. depicts flow cytometry data analyzing CD56 and CD3 markers on leukocytes. depicts data demonstrating that sialidase treatment potentiates rituximab- induced complement-dependent cytotoxicity (CDC). depicts data showing that Ramos cells have higher levels of Siglec-9 ligands than Daudi cells. depicts data demonstrating that sialidase potentiates rituximab in a complement-dependent .
DETAILED DESCRIPTION Provided are conjugates including a targeting moiety that binds to a cell surface molecule of a target cell and a target cell surface-editing enzyme. Also provided are compositions and kits that include the ates, as well as methods of using the conjugates. Methods of making conjugates are also provided.
Before the conjugates, compositions and methods of the t disclosure are described in greater detail, it is to be understood that the conjugates, compositions and s are not limited to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the conjugates, compositions and methods will be d only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, n the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the conjugates, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the r ranges and are also encompassed within the conjugates, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the conjugates, compositions and methods.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the t in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all cal and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the conjugates, compositions and methods belong. Although any conjugates, compositions and methods r or equivalent to those described herein can also be used in the practice or g of the conjugates, compositions and methods, representative illustrative conjugates, compositions and methods are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual ation or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or s in connection with which the publications are cited. The on of any publication is for its sure prior to the filing date and should not be construed as an admission that the present conjugates, itions and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural nts unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as y, l! “only” and the like in tion with the recitation of claim elements, or use of a “negative” limitation.
It is iated that certain features of the conjugates, compositions and methods, which are, for clarity, described in the context of te embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the conjugates, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be ed separately or in any suitable sub-combination.
All ations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was dually and explicitly disclosed, to the extent that such combinations embrace le ses and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present conjugates, compositions and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events d or in any other order that is lly possible.
CONJUGATES As summarized above, aspects of the present disclosure include conjugates. In certain aspects, the conjugates include a targeting moiety that binds to a cell surface molecule of a target cell, and a target cell e-editing enzyme. ing Moieties According to certain embodiments, a conjugate of the present disclosure es a ing moiety. The targeting moiety may vary and may be selected based, e.g., on the nature of the cell surface molecule on the target cell. Non-limiting examples of a targeting moiety that may be employed include an antibody, a ligand, an aptamer, a nanoparticle, and a small molecule.
In certain aspects, the targeting moiety specifically binds to the cell surface molecule. As used , a targeting moiety that “specifically binds to the cell surface molecule” or is “specific for the cell surface molecule” refers to a targeting moiety that binds the cell surface molecule with greater affinity than with other cell surface molecules.
According to n embodiments, the targeting moiety exhibits a binding affinity to the cell surface molecule of a Kd of less than or equal to about 10'5 M, less than or equal to about '6 M, or less than or equal to about 10'7 M, or less than or equal to about 10'8 M, or less than or equal to about 10'9 M, 10'10 M, 10'11 M, or 10'12 M or less. Such affinities may be readily determined using conventional techniques, such as by equilibrium dialysis, surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer), radioimmunoassay, or by another method.
According to certain embodiments, the targeting moiety is an antibody. The terms ody" and oglobulin” include antibodies or immunoglobulins of any isotype (e.g., lgG (e.g., lng, lgG2, lgGS, or lgG4), lgE, lgD, lgA, lgM, etc.), whole dies (e.g., antibodies composed of a er which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies; fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the cell surface molecule of the target cell, including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human dies, humanized antibodies (e.g., zed whole antibodies, humanized half antibodies, or humanized antibody fragments); and fusion proteins comprising an antigen-binding portion of an antibody and a tibody protein. The antibodies may be detectably labeled, e.g., with an in vivo imaging agent, a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
In certain aspects, when the targeting moiety is an antibody, the antibody may be a therapeutic antibody even in the absence of the target cell e-editing , e.g., an antibody having efficacy on its own in the ent of cancer (e.g., via antibody-dependent cellular cytotoxicity and/or another mechanism), an immune-related disorder, an endothelial cell-related disorder, or the like. For example, the antibody may be a eutic antibody that specifically binds to a tumor-associated cell surface molecule or a tumor-specific cell surface le.
Non-limiting examples of antibodies that specifically bind to a tumor-associated cell e molecule or a tumor-specific cell surface molecule which may be employed in a ate of the present disclosure include Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, tumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, lratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Radretumab, rumab, Rilotumumab, Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, tumab, Vesencumab, Votumumab, Zalutumumab, Flanvotumab, mab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, omab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, mab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, lndatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, ximab, ximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab, zumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, uzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, tuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, zumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or an antigen-binding t thereof. As used herein, “variant” is meant the antibody binds to the particular antigen (e.g., HER2 for trastuzumab) but has fewer or more amino acids than the parental antibody, has one or more amino acid substitutions relative to the parental antibody, or a combination thereof.
In certain aspects, the targeting moiety is a therapeutic antibody set forth in Table 1 below approved for treating cancer, or an antigen-binding variant thereof. Also provided in Table 1 is the corresponding tumor-associated cell surface molecule or tumor-specific cell surface molecule to which the therapeutic antibody ically binds, as well as the type of cancer for which the dy is ed for treatment.
Table 1 — Antibodies approved for treating cancer Cell Cancer Types Antibody surface molecule BCR-ABL CML ib, Dasatinib ALL Nilotinib, Bosutinib Ponatinib CD19 ALL Blinatumomab CD20 NHL, CLL Rituximab B-cell NHL Ofatumumab pre-B ALL ritumomab 131I-Tositumomab CD30 Hodgkin’s lymphoma Brentuximab vedotin CD33 AML Gemtuzumab ozogamicin CD52 CLL Alemtuzumab CTLA-4 Unresectable or metastatic umab melanoma EGFR CRC Cetuximab Head and Neck Panitumumab EpCAM Malignant ascites Catumaxomab HER2 Breast zumab Pertuzumab PAP Prostate Sipuleucel-T PD-1 Metastatic melanoma NSCLC Nivolumab Pembrolizumab VEGF Breast, Cervical Bevacizumab CRC, NSCLC RCC, Ovarian Glioblastoma WO 06034 Cell Cancer Types Antibody surface molecule VEGF-Fl2 Gastric Ramucirumab NSCLC Abbreviations for Table 1 are as follows: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; BCR-ABL, breakpoint cluster region Abelson tyrosine kinase; CLL, chronic lymphocytic leukemia; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; HER2, human epidermal growth factor receptor 2; NHL, non-Hodgkin’s lymphoma; NSCLC, non-small cell lung cancer; PAP, tic acid phosphatase; PD-1, mmed cell death receptor 1; R00, renal cell carcinoma; VEGF, vascular endothelial growth factor; VEGF-R2, vascular endothelial growth factor receptor 2.
In some embodiments, the ing moiety is a therapeutic antibody set forth in Table 2 below or an antigen-binding variant f. Also provided in Table 2 is the corresponding tumor-associated cell surface le or specific cell surface molecule to which the eutic antibody specifically binds, as well as an example cancer type which may be treated using a conjugate that includes the antibody.
Table 2 — Additional antibodies, cell surface molecules, and cancer types Cell surface Cancer types Antibody molecule A2aFl NSCLC PBF-509 AKAP4 NSCLC Preclinical BAGE Glioblastoma Preclinical Ovarian BORIS Prostate, Lung Preclinical Esophageal CD22 ALL Epratuzumab Moxetumomab Cell e Cancer types Antibody molecule Inotuzumab ozogamicin CD73 Advanced solid tumors MEDI9447 CD137 Advanced solid tumors Urelumab PF-O5082566 CEA CRC PANVACTM Ad5-[E1-, E2b-]-CEA(6D) CS1 Multiple myeloma Elotuzumab CTLA-4 Malignant mesothelioma imumab EBAGQ Bladder Preclinical EGF NSCLC CIMAvax EGFR NSCLC Necitumumab GAGE Cervical Preclinical GD2 Neuroblastoma Dinutuximab, hu3F8 blastoma hu14.18-|L-2, TSBsAb Melanoma other solid tumors anti-GD2 CAR GD2—KLH gp100 Melanoma gp100:209-217(210M) HPV—16 Cervical HPV—16 (E6, E7) SCCHN TG4001, Lm-LLO-E7 pNGVL4a-CRT/E7, IND-3112 HSP105 CBC Preclinical Bladder IDH1 Glioma |DH1(R132H) 42 Idiotype NSCLC, Breast Racotumomab (NeuGcGM3) Melanoma |DO1 Breast, Melanoma Indoximod |NCBO24360 Cell surface Cancer types Antibody molecule NSCLC ID01 peptide vaccine KIR Lymphoma Lirilumab LAG-3 Breast, Hemato- BMS—986016 logical, Advanced solid tumors IMP321 LY6K Gastric LY6K-177 peptide SCCHN LY6K, CDCA1, IMP3 3 Melanoma recMAGE-AB NSCLC Zastumotide MAGE—CZ Gastric, Melanoma Preclinical Multiple myeloma MAGE-D4 CBC Preclinical Melan-A Melanoma MART-1 (26-35, 27L) MET NSCLC Onartuzumab Tivantinib MUC1 NSCLC, Breast Tecemotide, TG401O Prostate PANVACTM MUC4 Pancreatic Preclinical MUC16 n Abagovomab Oregovomab NY-ESO-1 Ovarian NY-ESO-1/ISCOMATRIXTM Melanoma rV-NY-ESO-1; rF-NY-ESO-1 PD-1 B-cell ma zumab ma, CRC AMP-224, AMP-514 PD-L1 NSCLC, RCC EMS-936559, izumab Bladder, Breast Durvalumab, Avelumab Melanoma, SCCHN PRAME NSCLC Preclinical Cell surface Cancer types Antibody molecule PSA Prostate PROSTVAC®-VF ROR1 CLL, Pancreatic Preclinical Lung, Breast SialyI-Tn Breast Theratope SPAG-9 Prostate, CBC nical NSCLC, Ovarian SSX1 Prostate Preclinical Multiple myeloma in Melanoma EMD640744 Glioma, Solid tumors Trivalent peptide vaccine Tripeptide vaccine rase Pancreatic Tertomotide TIM-3 Melanoma, NHL NSCLC Preclinical VISTA Melanoma, Bladder Preclinical WT1 Ovarian, Uterine, AML WT1 peptide e Multiple myeloma XAGE-f b Prostate DC-based tumor vaccine 5T4 RCC, CRC TroVax® Prostate Naptumomab estafenatox Abbreviations for Table 2 are as follows: A2aR, adenosine A2a receptor; AKAP4, A kinase anchor protein 4; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; BAGE, B melanoma antigen; BORIS, brother of the regulator of imprinted sites; CEA, carcinoembryonic antigen; CLL, chronic lymphocytic leukemia; CRC, colorectal ; CS1, CD2 subset 1; CTLA-4, xic T-lymphocyte-associated antigen 4; EBAGQ, estrogen receptor binding site associated antigen 9; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; GAGE, G antigen; GD2, disialoganglioside GD2; gp100, glycoprotein 100; HPV-16, human papillomavirus 16; HSP105, heat-shock protein 105; IDH1, rate dehydrogenase type 1; IDO1, indoleamine—2,3-dioxygenase 1; KIR, killer cell globulin-like receptor; LAG-3, lymphocyte activation gene 3; LYGK. lymphocyte antigen 6 complex K; MAGE-As, melanoma antigen 3; MAGE-C2, melanoma antigen 02; MAGE—D4, melanoma antigen D4; Melan-A/MART-1, melanoma n recognized by T-cells 1; MET, N-methyl—N’—nitrosoguanidine human osteosarcoma transforming gene; MUC1, mucin 1; MUC4, mucin 4; MUC16, mucin 16; NHL, non-Hodgkin lymphoma; NY—ESO-1, New York esophageal squamous cell carcinoma 1; PD-1, programmed cell death receptor 1; PD-L1, programmed cell death receptor ligand 1; PRAME, preferentially sed antigen of melanoma; PSA, prostate specific antigen; RCC, renal cell carcinoma; ROR1, receptor tyrosine kinase orphan receptor 1; SCCHN, squamous cell oma of the head and neck; SPAG-Q, sperm-associated antigen 9; SSX1, synovial sarcoma X-chromosome breakpoint 1; TIM-3, T-cell immunoglobulin domain and mucin domain-3; VISTA, V-domain immunoglobulincontaining suppressor of T-cell activation; WT1, Wilms’ Tumor-1; XAGE-1b, X chromosome antigen 1b.
