JP7654710B2 - System for detecting the presence, location and/or amount of amyloid deposits in the heart, system for determining the effectiveness of removal of amyloid deposits in the heart and system for monitoring disease progression of amyloid deposits in the heart - Patents.com - Google Patents

Description

[Reference to sequence listing submitted via EFS-WEB]
The contents of the ASCII file of the Sequence Listing entitled "8441-0018-1-ST25" submitted electronically with this application via EFS-Web was created on January 22, 2019, is 16.9 kb in size, and is incorporated herein by reference in its entirety.

[Priority claim]
This application claims priority to U.S. Provisional Patent Application No. 62/753,410, filed October 31, 2018, the entire contents of which are incorporated herein by reference.

The present invention relates to humanized and chimeric (e.g., mouse-human) antibodies and antigen-binding fragments thereof linked or conjugated with detectable molecules, and methods of using them to detect and image amyloid deposits.

The following description is provided solely to aid the reader in understanding the present invention and is not admitted to describe or constitute prior art.

Natural antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one disulfide bond. The number of additional disulfide bonds between the heavy chains varies among the various antibody isotypes. The simplest isotype is IgG, which contains only two light chains and two heavy chains, with the two heavy chains linked by two disulfide bonds. Each heavy chain has a variable domain (VH) at one end and several adjacent constant domains. Each light chain has a variable domain ( _VL ) at one end and a constant domain at the other end. Each variable domain of an antibody's light and heavy chains contains three segments called complementarity determining regions (" _CDRs ") or hypervariable regions. Each CDR of a light chain, together with the corresponding CDR of an adjacent heavy chain, forms the antigen-binding site of the antibody. There are two main types of light chains, kappa and lambda, depending on their constant regions. Both kappa and lambda light chains can bind to any of a variety of heavy chain types.

Amyloid light chain amyloidosis (AL amyloidosis, AL or ALA), also called primary amyloidosis, is the most common form of systemic amyloidosis in the United States. The term "amyloidosis" refers to a cluster of diseases that share a common feature: extracellular deposition of pathological insoluble fibrillar proteins in organs and tissues (Rodney et al., NEJM, 25:898). Amyloidosis is caused by the malfunction of a person's antibody-producing cells, leading to the production of abnormal protein fibrils that aggregate to form insoluble amyloid deposits in organs and tissues. The type of amyloidosis is determined by the nature of the precursor protein that forms the fibrillar deposits. In primary amyloidosis, the fibrils contain fragments of immunoglobulin light chains, and in secondary amyloidosis, the fibrils contain amyloid A protein. The modern classification of amyloidosis is based on the nature of the precursor plasma protein that forms the fibrillar deposits.

The precursor plasma proteins are diverse and unrelated to each other. Nevertheless, all precursor deposits give rise to amyloid deposits that share a common typical β-pleated sheet structure that is responsible for the typical staining properties of fibrillar deposits. The final stage in the development of amyloidosis is the deposition of amyloid fibrils in the patient's organs. Mortality from amyloidosis is high, with the current 5-year survival rate being approximately 28%.

So far, treatment of AL has been directed at reducing the synthesis of amyloidogenic precursor light chains by attacking dysfunctional cells through conventional or high-dose cytotoxic chemotherapy. However, despite improved prognosis with elimination of the offending plasma cell clone, mortality remains high due to multiple organ failure caused by persistent deposition of insoluble amyloid fibrils. This treatment has two drawbacks. First, fibrillar deposits are often asymptomatic until substantial deposition has occurred; therefore, treatment is unlikely to occur before substantial deposits have developed. Second, this treatment is at best only effective in halting the production of precursor abnormal proteins, but is ineffective in removing existing deposits, and the prognosis of AL patients remains very poor due to the persistence (or progression) of pathological deposits (Solomon et al., Int. J. Exp. Clin. Invest., 2:269).

As such, therapeutic targeting and removal of amyloid deposits is an area of great medical interest. Concomitantly, there is also great interest in detecting the presence and location of amyloid deposits and monitoring their removal as an adjunct to the targeting and removal described above. The compositions and methods described herein fulfill this need for detecting such presence and location.