In some embodiments, the targeting moiety is a therapeutic antibody set forth in Table 3 below or an antigen-binding variant thereof. Also ed in Table 3 is the corresponding tumor-associated cell surface molecule or tumor-specific cell surface molecule to which the therapeutic dy specifically binds.
Table 3 — Additional antibodies and ponding cell surface molecules obinutuzumab ipilimumab CTLA-4 tremelimumab CTLA-4 cetuximab EGFR necitumumab EGFR panitumumab EGFR zalutumumab EGFR lomab EpCAM (17-1A) farletuzumab FR-alpha pertuzumab Her2 trastuzum ab Her2 lotumumab figitumumab IGF-1 ganitumab IGF1 R durvalumab |GG1K bavituximab Phosphatidylserine onartuzum ab scatter factor receptor ki nase bevacizumab VEGF-A ramucirumab VEGFR2 In some ments, a conjugate of the present disclosure includes a therapeutic antibody as the targeting moiety selected from trastuzumab, cetuximab, daratumumab, girentuximab, panitumumab, ofatumumab, rituximab, and antigen-binding variants thereof.
In certain aspects, a conjugate of the present disclosure includes a therapeutic antibody as the targeting moiety, and the therapeutic antibody is trastuzumab or a HERZ- binding variant thereof. The heavy and light chain amino acid sequences of trastuzumab are known and provided in Table 4 below.
Table 4 — zumab heavy and light chain amino acid seguences Trastuzumab Light Chain (SEQ DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW ID NO: 1) YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD SLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR Trastuzumab Heavy Chain (SEQ EVQLVESGGGLVQPGGSLRLSCAASGFNI KDTYI H ID NO: 2) WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGF YAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH KEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K When the targeting moiety is an antibody, the target cell surface-editing enzyme may be conjugated to any suitable region of the antibody. In certain aspects, the targeting moiety is an antibody having a light chain polypeptide, and the target cell surface-editing enzyme is conjugated to the light chain, e.g., at the C-terminus or an internal region of the light chain. ing to certain embodiments, the targeting moiety is an dy having a heavy chain polypeptide, and the target cell surface-editing enzyme is conjugated to the heavy chain, e.g., at the C-terminus or an internal region of the heavy chain. If the antibody having a heavy chain includes a fragment crystallizable (Fc) region, the target cell surface- editing enzyme may be conjugated to the Fc region, e.g., at the C-terminus or an internal region of the Fc region.
According to certain embodiments, the ing moiety is a ligand. As used herein, a “ligand” is a substance that forms a complex with a biomolecule to serve a biological purpose. The ligand may be a substance ed from a circulating factor, a secreted factor, a cytokine, a growth factor, a hormone, a peptide, a polypeptide, a small molecule, and a nucleic acid, that forms a complex with the cell surface molecule on the surface of the target cell. In certain aspects, when the targeting moiety is a ligand, the ligand is ed in such a way that complex ion with the cell surface molecule , but the normal biological result of such complex formation does not occur. In certain aspects, the ligand is the ligand of a cell surface receptor t on the target cell. Cell surface receptors of st include, but are not d to, receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (non-RTKs), growth factor receptors, etc. When the conjugates of the present disclosure include a ligand as the targeting , the target cell surface-editing enzyme may be conjugated to any suitable region of the ligand, e.g., a region of attachment that does not interfere or substantially interfere with the ability of the ligand to bind (e.g., specifically bind) the target cell surface molecule.
In certain aspects, the targeting moiety is an aptamer. By “aptamer” is meant a c acid (e.g., an oligonucleotide) that has a specific g affinity for the target cell surface molecule. Aptamers exhibit certain ble properties for targeted delivery of the target cell e-editing enzyme, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Aptamers that bind to cell surface molecules are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C). Aptamers that find use in the conjugates of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1):39-45; Sun et al. (2014) Mol. Ther. Nucleic Acids 3:e182; and Zhang et al. (2011) Curr. Med. Chem. 18(27):4185-4194.
According to certain embodiments, the targeting moiety is a nanoparticle. As used , a “nanoparticle" is a le having at least one dimension in the range of from 1 nm to 1000 nm, from 20 nm to 750 nm, from 50 nm to 500 nm, including 100 nm to 300 nm, e.g., 120-200 nm. The nanoparticle may have any suitable shape, including but not d to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder- shaped, lical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped, prism-shaped, or any other suitable geometric or non- geometric shape. In certain aspects, the nanoparticle includes on its surface one or more of the other targeting moieties described , e.g., dies, ligands, aptamers, small molecules, etc. Nanoparticles that find use in the ates of the present disclosure include those described in Wang et al. (2010) Pharmacol. Res. 62(2):90-99; Rao et al. (2015) ACS Nano 9(6):5725-5740; and Byrne et al. (2008) Adv. Drug Deliv. Rev. 60(15):1615-1626.
In certain aspects, the targeting moiety is a small molecule. By “small molecule” is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain aspects, the small molecule is not made of repeating lar units such as are t in a polymer. In certain aspects, the target cell surface molecule is a receptor for which the ligand is a small molecule, and the small molecule of the conjugate is the small molecule ligand (or a derivative thereof) of the receptor. Small molecules that find use in targeting a conjugate to a target cell of interest are known. As just one example, folic acid (FA) derivatives have been shown to effectively target certain types of cancer cells by binding to the folate receptor, which is overexpressed, e.g., in many epithelial tumors. See, e.g., e et al. (2015) Ther. Adv. Med. Oncol. 06—218. In another example, the small molecule 2 has proven to be effective in targeting cancer cells. See, e.g., Hashim et al. (2014) Molecular Oncology 8(5):956—967.
Sigma-2 is the small molecule ligand for sigma-2 receptors, which are overexpressed in many proliferating tumor cells including pancreatic cancer cells. In certain aspects, a conjugate of the present disclosure includes a small molecule as the targeting moiety, in which it has been demonstrated in the context of a small le drug conjugate (SMDC) that the small molecule is ive at targeting a conjugate to a target cell of interest by binding to a cell e molecule on the target cell.
Target Cell e Editing Enzymes As summarized above, the conjugates of the present disclosure include a target cell surface-editing enzyme. As used herein, a “target cell e-editing enzyme” is an enzyme which, upon binding of the targeting moiety to the corresponding cell e molecule of the target cell, effects a structural change in one or more molecules on the surface of the target cell. In certain aspects, the structural change is to the cell surface molecule to which the targeting moiety binds. In other s, the structural change is to a molecule on the surface of the target cell other than the cell surface molecule to which the targeting moiety binds.
In certain aspects, the target cell surface-editing enzyme is a wild-type enzyme (that is, an enzyme found in nature). In other aspects, the enzyme is not a wild-type enzyme.
For example, the target cell e-editing enzyme may be a non-natural derivative of a wild-type enzyme. Such derivatives at least partially retain the enzymatic activity of the corresponding wild-type enzyme. Enzyme derivatives that may be ed include those that have fewer amino acids or more amino acids than the corresponding wild-type enzyme.
Alternatively, or additionally, the enzyme derivatives may include one or more amino acid substitutions or amino acid modifications relative to the ponding wild-type .
An example of a structural change effected by a target cell surface-editing enzyme in a molecule on the surface of the target cell is the cleavage of the molecule. In certain aspects, the molecule cleaved by the target cell surface-editing enzyme is a polymer. Cell surface rs which may be cleaved (e.g., degraded) by the target cell surface-editing enzyme include, but are not limited to, polypeptides, polysaccharides, roteins, and the like. For e, the target cell surface—editing enzyme may be a protease that cleaves polypeptides (or a subgroup of interest thereof) on the surface of the target cell. In certain aspects, the polymer cleaved by the target cell surface-editing enzyme is a polysaccharide (or “glycan”), that is, a molecule containing ccharides linked glycosidically. In such embodiments, the target cell surface-editing enzyme may be, e.g., a glycoside hydrolase (e.g., a sialidase).
According to certain embodiments, the cell surface-editing enzyme cleaves (e.g., hydrolyzes) a terminal residue of a molecule (e.g., a polymer) on the e of the target cell. In certain aspects, the terminal residue is present in a molecule selected from a oligosaccharide, a polysaccharide, a glycoprotein, a glycolipid, and a ganglioside. In some embodiments the terminal residue is a terminal sialic acid residue. When the terminal residue is a terminal sialic acid residue, the cell e-editing enzyme may be a sialidase (or a derivative thereof as described above), which cleaves the glycosidic linkages of sialic (neuraminic) acids, releasing terminal sialic acid es from oligosaccharides, polysaccharides, glycoproteins, glycolipids, and other ates.
Sialidases which may be ed in the conjugates of the present disclosure include, but are not limited to, prokaryotic sialidases and eukaryotic ases. Prokaryotic sialidases that may be employed include bacterial sialidases. One example of a bacterial sialidase that finds use in the conjugates of the present disclosure is Salmonella typhimurium sialidase (e.g., UniProtKB - P29768). Another example of a ial sialidase that finds use in the conjugates of the present sure is Vibrio cholera sialidase (e.g., UniProtKB - POC6E9). Eukaryotic sialidases that may be employed include, e.g., mammalian sialidases and non-mammalian eukaryotic sialidases. Mammalian sialidases (or mammalian neuraminidases) of interest include those from primates, e.g., human or non-human neuraminidases. In n aspects, the ase is a human sialidase.
According to certain embodiments, the human sialidase is selected from human neuraminidase 1 (e.g., UniProtKB - 099519), human neuraminidase 2 (e.g., tKB - Q9Y3R4), human neuraminidase 3 (e.g., UniProtKB - Q9UQ49), and human neuraminidase 4 (e.g., UniProtKB - ). It will be tood that the sialidase may be a derivative of any of the wild-type sialidases above, such as truncated derivatives, derivatives that include more amino acids than the corresponding wild-type ase, derivatives that include one or more amino acid substitutions (e.g., one or more conservative substitutions, one or more nservative substitutions, a substitution of a natural amino acid with a non-natural amino acid, and/or the like), etc. The derivatives retain at least a portion of the glycoside hydrolase activity of the al wild—type sialidase.
Table 5 — Amino acid seguences of example sialidases Salmonella VFKAEGEHFTDQKGNTIVGSGSGGTTKYFRI PAMCTTS urium sialidase FADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYND (SEQ ID NO: 3) RVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYR DWDLVLYKSTDDGVTFSKVETNIHDIVTKNGTISAM LG GVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSL PSGYCEGFGSENN|IEFNASLVNNIRNSGLRRSFETKDFGKTWT EFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDY TRSDISLYAHNLYSGEVKLIDDFYPKVGNASGAGYSCLSYRKNV DKETLYVVYEANGSIEFQDLSRHLPVIKSYN Vibrio cholerae ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPA sralldase (SEQ ID WLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGG NO: 4) MlTNYYANGTQRVLPlISLDSSGNLVVEFEGQTGRTVLATGTAAT EYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGN GSSNTDGVAAYRDIKFEIQGDVIFRGPDRIPSIVASSVTPGVVTA FAEKRVGGGDPGALSNTNDI ITRTSRDGGITWDTELNLTEQINV SDEFDFSDPRPIYDPSSNTVLVSYARWPTDAAQNGDRI KPWMP NGIFYSVYDVASGNWQAP|DVTDQVKERSFQIAGWGGSELYRR NTSLNSQQDWQSNAKIRIVDGAANQIQVADGSRKYVVTLSIDES GGLVANLNGVSAPI ILQSEHAKVHSFHDYELQYSALNHTTTLFV DGQQITTWAGEVSQENNIQFGNADAQIDGRLHVQKIVLTQQGH N LVEFDAFYLAQQTPEVEKDLEKLGWTKIKTGNTMSLYGNASV NPGPGHGITLTRQQNISGSQNGRLIYPAIVLDRFFLNVMSIYSDD TGSTLP|PFRWKSSSILETLEPSEADMVELQNGDLLLT ARLDFNQIVNGVNYSPRQQFLSKDGGITWSLLEANNANVFSNIS TGTVDASITRFEQSDGSHFLLFTNPQGNPAGTNGRQNLGLWFS FDEGVTWKGPIQLVNGASAYSDIYQLDSENAIVIVETDNSNMRIL RMPITLLKQKLTLSQN According to certain embodiments, the target cell surface-editing enzyme edits (e.g., cleaves all or a portion of) a ligand on the surface of the target cell. In some embodiments, the ligand is a ligand of an immune receptor. Immune receptor ligands of interest include, but are not limited to, ligands of inhibitory immune receptors. In certain aspects, the target cell surface-editing enzyme cleaves a ligand of an inhibitory immune receptor, where the inhibitory immune receptor is present on a cell ed from a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a basophil, and an eosinophil. By way of example, the ligand on the surface of the target cell edited by the target cell surface-editing enzyme may be a ligand for a sialic acid-binding lg- like lectin (Siglec) receptor, e.g., Siglec 7, Siglec 9, and/or the like. According to n embodiments, such a ligand is a sialoglycan.
In n aspects, a structural change in a molecule on the surface of the target cell effected by the target cell surface-editing enzyme is the oxidation of the molecule.
In some embodiments, the structural change in a molecule on the surface of the target cell effected by the target cell surface-editing enzyme is the reduction of the molecule.
In certain aspects, the target cell surface-editing enzyme s a ural change in a molecule on the surface of the target cell by adding a moiety to the molecule. For example, the target cell surface-editing enzyme may be a transferase that transfers a functional group to the molecule from a donor molecule. In some embodiments, the target cell surface-editing enzyme is a kinase that adds a phosphate group to the le on the surface of the target cell.
According to some embodiments, the target cell surface-editing enzyme effects a structural change in a molecule on the surface of the target cell by removing a moiety from the molecule. For example, the target cell surface-editing enzyme may be a transferase that ers a functional group from the molecule to an acceptor molecule. In some embodiments, the target cell surface-editing enzyme is a phosphatase that removes a phosphate group from the molecule on the surface of the target cell.
Tar et Cells The targeting moiety and target cell surface-editing enzyme may be selected based on the cell to be targeted. According to certain embodiments, the target cell is selected from a cancer cell, an immune cell, an endothelial cell, and an epithelial cell. Target cells of interest include, but are not limited to, cells that are relevant to a particular disease or condition. For example, the target cell may be a normal oning cell (e.g., a normal functioning immune cell, etc), and the cell surface editing enzyme modulates the function of the cell in a manner that is therapeutic to an individual in need thereof, e.g., boosts a function of the cell that is cial in treating a disease in an individual.
In other aspects, the target cell is not a normal cell. Non-normal target cells of interest e, but are not limited to, cancer cells. By “cancer cell” is meant a cell exhibiting a neoplastic cellular ype, which may be characterized by one or more of, for e, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate tor of cellular transformation. “Cancer cell" may be used interchangeably herein with "tumor cell”, “malignant cell” or “cancerous cell", and encompasses cancer cells of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like. In certain aspects, the cancer cell is a carcinoma cell. According to certain ments, the cancer cell is selected from a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, a colon cancer cell, and a cancer cell of any of the cancer types set forth in Tables 1 and 2 above.
In certain aspects, when the target cell is a cancer cell, the molecule on the surface of the cancer cell to which the targeting moiety binds is a tumor-associated cell surface molecule or a tumor-specific cell surface molecule. By “tumor-associated cell surface molecule” is meant a cell surface molecule expressed on ant cells with limited expression on cells of normal tissues, a cell surface molecule expressed at much higher density on malignant versus normal cells, or a cell surface molecule that is developmentally expressed.
When the target cell is a cancer cell, the cancer cell may s a tumor-associated cell surface le or tumor-specific cell e molecule to which the targeting moiety binds. In certain aspects, such a cell surface molecule is ed from HER2, CD19, CD22, CD30, CD33, CD56, CD66/CEACAM5, CD70, CD74, CD79b, CD138, Nectin-4, Mesothelin, Transmembrane glycoprotein NMB (GPNMB), Prostate-Specific Membrane Antigen (PSMA), SLC44A4, CA6, CA-IX, an integrin, C-X-C ine receptor type 4 (CXCR4), cytotoxic T-Iymphocyte-associated protein 4 (CTLA-4), neuropilin-t (NRPt), matriptase, any cell surface molecule set forth in Tables 1, 2, and 3 above, and any other tumor-associated or tumor—specific cell surface molecules of interest.
Methods of Making Con'ugates Methods of making the conjugates of the present sure are also provided.
In cases where one wishes to produce the targeting moiety and/or the target cell surface-editing enzyme (e.g., because a particular targeting moiety and/or target cell surface-editing enzyme is not cially available), the methods may include producing one or both of the targeting moiety and target cell surface-editing enzyme.
When a component of the desired conjugate (that is, the targeting moiety or target cell surface-editing enzyme) is a peptide or ptide, recombinant methods can be used to produce the component. For example, a DNA ng a component of the desired conjugate can be inserted into an expression vector. The DNA encoding the component may be operably linked to one or more control sequences in the expression vector that ensure the expression of the component. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous ers), signal ces, enhancer elements, and transcription termination sequences. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting prokaryotic or eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the component.
When a component of the desired conjugate (that is, the targeting moiety or target cell surface-editing enzyme) is a e or ptide, the component may be produced using a chemical e synthesis technique. Where a polypeptide is chemically sized, the synthesis may proceed via liquid-phase or solid-phase. Solid phase polypeptide synthesis (SPPS), in which the C-termlnal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence, is an example of a suitable method for the chemical synthesis of a component of the desired ate. Various forms of SPPS, such as Fmoc and Boc, are ble for sizing the component. Briefly, small insoluble, porous beads may be treated with functional units on which peptide chains are built. After ed g of coupling/deprotection, the free N-terminal amine of a solid-phase attached is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The peptide remains immobilized on the solid-phase and undergoes a tion process before being cleaved off.
Once synthesized r chemically or recombinantly), the component can be purified according to standard procedures of the art, including ammonium sulfate WO 06034 precipitation, affinity columns, column chromatography, high mance liquid chromatography (HPLC) purification, gel electrophoresis, and the like.
Once the targeting moiety and target cell surface-editing enzyme are ed, a variety of conjugation strategies are available, and a particular method may be selected based on the nature/type of targeting moiety and target cell surface-editing enzyme in the desired conjugate (e.g., based on available, or ed, reactive functional groups in the ing moiety and target cell surface-editing enzyme). Bioconjugation strategies that find use in stably associating a targeting moiety and a target cell surface-editing enzyme to produce a conjugate of the present disclosure include those described in Hermanson, “Bioconjugate Techniques,” Academic Press, 2nd edition, April 1, 2008, Haugland, 1995, Methods Mol. Biol. 45:205-21; Brinkley, 1992, Bioconjugate Chemistry 3:2, and elsewhere.
According to certain embodiments, the targeting moiety and target cell surface- editing enzyme are directly conjugated to each other — that is, the components of the conjugate are conjugated to each other without the use of a linker. In other aspects, the targeting moiety and target cell surface-editing enzyme are conjugated to each other via a linker. Any suitable linker(s) may be employed. Linkers that find use in the conjugates of the t disclosure include ester linkers, amide linkers, maleimide or maleimide-based linkers; valine-citrulline linkers; hydrazone linkers; N-succinimidyl(2-pyridyldithio)butyrate (SPDB) linkers; Succinimidyl(N-maleimidomethyl)cyclohexanecarboxylate (SMCC) linkers; vinylsulfone-based linkers; linkers that include polyethylene glycol (PEG), such as, but not limited to tetraethylene ; linkers that include propanoic acid; linkers that include caproleic acid, and linkers ing any combination thereof. In certain aspects, the linker includes polyethylene glycol (PEG). In some embodiments, the linker is a peptide linker.
The peptide linker may be flexible or rigid. Peptide linkers of interest include, but are not limited to, those described in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369. In certain s, when the linker is a e linker, the ate is a fusion protein. When the conjugate is a fusion protein, the present sure r provides nucleic acids that encode such fusion proteins, expression vectors that include such nucleic acids operably linked to a promoter, and host cells (e.g., mammalian host cells) that include such fusion proteins, nucleic acids, and/or expression vectors. In n aspects, the linker is serum- stable. Serum-stable linkers are known and include, e.g., linkers that e PEG, sulfone linkers (e.g., phenyloxadiazole sulfone linkers (see Patterson et al. (2014) Bioconj. Chem. (8):1402-7)), and the like.
Numerous strategies are ble for linking the targeting moiety and target cell surface-editing enzyme via a linker. For e, one component of the conjugate may be derivatized by covalently attaching a linker to the component, where the linker has a functional group capable of reacting with a “chemical ” on that component, and where the linker has a second functional group capable of reacting with a “chemical handle” on the other ent. The functional groups on the linker may vary and may be selected based on compatibility with the chemical s on the components of the desired conjugate.
The conjugate components may already include a functional group useful for reacting with a functional group of the linker, or such a onal group may be provided to one or both components of the desired conjugate. Functional groups that may be used to bind components of the conjugates to a linker include, but are not limited to, active esters, isocyanates, imidoesters, ides, amino groups, aldehydes, ketones, photoreactive groups, maleimide groups, alpha-halo-acetyl groups, epoxides, azirdines, and the like. ts such as iodoacetamides, maleimides, benzylic halides and bromomethylketones react by lation of thiols to generate stable thioether products. For example, at pH 6.5- 7.5, maleimide groups react with sulfhydryl groups to form stable thioether bonds. Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the ic halide by the nucleophile. Because the thiolate anion is a better nucleophile than the neutral thiol, cysteine is more reactive above its pKa (~8.3, depending on protein structural context). Thiols also react with certain amine-reactive reagents, including isothiocyanates and imidyl esters. The TS-Link series of ts are available for reversible thiol modification.
With respect to amine reactive groups, primary amines exist at the N-terminus of polypeptide chains and in the side-chain of lysine (Lys, K) amino acid residues. Among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic, making them ready targets for conjugation with several reactive groups. For example, NHS esters are ve groups formed by carbodiimide-activation of carboxylate les. NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The on releases N-hydroxysuccinimide (NHS). Also by way of example, imidoester crosslinkers react with primary amines to form amidine bonds. lmidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases. As such, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine ion is favored n pH 8-10.
Numerous other synthetic al groups will form chemical bonds with primary amines, including but not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, des, glyoxals, epoxides, oxiranes, carbonates, aryl halides, carbodiimides, anhydrides, and fluorophenyl esters. Such groups conjugate to amines by either acylation or alkylation.