Described herein are compositions and methods for detecting the presence, location and amount of amyloid deposits, including those resulting from primary (AL) amyloidosis. The compositions described herein include a humanized or chimeric antibody or fragment thereof ("antigen-binding fragment") that specifically binds to amyloid deposits (e.g., amyloid light chain fibrils) and is linked to a detectable molecule. The methods described herein include administering the above-described composition to a subject suspected of having amyloid deposits, and detecting the presence, amount and/or location of the amyloid deposits by detection of the detectable molecule by diagnostic imaging.

The method also includes a method of stratifying a patient for treatment with a humanized or chimeric antibody described herein based on the affinity of the diagnostic composition for amyloid deposits. The method also includes a step of determining an appropriate dose of a humanized or chimeric antibody for treatment described herein based on the affinity of the diagnostic composition for amyloid deposits. That is, an appropriate dose for a patient can be determined based on the detected affinity (uptake) of the labeled antibody or antibody fragment. A patient exhibiting a strong affinity for the labeled antibody or antigen-binding antibody fragment may require a smaller amount of the therapeutic antibody or antigen-binding antibody fragment than a patient exhibiting a weak affinity for the labeled antibody or antigen-binding antibody fragment. Thus, the present invention provides a method for determining an appropriate dose of a humanized or chimeric antibody for treatment described herein, comprising administering to a patient a labeled antibody or antibody fragment described herein, determining the affinity of the labeled antibody or antibody fragment for amyloid deposits, and administering a dose or series of doses of the chimeric or humanized antibody or antibody fragment based on the strength of the affinity.

In some embodiments, the antibody described herein included in the diagnostic composition has a _VK region comprising SEQ ID NO: 47 and a _VH region comprising SEQ ID NO: 48. In some embodiments, the antibody has a constant region derived from human IgG1. In some embodiments, the antibody binds to amyloid fibrils with higher affinity than the mouse equivalent. In some embodiments, the antibody binds to an epitope expressed by the beta-pleated sheet structure of amyloid fibrils with higher affinity than a mouse antibody having a _VK region of SEQ ID NO: 36 and a _VH region of SEQ ID NO: 35. Also, in some embodiments, the antibody binds to kappa and lambda amyloid fibrils in vivo.

In another aspect, the present invention provides a composition comprising a humanized or chimeric antibody described herein linked to a detectable label or molecule and a pharma- ceutically acceptable carrier.

Chimeric antibodies useful in the present methods and compositions may be produced by co-transfection of vector constructs 11-1F4VK.pKN100 and 11-F4VH.pG1D200 in mammalian cells, or by transfection of supervector construct pG1KD200-11-1F4 in mammalian cells. In some embodiments, co-transfection of vector constructs 11-1F4VK.pKN100 and 11-F4VH.pG1D200 or transfection of supervector construct pG1KD200-11-1F4 occurs in COS cells. Antibodies produced in this manner are referred to as "chimeric 11-1F4 antibodies." Fragments of chimeric 11-1F4 antibodies are also useful in the present methods and compositions, as described elsewhere herein.

In some embodiments, the detected amyloid deposits result from primary amyloidosis. In some embodiments, the primary amyloidosis involves involvement of at least one organ or tissue selected from the group consisting of the heart, kidney, liver, lungs, gastrointestinal tract, nervous system, musculoskeletal system, soft tissue, and skin, and the deposits are detected in one or more of those organs.

In one aspect, the invention provides a method of detecting or imaging amyloid deposits comprising administering to a patient diagnosed with or suspected of having an amyloid deposition disease a diagnostically effective amount of a humanized or chimeric antibody or antigen-binding fragment thereof linked to a detectable label or marker, and detecting the presence, location and/or amount of the detectable label bound to amyloid deposits in the patient by imaging. In this aspect, the antibody or antigen-binding fragment may have a variable heavy chain (VH) having complementarity determining regions (CDRs) H1 comprising SEQ ID NO: 52, CDRH2 comprising SEQ ID NO: 53, and CDRH3 comprising SEQ ID NO: 54, and a variable light chain ( _VK ) having a CDRL1 comprising SEQ ID NO: 49, CDRL2 comprising _SEQ ID NO: 50, and CDRL3 comprising SEQ ID NO: 51. In another aspect, the invention provides a composition comprising the antibody or antibody fragment described above linked to a detectable molecule and a pharma- ceutically acceptable carrier.