According to one embodiment, the chemical handle on the targeting moiety, target cell surface-editing enzyme, or both, is provided by incorporation of an ral amino acid having the chemical handle into the ent. The unnatural amino acid may be incorporated via chemical synthesis or recombinant approaches, e.g., using a le orthogonal amino acyl tFlNA synthetase-tRNA pair for incorporation of the unnatural amino acid during translation in a host cell. The functional group of an unnatural amino acid present in the component may be an azide, alkyne, alkene, amino-oxy, ine, aldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl , boronic acid, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa).
In other aspects, the chemical handle on the targeting moiety, target cell surface- editing enzyme, or both, is provided using an approach that does not involve an ral amino acid. For example, a ent containing no unnatural amino acidls) could be conjugated to a linker by utilizing, e.g., nucleophilic functional groups of the component (such as the inal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a linker construct g a reactive leaving group or other electrophilic group.
In certain s, the target cell surface-editing enzyme is site-specifically conjugated to the targeting moiety, the targeting moiety is site-specifically conjugated to the target cell surface-editing enzyme, or both. In some ments, site-specific conjugation is achieved by incorporating an unnatural amino acid having the reactive functional group at a predetermined location in the targeting moiety and/or target cell surface-editing enzyme.
Details for site-specific incorporation of unnatural amino acids into proteins can be found, e.g., in Young & Schultz (2010) J. Biol. Chem. 285:11039-11044.
In n aspects, the targeting moiety has a C-terminal aldehyde tag, and site- ic conjugation is achieved by reacting the C-terminal aldehyde with aminooxy— tetraethyleneglycol-azide (aminooxy-TEG-Ng), followed by reacting with a bicyclononyne-N- hydroxysuccinimde ester (BCN-NHS)-labeled target cell surface-editing enzyme. This example embodiment is bed in more detail in the Experimental section below.
In certain aspects, the targeting moiety has a C-terminal aldehyde tag, and site specific conjugation is ed by reacting the C-terminal aldehyde with aminooxy— tetraethyleneglycoI-azide (aminooxy-TEG-Ns). A target cell surface editing enzyme has an aldehyde tag sequence (SLCTPSRGS), and site-specific conjugation is achieved by reacting aldehyde tag cysteine with Dibenzocyclooctyne-tetrapolyethyleneglycol-maleimide (DBCO-PEG4-maleimide) followed by reaction with the TEG-Na-labeled targeting moiety.
This example embodiment is described in more detail in the Experimental section below.
Accordingly, aspects of the present disclosure include methods that include conjugating a target cell surface-editing enzyme to a targeting moiety that binds to a cell surface le on the surface of a target cell. One or more {e.g., two or more, three or more, four or more, etc.) target cell surface-editing enzymes may be conjugated to the targeting moiety. The targeting moiety and target cell surface-editing enzyme may be any of the targeting moieties and target cell e-editing enzymes described herein. As just one example, in some embodiments, the target cell surface-editing enzyme is a sialidase (e.g., any of the sialidases described herein) and the targeting moiety is an dy (e.g., any of the antibodies described herein, ing, by way of e, an anti-HER2 dy (e.g., trastuzamab), cetuximab, daratumumab, girentuximab, panitumumab, ofatumumab, mab, etc.). As noted above, the conjugation may be site-specific (e.g., via a functional group of a non-natural amino acid at a predetermined position) with respect to the targeting moiety, the target cell surface-editing enzyme, or both.
ITIONS Also provided are compositions that include a conjugate of the present disclosure.
The compositions may include any of the conjugates described herein (9.9., a conjugate having any of the targeting moieties and target cell surface-editing enzymes described herein). In n aspects, the compositions include a conjugate of the present disclosure present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgClg, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine—N'—(2— ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl—B-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a ent (e.g., a non-ionic detergent such as Tween-20, etc.), a ribonuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.
Pharmaceutical compositions are also provided. The pharmaceutical compositions include any of the conjugates of the present disclosure, and a pharmaceutically able carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the conjugate. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect cial or desired eutic ding preventative) results, such as a ion in a m of a e or disorder associated with the target cell or a population thereof, as compared to a control.
An effective amount can be administered in one or more administrations.
A ate of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the conjugate can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.
Formulations of the conjugates of the present disclosure suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient ing to a selected route of administration.
In pharmaceutical dosage forms, the conjugates can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds, e.g., an ancer agent (including but not d to small molecule anti-cancer agents), an immune checkpoint inhibitor, and any combination thereof. The following methods and rs/excipients are merely examples and are in no way limiting.
For oral preparations, the conjugate can be used alone or in combination with appropriate ves to make tablets, powders, es or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or ium stearate; and if desired, with diluents, buffering , moistening agents, preservatives and flavoring agents.
The conjugate can be formulated for parenteral (e.g., intravenous, intra—arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration. In certain aspects, the conjugate is formulated for injection by dissolving, ding or emulsifying the conjugate in an aqueous or non- aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or ene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and vatives.
Pharmaceutical compositions that include the conjugate may be prepared by mixing the ate having the desired degree of purity with al physiologically acceptable carriers, excipients, stabilizers, tants, s and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to ents at the dosages and concentrations ed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium de, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, alanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as ose, sucrose, lactose, e, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N- methylglucosamine, galactosamine, and inic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).
The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form tituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume d during lyophilization); however solutions comprising antibacterial agents may be used for the tion of pharmaceutical compositions for parenteral administration.
An aqueous formulation of the conjugate may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid s. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
A ty agent may be included in the formulation to modulate the ty of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as ations f.
In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
A surfactant may also be added to the ation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. e surfactants include polyoxyethylensorbitan fatty acid esters ), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, ic), and sodium dodecyl sulfate (SDS).
Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20“") and polysorbate 80 (sold under the trademark Tween 80“”). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188W. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM. e concentrations of surfactant may range from about 0.001% to about 1% w/v.
A Iyoprotectant may also be added in order to protect the conjugate against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 nM.
In some embodiments, the pharmaceutical composition includes a conjugate of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, , m—cresol, p-chlor—m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
METHODS As summarized above, methods of using the conjugates of the present disclosure are also provided. In certain aspects, the methods of the present disclosure include administering to an individual in need thereof a therapeutically effective amount of any of the ates of the t disclosure, or any of the pharmaceutical compositions of the present disclosure.
In certain aspects, the administering modulates an immune pathway in the individual. For example, the administering may modulate an immune pathway selected from an inhibitory immune or pathway, a complement pathway, a paired immunoglobulin- like type 2 receptor (PILR) pathway, and a natural-killer group 2, member D protein (NKG2D) pathway. In certain aspects, the target cell es a ligand on its surface, and the stering results in g of the ligand by the target cell surface—editing enzyme.
The ligand may be edited in any manner bed elsewhere herein. According to certain embodiments, the editing of the ligand ses cleavage of all or a portion of the ligand.
As just one example, the ligand may be a lycan, the target cell surface-editing enzyme may be a sialidase, and the editing may e cleavage of a terminal sialic acid residue of the sialoglycan. The sialidase of the conjugate may be a bacterial sialidase, a mammalian neuraminidase, or the like. When the sialidase is a ian neuraminidase, the mammalian neuraminidase may be a human neuraminidase, e.g., a human neuraminidase selected from human neuraminidase 1, human neuraminidase 2, human neuraminidase 3, and human neuraminidase 4.
When the administering results in editing of a ligand on the target cell by the target cell surface-editing , the ligand may be a ligand of an inhibitory immune receptor. In n aspects, the ligand is a ligand of an inhibitory immune receptor present on an immune cell selected from the group consisting of: a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a basophil, and an eosinophil. In some embodiments, the inhibitory immune receptor is a sialic acid-binding lg- like lectin (Siglec) receptor.
In certain aspects, the methods of the present sure include administering the conjugate or pharmaceutical composition to an individual having cancer, e.g., to treat the cancer. Cancers which may be treated ing to the methods of the present disclosure e, but are not limited to, any of the cancers set forth in Tables 1 and 2 above. The conjugate may include a targeting moiety (e.g., a therapeutic antibody, such as any of the dies set forth in Tables 1, 2, and 3 above) that binds to a tumor-associated cell e molecule or tumor-specific cell surface molecule on the surface of a cancer cell of the individual. In some embodiments, the cancer cell is a carcinoma cell. According to certain embodiments, the cancer cell is selected from a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, a colon cancer cell, and a cancer cell of any of the cancer types set forth in Tables 1 and 2 above. In certain aspects, the cell surface molecule is human epidermal growth factor receptor 2 (HER2). When the cell e molecule is HER2, the ing may be, e.g., an anti-HERE antibody (e.g., trastuzamab or another suitable ER2 antibody).
In some embodiments, the administering includes administering a conjugate or pharmaceutical composition of the present disclosure, and the conjugate includes a targeting moiety that is an antibody. In certain aspects, the dual in need f has cancer, the targeting moiety of the conjugate is an antibody set forth in Table 1, and the methods are for treating (e.g., by ed antibody-dependent cellular cytotoxicity (ADCC)) the same or different type of cancer corresponding to the antibody as set forth in Table 1.
In certain aspects, the stering includes administering a conjugate or pharmaceutical composition of the present disclosure, and the conjugate includes a targeting moiety that is an dy. In some embodiments, the individual in need thereof has cancer, the targeting moiety of the conjugate is an antibody set forth in Table 2, and the methods are for treating (e.g., by enhanced antibody-dependent cellular cytotoxicity (ADCC)) the same or different type of cancer corresponding to the antibody as set forth in Table 2.
In some embodiments, the administering es administering a conjugate or pharmaceutical composition of the present disclosure, and the conjugate es a targeting moiety that is an antibody. In certain aspects, the individual in need thereof has cancer, the targeting moiety of the conjugate is an antibody set forth in Table 3, and the methods are for treating the cancer (e.g., by enhanced antibody-dependent cellular xicity (ADCC)).
In certain aspects, the administering includes administering a conjugate or pharmaceutical composition of the t disclosure, and the conjugate includes a targeting moiety that is an antibody ed from trastuzamab, cetuximab, daratumumab, girentuximab, mumab, ofatumumab, and rituximab.
The conjugates of the present disclosure are administered to the individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intra—tracheal, subcutaneous, intradermal, topical application, , intravenous, intra- arterial, nasal, oral, and other l and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the conjugate and/or the d effect. The conjugate may be administered in a single dose or in le doses. In some embodiments, the conjugate is administered orally. In some embodiments, the conjugate is administered via an inhalational route. In some embodiments, the conjugate is administered intranasally. In some embodiments, the ate is administered locally. In some embodiments, the conjugate is administered ocularly. In some embodiments, the conjugate is administered ranially. In some embodiments, the conjugate is stered intravenously. In some embodiments, the conjugate is administered by injection, e.g., for systemic delivery (e.g., intravenous infusion) or to a local site.
A variety of individuals are treatable according to the subject methods. Generally such individuals are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), ia (e.g., mice, guinea pigs, and rats), and es (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is a human.
By “treat” or ment” is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the individual, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the pathological condition being treated, such as disease or disorder associated with (e.g., caused by) the target cell or population thereof, where the editing of the surface of the target cell is beneficial. As such, treatment also includes situations where the ogical condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the individual no longer suffers from the pathological condition, or at least the symptoms that terize the pathological condition.
Dosing is dependent on severity and responsiveness of the disease state to be treated. Optimal dosing schedules can be calculated from measurements of conjugate lation in the body of the individual. The administering physician can ine optimum s, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of conjugate, and can generally be estimated based on E0503 found to be effective in in vitro and in vivo animal models, etc. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, , y or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
Following sful treatment, it may be ble to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, where the conjugate is administered in nance doses, once or more daily, to once every l months, once every six , once every year, or at any other suitable frequency.
The therapeutic methods of the present disclosure may include administering a single type of conjugate to an individual, or may include administering two or more types of conjugates to an individual (e.g., a cocktail of different conjugates), where the two or more types of conjugates may be designed to edit the surface of the same type or different types of target cells.
In certain aspects, a ate of the present sure is administered to the individual in combination with a second therapeutic agent as part of a combination therapy.
Such administration may include administering the conjugate and the second agent concurrently, or administering the conjugate and the second agent sequentially. In some embodiments, the individual has cancer, and the second therapeutic agent is an anti-cancer agent. Anti-cancer agents of interest include, but are not limited to, ancer antibodies (e.g., any of the antibodies set forth in Tables 1, 2, and 3 above), small molecule anti- cancer agents, or the like.
In some embodiments, the second therapeutic agent is a small le anti-cancer agent selected from erone, bendamustine, bexarotene, bortezomib, clofarabine, decitabine, exemestane, temozolomide, afatinib, axitinib, bosutinib, cabozantinib, crizotinib, dabrafenib, dasatinib, erlotinib, gefitinib, ibrutinib, ib, nib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, fenib, enzalutamide, fulvestrant, epirubicin, ixabepilone, bine, vismodegib, cabazitaxel, pemetrexed, azacitidine, carfilzomib, everolimus, temsirolimus, eribulin, omacetaxine, trametinib, lenalidomide, pomalidomide, romidepsin, vorinostat, brigatinib, ribociclib, midostaurin, telotristat ethyl, niraparib, cabozantinib, lenvatinib, rucaparib, granisetron, dronabinol, venetoclax, alectinib, cobimetinib, panobinostat, palbociclib, gene arepvec, lenvatinib, trifluridine and tipiracil, ixazomib, sonidegib, osimertinib, rolapitant, uridine triacetate, trabectedin, netupitant and palonosetron, stat, ibrutinib, olaparib, idelalisib, and ceritinib.
In certain aspects, the second therapeutic agent is an immune checkpoint inhibitor.
Immune checkpoint inhibitors of interest include, but are not limited to, inhibitors (e.g., antibodies) that target PD-1, PD-L1, CTLA-4, TIM3, LAG3, or a member of the B7 .
According to certain embodiments, the conjugate and the second therapeutic agent are administered according to a closing regimen ed for individual use. In some embodiments, the stration of the second therapeutic agent permits the conjugate administered to the individual to be administered according to a dosing regimen that involves one or more lower and/or less frequent doses, and/or a reduced number of cycles as compared with that utilized when the conjugate is stered without administration of the second therapeutic agent. In certain aspects, the administration of the conjugate permits the second eutic agent administered to the individual to be administered according to a dosing regimen that involves one or more lower and/or less frequent doses, and/or a d number of cycles as compared with that utilized when the second therapeutic agent is administered without administration of the conjugate.
In certain aspects, desired ve dosing ns for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in viva and/or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., so that a correlation is established), or alternatively in a particular individual of interest.
In certain aspects, one or more doses of the ate and the second therapeutic agent are administered to the individual at the same time; in some such embodiments, such agents may be administered present in the same pharmaceutical composition. In some ments, however, the conjugate and the second therapeutic agent are administered to the individual in different itions and/or at different times. For example, the conjugate may be administered prior to administration of the second therapeutic agent (e.g., in a ular cycle). Alternatively, the second therapeutic agent may be administered prior to stration of the conjugate (e.g., in a particular cycle). The second agent to be stered may be administered a period of time that starts at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more after the stration of the first agent to be administered.
KITS As summarize above, the present disclosure provides kits. ing to certain embodiments, the kits e any of the ates or compositions of the present disclosure. The kits find use, e.g., in practicing the methods of the present disclosure. For example, kits for practicing the subject methods may include a quantity of the compositions of the present disclosure, present in unit dosages, e.g., ampoules, or a multi-dosage format.
As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of a composition that includes a conjugate of the present disclosure. The term “unit ”, as used herein, refers to physically discrete units le as unitary dosages for human and animal subjects, each unit containing a predetermined ty of the composition calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the particular conjugate employed, the effect to be achieved, and the pharmacodynamics associated with the conjugate in the subject. In yet other embodiments, the kits may include a single multi dosage amount of the composition.
Components of the kits may be present in separate containers, or multiple components may be present in a single container. A suitable container includes a single tube (e.g., vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.
WO 06034 According to certain embodiments, a kit of the present disclosure includes instructions for using the composition to treat an individual in need f. The instructions may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file t on a suitable computer readable storage medium, e.g., le flash drive, DVD, CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a le substrate.
The ing examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL Materials and methods PBS buffer, DPBS buffer, DMEM, 640 media and nactivated fetal bovine serum were obtained from Corning-Mediatech. X-VIVO 15 serum-free medium was purchased from Lonza. LB agar, 2xYT and Antibiotic-Antimycotic were purchased from Fisher Scientific and 4-12% Bis-Tris gels for SDS-PAGE were purchased from Bio-Rad.
Heat-inactivated human male AB serum was purchased from Sigma-Aldrich. Human recombinant lL-2, human recombinant lL-4, and human recombinant lL-13 were purchased from end. Humanized er2—lgG with an aldehyde tag was a gift from Catalent Pharma Solutions (Emeryville, CA). Absorbance spectra were measured with a SpectraMax i3x (Molecular Devices). Pierce High-Capacity xin Removal Spin Columns, Pierce LAL Chromogenic Endotoxin Quantitation Kit and LDH cytotoxicity assay kit were ed from Thermo Fisher Scientific. Bicyclononyne—N-hydroxysuccinimide ester (BCN-NHS) and aminooxy-tetraethyleneglycol-azide (aminooxy-TEG-Na) were purchased from Berry & Associates, Inc. Dibenzocyclooctyne-tetrapolyethyleneglycoI-maleimide (DBCO-PEG4- ide) was purchased from Click Chemistry Tools. 2’-(4—Methylumbelliferyl)-or-D-N- acetylneuraminic acid (MuNeuNAc) was obtained from Biosynth International Inc. All other chemicals were purchased from Sigma-Aldrich and used without further purification.
The following antibodies and recombinant proteins were used: Human recombinant SiglecFc chimera, SiglecFc chimera, NKG2D-Fc chimera ns, AF488—Iabeled anti-Siglec-7 mAb (clone 194211) and blocking anti-NKG2D mAb (clone 149810) were purchased from R&D s. Fluorescein isothiocyanate (FITC)—labeled Sambucus nigra (SNA) lectin was obtained from EY Laboratories. AF647-Iabeled anti-Her2 mAb (clone 24D2), AF647-Iabeled anti-CD16 mAb (clone 3G8), AF647-Iabeled anti-CD56 mAb (clone , ng anti-Siglec-7 mAb (clone 87.7), blocking iglec-9 mAb (clone K8) were obtained from Biolegend. TRITC-labeled anti-Fc mAb was purchased from Jackson lmmunoresearch. FITC-Iabeled anti-CD3 mAb (clone 56) was purchased from Miltenyi Biotec. Humanized anti-Her2-lgG with C-terminal aldehyde-tag was a gift from Catalent Pharma Solutions (Emeryville, CA).
Cell lines and cell e: Breast cancer cells SKBRS, HCC-1954, MDA-MB-453, ZR1, BT-20, MDA-MB- 231, and MDA-MB-468 were obtained from American Type Culture Collection (ATCC).
SKBRS, HOG-1954, ZR1, and MDA-MB-468 were maintained in RPMI-1640 medium supplemented with 10 % heat-inactivated fetal bovine serum, plus 0.4 % Antibiotic- Antimycotic and L-glutamine (300 mg/L). MDA-MB-453, BT-20, and MDA-MB-231 were ined in DMEM medium mented with 10% heat-inactivated fetal bovine serum, plus 0.4 % Antibiotic-Antimycotic, L-glucose (4.5 g/L), L-glutamine (584 mg/L) and sodium pyruvate (110 mg/L).
Peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood bank donors and were isolated using Ficoll-Paque (GE Healthcare Life Sciences, GE1440- 02) density gradient separation. NK cells were isolated from PBMCs by negative selection using the MACS NK cell ion kit (Miltenyi Biotec, 130657) and LS columns (Miltenyi Biotec, 130401) according to the manufacturer’s protocol and cultured in X- VIVO 15 supplemented with 5 % heat-inactivated human male AB serum (Sigma—Aldrich), and 100 ng/mL recombinant human interleukin-2 (IL-2) (Biolegend) overnight before using.
NK cell enrichment was verified by flow try to result in > 95 % CD3- cells (see, ). Monocytes were isolated from PBMCs using the Pan Monocyte isolation kit (Miltenyi Biotec 130537). CD16+ monocytes were isolated from PBMCs using the CD16+ Monocyte isolation kit (Miltenyi Biotec 130765). After isolating fresh PBMCs, M1 and M2-polarized macrophages were ed by first plating freshly isolated PBMCs in serum-free RPMI a T75 flask r Scientific 1368065) at 37 °C in 5% 002 for 2 hours, then removing media and washing cells three times with phosphate buffered saline (PBS +Ca +Mg) to isolate monocytes. arized cells were generated by incubating remaining monocytes with 50 ng/mL recombinant human GM—CSF (PeproTech 300-03) for 6 days in RPMI + 20% heat inactivated fetal bovine serum, followed by 4 days incubation with 100 ng/mL bacterial Lipopolysaccharide (lnvivogen tlrl-3pelps) and 20 ng/mL inant human IFNy (PeproTech 300-0280) in RPMI with 10% heat-inactivated fetal bovine serum. M2-polarized macrophages were generated by incubating monocytes with 50 ng/mL recombinant human M-CSF (PeproTech 300-25) for 6 days in RPMI + 20% heat inactivated fetal bovine serum followed by 4 days incubation with 20 ng/mL recombinant human lL-13 (carrier-free) (Biolegend 571102) and 100 ng/mL recombinant human lL-4 er-free) (Biolegend 574004). Human v6 T cells were isolated from PBMCs by ve selection with the EasySepTM Human Delta T Cell Isolation Kit (Stemcell Tech 19255).
FACS analysis: Cells were incubated with sialidase, anti-Her2-lgG, anti-Her2-lgG-Sia, or PBS control for 1 hour at 37 °C. After three washes with PBS, cells were resuspended in cold PBS with 0.5% bovine serum albumin (BSA) containing the probe of : antibody, receptor-Fc fusion protein with secondary anti-Fc antibody pre-complexed in solution, or FITC-Iabeled SNA lectin. Cells and antibodies/fusion proteins were incubated for 30 mins at 4 °C in the dark. After three washes with PBS with 0.5% BSA, the cells were t up in PBS with 0.5% BSA then analyzed by flow cytometry. All flow cytometry data was analyzed using FlowJo v. 10.0 (Tree Star).
Expression and Purification of sialidase-s: Escherichia coli C600 transformed with plasmid pCVD364 containing the Vibrio cholerae sialidase gene was a gift from Prof. Eric R. Vimr, University of Illinois, Urbana— Champaign. Cells were grown in 2xYT media, supplemented with ampicillin (100 ug/mL) at 37 °C for 12 hours. After incubation, the cells were harvested by fugation at 4, 700 x g for 10 min. And the pellet was resuspended in osmotic shock buffer (20 % sucrose, 1 mM WO 06034 EDTA, 30 mM Tris-HCI, pH 8.0) and shaken gently for 10 min at room temperature. The cells were collected by centrifugation (9,000 x g for 10 min) and the pellets were resuspended in ice-cold pure water. After a 10 min incubation at 4 °C, the supernatant was obtained by centrifugation at 9,000 x g for 10 min. To purify the protein, the sample was further trated using an Amicon ultrafiltration device (membrane molecular mass cutoff, 30, 000 Da), reconstituted in 0.02 M Tris-HCl buffer (pH 7.6), and loaded onto a HitrapQ-HP exchange column (GE Healthcare Life Sciences, 1701). The protein was eluted with a gradient of NaCI in 0.02 M Tris-HCI buffer (pH 7.6) at a flow rate of mL/min. The protein fractions with expected molecular mass as determined by SDS- PAGE stained with Coomassie brilliant blue were collected and pooled. Endotoxins were removed using high-capacity endotoxin removal spin kit (Thermo Fisher Scientific, 88275) and the endotoxin concentration of the sample was determined by LAL chromogenic xin quantitation kit (Thermo Fisher Scientific, .
The Salmonella typhimurium sialidase gene was cloned into a pET151 vector with an N-terminal Hexahisitidine tag and inal aldehyde tag (SLCTPSRGS) and transformed into BL21(DE3) competent E. coli (NEB C2527H). Cells were grown in 2XYT media, supplemented with ampicillin (100 ug/mL) at 37 °C for until they reached an optical density of 0.6, then 0.3 mM IPTG was added and the cells were grown at 37 °C shaking for 16 hours. After incubation, the cells were harvested by centrifugation at 4, 700 x g for 10 min. And the pellet was ended in 50 mL lysis buffer (phosphate buffered saline (Fisher ific MT21040cv) + 150 mM NaCl + 10 mM imidazole. A protease inhibitor tablet (Sigma 5892970001) and 1 0L of nuclease (Thermo Scientific- Pierce 88702) was added and the cells in lysis buffer were ted at 4 °C shaking for 2 hours. Cells were lysed via homogenizer and purified using nickel-NTA resin (Thermo Fisher 88221) with 250 mM imidazole elution. The protein fractions with ed molecular mass as determined by SDS-PAGE stained with sie brilliant blue were collected and pooled. Endotoxins were d using high-capacity endotoxin removal spin kit (Thermo Fisher Scientific, 88275) and the endotoxin tration of the sample was determined by LAL chromogenic endotoxin quantitation kit (Thermo Fisher Scientific, 88282).
Activity assay of sialidases using MuNeuNAc: 0L of sialidase (30-60 nM in DPBS buffer with Ca2+ and Mg”, pH 7.0) was added to 50 uL solution containing 0.1 mM 2’-(4-methylumbelliferyl)-0i-D-N-acety|neuraminic acid (MuNeuNAc, Biosynth International Inc.) in DPBS buffer with Ca2+ and Mg2+ (pH 7.0). After incubation for 10 min at 37 °C, the mixture was d with 150 uL of 0.1 M glycine-NaOH buffer, pH 10.4. Fluorescence was read with a fluorescence spectrophotometer (excitation 360 nm; emission 440 nm). Activity is reported as U/mg, where a unit is defined as the amount of enzyme required to release 1 umol of methylumbelliterone per minute in DPBS buffer, pH 7.
Preparation of anti-Her2-lgG-Sia: Purified Vibrio cholerae sialidase (2 mg/mL in DPBS buffer with Ca2+ and Mg”, pH 7.0) was reacted with 12 equivalent of bicyclononyne-N-hydroxysuccinimide ester (BCN- NHS) at 4 °C overnight. Excess linker was removed using a PD-10 Desalting Column (GE care Life Sciences, 1701). The degree of labeling was ined by ESl-MS (see, FIG. GB). Humanized anti-Her2-lgG with C-terminal aldehyde-tag was produced as described previously. er2-lgG-Sia was prepared by first coupling anti-Her2-lgG with C—terminal aldehyde-tag (120 (M) to aminooxy-tetraethyleneglycol-azide (aminooxy—TEG- N3) (10 mM) in 100 mM ammonium acetate buffer, pH 4.5, at 37 °C for 10 days, followed by buffer-exchange into DPBS buffer with Ca2+ and Mg2+ (pH 7.0) using a PD-10 Desalting Column (GE Healthcare Life Sciences, 1701). The resulting conjugate was then coupled to labeled sialidase at 1:28 molar ratio at 120 mg/mL total protein concentration in DPBS buffer with Ca2+ and Mg2+ (pH 7.0). After a 3 day incubation at room ature, anti-Her2—lgG-Sia was purified by size ion chromatography Superdex 200. The purified product was analyzed by SDS-PAGE gel and ESI-MS. ed Salmonella typhimurium sialidase (3 mg/mL in DPBS buffer with Ca2+ and Mg“, pH 7.0) was reacted with 20 lent of DBCO-PEG4-Maleimide at 4 °C overnight.
Excess linker was removed using a PD-10 desalting column (GE Healthcare Life Sciences, 17—0851-01). The degree of labeling was ined by ESl-MS (see FIG. GB). Humanized er2—IgG with C-terminal aldehyde-tag was produced as described previously. Anti- gG-Sia was prepared by first coupling anti-Her2-lgG with C-terminal aldehyde-tag (120 (M) to xy-tetraethyleneglycol-azide (aminooxy-TEG—Ng) (10 mM) in 100 mM ammonium acetate buffer, pH 4.5, at 37 °C for 10 days, followed by buffer-exchange into DPBS buffer with Ca2+ and Mg2+ (pH 7.0) using a PD-10 Desalting Column (GE Healthcare Life Sciences, 1701). The resulting conjugate was then coupled to labeled sialidase at 1:14 molar ratio at 25 mg/mL total protein concentration in DPBS buffer with Ca2+ and Mg2+ (pH 7.0). After a 3 day incubation at room temperature, er2—lgG-Sia was purified by size exclusion chromatography Superdex 200. The purified product was analyzed by SDS-PAGE gel and ESl-MS.
Cell cytotoxicity assay: Antibody-dependent cellular cytotoxicity (ADCC) was analyzed by measuring lactate dehydrogenase (LDH) e from breast cancer cells as a result of ADCC activity of peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, CD16+ monocytes, M1 macrophages, or M2 macrophages. Tumor cells (target cells) were co-incubated with PBMCs, NK cells, monocytes, or macrophages (effector cells) at various effector/target (E/T) ratios in the presence or absence of sialidase or mAbs in triplicate. In a typical experiment, 100 uL of or cells were added to a om 96-well plate containing 100 uL of target cells at 2 x 105 cells/mL. After 4 hours, supernatants were collected, and LDH release was measured using a LDH cytotoxicity assay kit (Thermo Fisher Scientific, 88954) according to the manufacturer’s protocol. The absorbance at 490 nm was measured with a SpectraMax i3x (Molecular s). Specific lysis was calculated as 100 x (experimental - effector cells spontaneous release - target cells spontaneous e) / t cells maximum e - target cells neous release).
Fluorescence microscopy: For visualization of HER2—specific enzymatic activity of the conjugate: cells were incubated with various concentrations of anti-Her2-IgG-Sia in PBS buffer for 1 hour at 37 °C. After washes with PBS, cells were then fixed with 4% formaldehyde at room temperature for 20 min. The fixed cells were washed with 0.5% BSA in PBS three times, ed by blocking in PBS with 0.5% BSA for 1 hour. Cells were ted with FITC- labeled SNA (1:100) and AF647-labeled anti-Her2 antibody (1:100) in 0.5% BSA in PBS for min at room temperature in the dark with gentle shaking. After washing thrice with 0.5% BSA in PBS, DAPl (1:1250 dilution from a 10 mM stock) was added right before imaging with a Nikon A1R+ Resonant Scanning Confocal Microscope.
For visualization of NK-tumor cell synapses: tumor cells were incubated with 6 nM anti-Her2—lgG or 6 nM anti-Her2—lgG-Sia in PBS buffer for 1 hour at 37 °C. y isolated NK cells were added to tumor cells at an E/T ratio of 2:1 and incubated together for 15 min at 37 °C. After washing with PBS, cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature. The fixed cells were washed with 0.5% BSA in PBS three times, followed by blocking in PBS with 0.5% BSA for 1 hour. Cells were incubated with a e of Iabeled anti-Siglec 7 (1:100), TRITC-Iabeled anti-Fc (1 :400), and AF647-Iabeled anti-CD16 (1:100) in PBS buffer for 30 min at room temperature in the dark with gentle g. After washing thrice with 0.5% BSA in PBS, DAPI (1:1250 dilution from a 10 mM stock) was added right before imaging with a Nikon A1 R+ Resonant Scanning al Microscope. tical is: Statistical analyses were conducted with Prism 6. Data are shown as mean 4; SD of triplicate experiments, and significance was determined using a t-test, unless otherwise noted. **= p < 0.005, *= p < 0.05, and a p value > 0.05 was considered significant.
Introduction When sufficiently abundant, glycans terminating in sialic acid residues create a signature of “healthy self” that suppresses immune activation via several pathways — through tment of complement factor H and subsequent down-regulation of the alternative complement cascade, for example, and by recruitment of immunosuppressive sialic acid-binding lg-like lecfins cs) found on most types of leukocytes to the immunological synapse. Sialylation status plays an important role in a cell’s ability to trigger or evade immunological recognition. lation of sialylated glycans has been correlated with poor prognosis and sed immunogenicity of tumors. Hypersialylation of cancer cells may contribute to evasion of immune surveillance by NK cells, the major mediators of antibody-dependent cell-mediated cytotoxicity (ADCC). Dense populations of sialylated glycans can t NK cell-associated Siglec-7 and/or Siglec-9 to the immune synapse (. Like PD-1, these Siglecs possess a cytosolic immunoreceptor tyrosine-based inhibitory (ITIM) motif that mediates suppression of signals from activating NK cell receptors (. Engineered hypersialylation of tumor targets is protective from innate NK cell killing as well as ADCC in a Siglec-7—dependent manner. Likewise, enzymatic removal of sialic acids by treatment of tumor cells with sialidase potentiates NK cell-mediated killing, as does inhibition of Siglec-7 or -9 with blocking antibodies. Sialylation of cancer cell glycans also disrupts the interaction of the NK-activating or, natural killer group 2D (NKGED), with its cognate ligands, WO 06034 2017/040411 thus reducing NK-activating signals from tumor cells (. Conversely, l of cell- surface sialic acids enhances NK cell activation by increasing NKG2D-Iigand binding. Thus, during the microevolutionary process of tumor progression, hypersialylation provides a selective advantage by reducing NK ting signals while ing NK inhibitory signals emanating from the immune synapse.
An immune evasion strategy targeting NK-activating receptors and ibitory receptors using sialic acids is schematically illustrated in In sialic acid- overexpressing cancer cells, hypersialylated glycans interact with NK inhibitory receptors, g to tion of NK cells activation. Removal of cell-surface sialic acids by antibody- sialidase conjugate abolishes the interaction of sialylated glycans and NK-inhibitory receptors, and increases the binding n NK-activating receptor and its ligands, thereby enhancing the tumor cell susceptibility to NK cell-mediated ADCC.
It was reasoned that tumor-specific desialylation could be a powerful intervention that potentiates tumor cytolysis by NK cells. It is reported here that an antibody-enzyme conjugate (AEC) can selectively edit the tumor cell glycocalyx and potentiate NK cell killing by ADCC. a therapeutically important mechanism harnessed by many antibody cancer drugs. A recombinant sialidase was chemically fused to the HEFl2-targeting therapeutic monoclonal antibody trastuzumab. The antibody-sialidase conjugate ylated tumor cells in a HERZ-dependent manner, destroyed ligands for inhibitory Siglecs while ing NKG2D binding, and amplified NK cell killing compared to trastuzumab alone (.
Example 1 — Suitability of V. cholera and S. urium sialidases To identify suitable sialidases for the trastuzumab AEC, a panel of enzymes were expressed and purified as described previously ( and B) and the Vibrio cholera and ella typhimurium sialidases were identified as well suited for this purpose. V. cholerae and S. typhimurium sialidases were expressed and purified as described usly. The purity of protein was ined by SDS—PAGE gel and ESl-MS (, 2E, and FIG 3B, and FIG 7A, 7F). Approximately 15 mg of enzymes were purified from 1 liter of cultured cells, with an in vitro hydrolytic activity of more than 10 U/mg for V. cholerae and 114 for S. typhimurium as measured with the fluorogenic substrate 2’-(4— methylumbelliferyl)-d-D-N-acetylneuraminic acid (MuNeuNAc) as previously reported, where a unit is defined as the amount of enzyme required to release 1 umol of methylumbelliferone per minute in DPBS buffer, pH 7.. To determine if V. cholerae sialidase could efficiently remove sialic acids from cell-surface glycans, its effects on cell surface labeling was tested with FlTC-labeled Sambucus nigra agglutinin (SNA). As well, the effects of V. cholerae treatment on cell labeling were evaluated with receptor-Fc chimeras comprising the ectodomains of Siglec-7, Siglec—9 or NKG2D. ylation of various tumor cell lines by sialidase at 37 °C for 1 hour significantly reduced binding of SNA as well as SiglecFc and Siglec-Q—Fc as (. With a decrease in SNA binding, an increase in binding capacity of NKG2D-Fc chimera was observed for most breast cancer cell lines after sialidase treatment (). ation and characterization of antibody-sialidase conjugates is shown in tically illustrates the preparation of antibody-Vibrio cholerae sialidase conjugates. shows GE analysis of sialidase, trastuzumab, and sialidase- trastuzumab conjugate under non-reducing (lanes 3, 4, and 5) and ng conditions (lanes 6, 7, and 8), visualized by coomassie staining. Pre-stained protein ladder: lanes 1, 2, and 9. FIG 20 shows ESl-MS of antibody ase conjugate with Vibrio cholerae sialidase. schematically illustrates the preparation of antibody-Salmonella typhimurium sialidase conjugates. shows GE analysis of sialidase. DBCO- modified sialidase, trastuzumab, and trastuzumab-sialidase conjugate under non-reducing conditions (lanes 3, 4, 5, and 6) and trastuzumab and trastuzumab-sialidase conjugate under reducing conditions (lanes 7 and 8), ized by coomassie staining. Pre-stained protein ladder: lanes 1, 2, and 9. shows ESl-MS of antibody-sialidase conjugate with Salmonella urium sialidase. shows the characterization of a panel of sialidases. depicts activity of sialidases on the ate 2’-(4-methylumbelliferyl)-or-D-N-acetylneuraminic acid (MuNeuNAc). SDS-PAGE analysis of wild-type human neuraminidase 2, human neuraminidase 3, V. cholerae sialidase, S. typhimurium sialidase, C. perfringens sialidase, and A. ureafaciens sialidase is shown in FIG. SB, visualized by sie staining. FIG SC shows flow cytometry of Siglec 9 ligand cleavage by V. cholerae, S. urium, and human Neuraminidase 2 from ZR1 breast cancer cells. Siglec-7 ligands on BT-2O cells are efficiently removed after treatment with V. cholerae sialidase, as shown in .
Analysis of cell-surface ation levels of different breast cancer cell lines with or without sialidase treatment is shown in . Ligand levels of Siglec-7 on ent breast cancer cell lines with or without sialidase treatment is shown in . Ligand levels of Siglec-9 on different breast cancer cell lines with or without ase treatment is shown in . Ligand levels of NKG2D on different breast cancer cell lines with or without sialidase treatment is shown in .
Example 2 — Removal of cell-surface sialic acids e susceptibility to ADCC To demonstrate that removal of cell-surface sialic acids can e their susceptibility to NK cell-mediated ADCC, ADCC assays were performed with SKBR3 (HER2 3+), -453 (HER2 2+) and BT-20 (HER2 1+) cell lines with and without the sialidase treatment in the presence of 30 nM trastuzumab using purified human peripheral blood NK cells. An approximate 5%-100°/o se in maximal cell killing was observed in trastuzumab-directed ADCC with various sialidase-treated cell lines (. To te that the enhanced ADCC was due to sialidase enzymatic activity, the hydrolytic activity assay and ADCC assay using a heat-inactivated V. cholerae ase was also med.
Inactivation of V. cholerae sialidase by heating to 80 °C for 20 minutes led to the loss of hydrolytic activity against sialic acid containing s as well as the loss of the enhancement in ADCC (. It was expected that by conjugating sialidase to trastuzumab, increased local tration of sialidase on the cell-surface would provide proximity-enhanced activity and further potentiate the effect as well as limit the promiscuity of the enzymatic activity in a tissue-specific manner.
Shown in is cytotoxicity of isolated peripheral blood NK cells from healthy donors against BT-20 breast cancer cells alone (no treatment), in the presence of anti-Her2— IgG (Tras) or in the presence of ER2—IgG and human neuriminidase 2 (Neu2), human neuriminidase 3 (Neu3), Vibrio cholerae sialidase (VCSia), Salmonella typhimurium sialidase (STSia), Arthrobacter ureafaciens sialidase (AUSia), or C/ostridium perfringens sialidase, (CPSia). s cytotoxicity of isolated peripheral blood NK cells from healthy donors against different breast cancer cells in the absence or presence of sialidase (30 nM), anti-Her2—IgG (30 nM) or a mixture of sialidase (30 nM) and anti-Her2—IgG (30 nM) at E/T ratios of 2:1 and 4:1. *P < 0.05, **P< 0.005. shows the characterization of ype and heat-inactivated Vibrio cholerae sialidase. xicity of isolated peripheral blood NK cells against BT-20 cells in the absence or presence of anti-Her2—IgG (30 nM), sialidase (30 nM), a mixture of anti-Her2— IgG (30 nM) and sialidase (30 nM), heat—inactivated ase (HI—Sialidase 30nM), or a mixture of anti—Her2—lgG (30 nM) and heat—inactivated sialidase (30 nM) at an E/T ratio of 4:1 is shown in . Hydrolytic activities of wild-type and heat-inactivated V. cholerae sialidase using 2’-(4-methylumbelliferyl)-or-D-N-acetylneuraminic acid (MuNeuNAc) is shown in FIG. GB. Levels of Sambucus nigra lectin (SNA) ligands on BT-2O cells with or without 30 nM wild-type sialidase or heat-inactivated sialidase ent is shown in . Levels of Siglec-7 s on BT-2O cells with or without 30 nM wild-type sialidase or heat—inactivated sialidase treatment is shown in . Levels of Siglec-Q ligands on BT- cells with or without 30 nM wild-type sialidase or heat-inactivated sialidase treatment is shown in . Levels of NKG2D ligands on BT-20 cells with or without 30 nM wild-type sialidase or heat-inactivated sialidase treatment is shown in . **P < 0.005, NS: not significant.
Exam pie 3 — Preparation and characterization of antibody-sialidase conjugates A key concern in designing the sialidase-trastuzumab AEC was to identify a site for enzyme conjugation that would not undermine g to chRlll (CD16), the interaction that initiates ADCC. Inspiration from the field of antibody-drug conjugates (ADCs) was taken where sites of attachment have been tailored to avoid interference with immune effector ons. Accordingly, sialidase was chosen to link near the C-terminus of trastuzumab’s heavy chain, far from the CH2 domain at which FcyRIII binds. The aldehyde tag method for site-specific conjugation was used based on precedents of its use in the assembly of protein-protein chemical fusions as well as site-specific antibody-drug conjugates. zumab (anti-Her2-lgG) bearing a C-terminal aldehyde tag was obtained as previously described. The functionalized antibody was first coupled to aminooxy— tetraethyleneglycol-azide (aminooxy-TEG-Ns) (). In parallel, sialidases were prepared. V. cholerae ase was randomly functionalized on lysine residues with bicyclononyne-N-hydroxysuccinimide ester (BCN-NHS). After an overnight reaction, excess linker was removed and the extent of BCN—NHS modification of sialidase was ined by ESl-MS (). Finally, trastuzumab adorned with the azide—functionalized linker was conjugated to BCN-functionalized V. cholerae sialidase via copper-free click chemistry (). The desired conjugate was purified using a xclusion column and its nt molecular weight (anti-Her2—IgG-Sia, ca. 312 kDa) was confirmed by SDS—PAGE ().
ESl-MS analysis med that the sialidase was covalently linked to the heavy chain of trastuzumab ( and ). ase activity of the final AEC was evaluated using the genic substrate MuNeuNAc. More than 85% enzymatic activity remained after the al conjugation process (. Alternatively, S. typhimurium sialidase was site- specifically conjugated to the cysteine on the C-terminal aldehyde tag by ng with DBCO-PEG4-Maleimide overnight; following this excess linker was removed and the conjugation of DBCO to S. typhimurium sialidase was ined to be te by ESI- MS (). Finally trastuzumab with azide linker was conjugated to DBCO- functionalized S. typhimurium sialidase via copper-free click chemistry (). The desired conjugate was purified using a size—exclusion column and its apparent molecular weight (anti_HER2-lgG-StSia, ca 240 kDa) was confirmed by SDS-PAGE (). ESI- MS is confirmed that the sialidase was covalently linked to the heavy chain of trastuzumab ( and ). Sialidase activity of the final AEC was evaluated using the genic substrate MuNeuNAc. A slight increase in tic activity compared to free aldehyde-tagged sialidase resulted after the chemical conjugation process to the free cysteine on the C-terminal tag (. shows ESl-MS spectra of sialidase, anti-Her2-lgG and its conjugates. ESl-MS spectrum of purified Vibrio cholerae sialidase is shown in . ESl-MS spectrum of V. ae ase labeled with BCN-NHS at 1:12 molar ratio is shown in . ESl-MS spectrum of anti-Her2-lgG with inal aldehyde tag is shown in . ESl-MS spectrum of anti-Her2-lgG with C-terminal aldehyde tag conjugated with aminooxy-TEG- azide is shown in . ESl-MS spectrum of anti-Her2-lgG-Sia is shown in .
ESl-MS of purified Salmonella typhimurium sialidase is shown in . ESl-MS of S. typhimurium labeled with DBCO_PEG4—Maleimide at a 1:20 molar ratio is shown in . ESl-MS spectrum of anti-HERZ-lgG-St-Sla shows the hydrolytic activities of V. cholerae sialidase and er2-lgG-Sia, as well as S. typhimurium ase and anti-Her2-lgG-Sz‘8ia t substrate 2’-(4- methylumbelliferyl)-or-D-N-acetylneuraminic acid (MuNeuNAc) To further demonstrate that anti-Her2—lgG-Sia is able to specifically remove sialic acid on HER2-expressing cells, SKBR3 (HER2 3+) and MDA-MB-468 (HER2 0) were incubated in the absence or presence of 6 nM or 60 nM anti-Her2-lgG-Sia. As shown in and , treatment with 6 nM anti-Her2-lgG-Sia for 1 h resulted in a selective desialylalion of SKBRS cells even in the presence of MDA-MB-468 cells (. However, this effect is dose-dependent. e sialic acid levels of SKBRS and MDA-MB-468 cells were both reduced with a treatment at 60 nM of er2-lgG-Sia for 1 h. This effect was quantified using flow cytometry on mixtures of cells treated with various concentrations of anti-Her2—lgG-Sia (). However, in another conjugate with a smaller sialidase lacking lectin domains HERZ-IgG-St—Sia) surface sialic acid levels of off-target HEFl2 O MDA- MB-468 cells remained untouched until much higher concentrations of about 1 uM anti- Her2—IgG-St—Sia conjugate (FIG QB). shows in vitro terization of trastuzumab and trastuzumab-sialidase conjugate with different HER2—expressing cancer cells. Cell-surface sialic acid on the HER2—high expressing cell line, SKBR3, can be selectively removed using 6 nM trastuzumab-sialidase conjugate. Scale bar, 25 um. shows SNA ligands on SKBR3 and MDA-MB-468 cells in the absence or presence of anti-Her2—lgG-Sia conjugate. SNA ligands on individual cultures of SKBRS and -468 cells in the absence or presence of anti-Her2—IgG-Sia ate is shown in A. Cells were incubated with 6 nM anti-Her2—lgG-Sia conjugate or PBS in RPMI- 1640 media for 1 hour at 37 °C and stained with FITC-Iabeled SNA lectin, AF647-Iabeled anti-Her2 and DAPI nuclear stain. SNA ligands on a mixture of SKBR3 and MDA-MB-468 cells in the absence or presence of anti-Her2—IgG-Sia conjugate is shown in FIG. foB.
SKBRS and MDA-MB-468 cells were mixed at a 1:1 ratio and cultured overnight. The cell es were incubated in the e or presence of 6 nM or 60 nM anti-Her2—lgG-Sia conjugate for 1 hour at 37 °C. Scale bar :25 pm.
To assess the effect of the antibody-sialidase ate on NK cell-mediated ADCC, cytotoxicity assays were performed using various breast cancer cell lines (SKBFIS, HER2 3+; HOG-1954, HER2 3+; MDA-MB-453, HER2 2+; ZR1, HER2 1+; BT-20, HER2 1+; MDA-MB-231, HER2 1+; MDA—MB-468, HER2 O) in the presence of anti-Her2—IgG or anti- Her2—lgG-Sia at effector/target (E/T) ratios of 4:1 and 8:1. In comparison to anti-Her2—lgG, anti-Her2—lgG-Sia demonstrated increases of 33 % to 140 % of l cell g with HER2 1+ cell lines 1, BT-20, and MDA-MB-231 (). In addition, BT-2O cells were exposed to purified human peripheral blood NK cells at various E/T ratios in the absence or ce of sialidase (30 nM), er2—IgG (30 nM), or anti-Her2—lgG-Sia (30 nM) (A). Sialidase treatment alone of BT-20 cells lines showed little NK cellmediated cytotoxicity at different E/T ratios. Compared to anti-Her2—lgG, anti-Her2-lgG-Sia showed significantly improved cytolysis at various ratios. At an E/T ratio of 4, the largest enhancement was observed: 46 % 1r 1 cytolysis for anti-Her2—IgG-Sia versus 21 % i 1 for anti-Her2—lgG. It was verified that ADCC was likely being mediated by NK cells as NK celldepleted PBMCs showed little cell lysis (B). shows cytotoxicity data of ed peripheral blood NK cells from healthy donors against different breast cancer cells in the presence of anti-Her2—IgG (30 nM) or anti-Her2—IgG-Sia (30 nM) at E/T ratios of 4:1 and 8:1. shows in vitro activity of trastuzumab and trastuzumab-sialidase conjugate against different HER2—expressing cancer cells. Cytotoxicity assays med with BT-2O cells in the absence or presence of sialidase (30 nM), anti-Her2—IgG (30 nM) and anti-Her2— IgG-Sia (30 nM) using NK cells are shown in A. Results of cytotoxicity assays performed with BT-20 cells in the absence or presence of sialidase (30 nM), anti-Her2-IgG (30 nM) and anti-Her2—IgG-Sia (30 nM) using NK cells-depleted PBMCs are shown in 128. The trend seen in the enhancement of ADCC correlated with Fc, Siglec-9—Fc and NKG2D-Fc binding is shown in 0-12F. Cytotoxic activity of NK cells against different HERZ-expressing cancer cells in the presence of ted concentrations of trastuzumab and trastuzumab-sialidase conjugate is shown in G-12J. Fluorescent microscopy analysis of Siglec-7 distribution on NK cells with trastuzumab or trastuzumab- sialidase conjugate treatments is shown in K. -7 displayed tment to the NK synapse with trastuzumab treatment. After removing sialic acids on SKBRS cells using trastuzumab-sialidase conjugate, Siglec-7 recruitment to the NK-tumor e is lost.
Scale bar, 10 um, **P< 0.005.
To assess the effect of the antibody-sialidase conjugate on cytotoxicity ed by monocytes and macrophages, xicity assays were performed using breast cancer cell lines (SKBR3, HER2 3+; BT-20, HER2 1+) in the presence of Vibrio cholerae sialidase alone (VCSia), anti-Her2—lgG (Tras), or anti-Her2—IgG-Sia (T-Sia) at various effector/target (E/T) . s the total monocyte population ted low overall killing of tumor cells, isolated CD16+ monocytes primarily expressing Siglecs 3, 7, and 9 demonstrated increases of about 100% upon treatment with the conjugate anti-Her2—IgG-Sia (T-Sia) compared to treatment with anti-Her2—IgG (Tras) (FIG 13). Differentiated M1 macrophages expressing Siglecs 3, 6, 7, and 9, and M2 macrophages expressing Siglecs 3, 5, 6, 7, 8, 9, , and 11 both appear to exhibit increases in cytotoxic killing of tumor cells with trastuzumab-sialidase conjugate as opposed to trastuzumab alone (FIG 13). yo T cell mediated xicity can also be potentiated by trastuzumab ase conjugate (T-Sia) rather than treating with trastuzumab (Tras) or sialidase (VCSia) alone ().
A depicts the siglec expression levels of a human isolated monocyte population as determined by flow try. 8 shows cytotoxicity data of isolated human monocytes against BT-2O breast cancer cells after 24 hours incubation with V. cholerae sialidase, anti-HER2-lgG (Tras), or anti-Her2-IgG-Sia (T-Sia) and an E:T ratio of 1:8. C depicts the siglec receptor expression levels of CD16+ monocytes isolated from the te population. D. Shows cytotoxicity data of CD16+ monocytes from healthy donors after four hours incubation with V. cholerae sialidase, ER2-IgG (Tras), or anti—Her2-lgG—Sia (T-Sia) and BT-2O breast cancer cells. E depicts the siglec receptor expression levels of isolated monocytes from healthy donors that have been differentiated into M1 macrophages as described previously. F shows cytotoxicity data from M1 macrophages differentiated from isolated monocytes from healthy donors after 24 hours tion with V. cholerae sialidase, anti-HER2-IgG (Tras), or anti-Her2- IgG-Sia (T-Sia) and SK-BR-3 breast cancer cells. G depicts the siglec receptor expression levels of isolated monocytes from healthy donors that have been entiated into M2 macrophages as bed previously. H shows xicity data from M2 macrophages differentiated from ed tes from healthy donors after 24 hours incubation with V. cholerae sialidase, anti-HER2-lgG (Tras), or anti-Her2-IgG-Sia (T-Sia) and SK-BR-S breast cancer cells. FIG 13| shows the CD16 expression level of the M2 macrophages from (13E and 13F).
Fig 14 depicts the cytotoxicity of isolated yo T cells in the presence of V. cholerae sialidase, anti-HER2-IgG (Tras), or anti-Her2-IgG-Sia (T-Sia) and SK-BR-3 cells at an E:T of 5:1. e 4 — Mechanisms of enhanced ADCC using antibody-sialidase con'ugate Previous studies have suggested that hypersialylation of cancer cells s in the reduced binding of activating receptor NKG2D as well as enhanced binding of inhibitory receptors, Siglec-7 and Siglec-Q, thus reducing NK-mediated cytotoxicity. To explore the mechanism of increased ADCC using trastuzumab-sialidase conjugate, fold increase of ADCC was correlated with receptor binding in various breast cancer cell lines. Cell lines with the highest increases of NK-mediated ADCC correlated with the highest levels of Siglec-7 and Siglec-9 binding (FIGs. 12C-12E). Treatment of BT-20 and ZR1 cells— those with the highest expression of Siglec-7 and Siglec-9 surface ligands—with trastuzumab—sialidase conjugate enhanced ADCC by more than 2 fold compared to zumab alone. In contrast, the conjugate d little significant improvement on ADCC of -453 cells, which have the lowest expression of Siglec-7 and Siglec-9 surface ligands. To further substantiate that anti-Her2-lgG-Sia was enhancing ADCC through a reduction in binding of inhibitory receptors Siglec-7 and Siglec-9 along with ement of the interaction with activating receptor NKG2D, ng antibodies against Siglec-7, Siglec-9 and NKG2D were used to specifically block ligand-receptor interactions.
Anti-Siglec-7 and iglec-Q antibodies at 5 ug/mL led to significantly enhanced NK cell cytotoxicity against BT-20 cells with anti-Her2-lgG, but not with a mixture of er2-lgG and sialidase (). In addition, blocking the NKG2D receptor showed a greater effect on ADCC in the mixture of er2-IgG and sialidase-treated cells compared to anti-Herz- IgG-mediated ADCC (). shows results relating to cytotoxicity of isolated peripheral blood NK cells from y donors against BT-20 cells with anti-Her2-lgG (30 nM) or a mixture of anti- Her2-lgG (30 nM) and sialidase (30 nM) in the absence or presence of 5 pg/mL blocking anti-NKG2D (clone 149810), anti-Siglec-7 (clone 87.7), anti-Siglec-9 (clone 89), a mixture of iglec-7 (clone 87.7) and anti-Siglec-9 (clone 89), or mouse lgG1 isotype antibody (clone 1) at an E/T ratio of 4:1. *P < 0.05, **P < 0.005, ns: not significant.
Example 5 — Comparison between antibody-sialidase coniugate and antibody alone In order to directly compare the ability of anti-Her2-IgG-Sia to direct ADCC versus anti-Her2—lgG alone, the dose response for cytotoxicity was measured using four ent breast cancer cell lines: SKBR3 (HER2 3+), 1 (HER2 1+), BT-20 (HER2 1+), MDA- MB-468 (HER2 0). Compared to anti-Her2-lgG, anti-Her2-lgG-Sia is more cytotoxic in all three xpressing cell lines at an E/T ratio of 4. For the HER2 3+ cell line, anti-Her2- lgG-Sia killed SKBR3 cells with an E050 of 76 i 14 pM, which was slightly better than anti- gG (E050 177 i 54 pM). While for HER2 1+ cell lines ZR1 and BT-20, the anti- Her2-lgG—Sia is ~ 10 times more cytotoxic than the anti-Her2-lgG (ZR1 cells: anti-Her2- lgG-Sia E050 135 i 47 pM, anti-Her2-lgG E050 1143 1r 274 pM; BT-20 cells: anti-Her2-lgG- Sia E050 170 i 34 pM, anti-Her2-lgG E050 1823 i 850 pM) (Fle. 12G-12J and Table 6).
Little lysis of the HER2 negative cell line MDA-MB-468 was observed for either anti-Her2- lgG or anti-Her2-lgG-Sia (]). Next, the difference between anti-Her2-lgG-Sia and a mixture of anti-Her2-lgG and ugated sialidase (anti-Her2-lgG/sialidase) was tested.
Anti—Her2—lgG—Sia showed lower EC50 in SKBRS cells (anti—Her2—lgG—Sia EC50 76 i 14 pM; anti—Her2—IgG/sialidase EC50 136 i 52 pM), ZR—75—1 cells (anti—Her2—IgG—Sia EC5O 135 i 47 pM; anti-Her2-lgG/sialidase E050 492 i 67 pM), and BT-20 cells (anti-Her2-lgG-Sia E050 WO 06034 170 : 34 pM; anti-Her2-lgG/sialidase E050 692 i 156 pM) (Table 6). The enhanced potency of the conjugate versus the mixture of anti-Her2-lgG and unconjugated sialidase is evidence of a proximity effect on the enzymatic activity.
Table 6: Cytotoxic activity of isolated NK cells against various human breast cancer cells induced by er2-lgG, a mixture of anti—Her2-lgG and sialidase, or anti-Her2-lgG-Sia conjugate. (N.D. none detected).
EC50 (pM) [Maximal killing (%) Cell “he HER2 level anti-Her2-lgG anti-Her2-lgG/sialidase anti-HerZ-lgG-Sia SKBR3 3+ 177:
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