In some embodiments of the above aspects, the antibody or antigen-binding fragment thereof may be a humanized antibody, and in other embodiments, the antibody or antigen-binding fragment thereof may be a chimeric antibody.

In some embodiments of the above aspects, the _VK region of the antibody or antigen-binding fragment may comprise SEQ ID NO:47 and the _VH region may comprise SEQ ID NO:48.

In some embodiments of the above aspects, the antibody or antigen-binding fragment may have a constant region derived from human IgG1. In some embodiments, the antibody may be a chimeric 11-1F4 antibody.

In another aspect, the invention provides a method for detecting an amyloid deposition disease in a patient suspected of having the disease by administering a labeled antibody or antigen-binding fragment thereof and detecting the presence of the label in the patient by imaging.

In some embodiments of this aspect, the humanized or chimeric 11-1F4 antibody has a constant region derived from human IgG1.

In another aspect, the invention provides a method of detecting the presence, location and/or amount of amyloid deposits in vivo in a patient suspected of having amyloid deposits, the method comprising the step of administering to the patient an antibody or antigen-binding fragment linked to a detectable molecule, wherein the antibody or antigen-binding fragment has a variable heavy chain (VH) having complementarity determining regions (CDRs) H1 comprising SEQ ID NO:53, CDRH2 comprising SEQ ID NO:53, and CDRH3 comprising SEQ ID NO:54, and a variable light chain ( _VK ) having a CDRL1 comprising SEQ ID NO:49, CDRL2 comprising SEQ ID NO:50, and CDRL3 comprising _SEQ ID NO:51. The method also comprises detecting the presence, location and/or amount of amyloid deposits by detection of a detectable label bound to the amyloid deposits by imaging.

In another embodiment, the invention provides a method of detecting the presence, location and/or amount of amyloid deposits in vivo in a patient suspected of having amyloid deposits, comprising administering to the patient an antibody or antigen-binding fragment linked to a detectable molecule, wherein the antibody or antigen-binding fragment has a _VK region comprising SEQ ID NO: 47 and a _VH region comprising SEQ ID NO: 48, and detecting the presence, location and/or amount of amyloid deposits bound to the amyloid deposits by detection of the detectable label by imaging.

In one embodiment of any of the above methods, the invention provides the above methods, wherein the method of detection is positron emission tomography (PET). In the embodiment where the method of detection is PET, the detectable label may be ¹²⁴ I or ⁸⁹ Zr. In another embodiment of the above method, the antibody is selected from chimeric or humanized 11-1F4, antigen-binding fragments thereof, and antibody CAEL-101. In yet another embodiment of the above method, the amyloid deposits occur in the heart.

In another aspect, the invention provides a composition for detecting the presence of amyloid deposits in a subject comprising an antibody or antigen-binding fragment linked to a detectable molecule, wherein the antibody or antigen-binding fragment has a variable heavy chain (VH) having complementarity determining regions (CDRs) H1 comprising SEQ ID NO:53, CDRH2 comprising SEQ ID NO:53, and CDRH3 comprising SEQ ID NO:54, and a variable light chain ( _VK ) having a CDRL1 comprising SEQ ID NO:49, CDRL2 comprising SEQ ID NO:50, and CDRL3 comprising _SEQ ID NO:51.

In another aspect, the invention provides a composition for detecting the presence of amyloid deposits in a subject comprising an antibody or antigen-binding fragment linked to a detectable molecule, wherein the antibody or antigen-binding fragment has a _VK region comprising SEQ ID NO:47 and a _VH region comprising SEQ ID NO:48.

In one embodiment of the above-mentioned composition, the detectable molecule is selected from ¹²⁴ I and ⁸⁹ Zr. In another embodiment of the above-mentioned composition, the antibody is selected from chimeric or humanized 11-1F4 or an antigen-binding fragment thereof and antibody CAEL-101. In yet another embodiment of the above-mentioned composition, the amyloid deposits occur in the heart.

In another aspect, the present invention provides a method for monitoring disease progression in a patient diagnosed with an amyloid deposition disease and having amyloid deposits. The method includes the steps of (a) administering to the patient a composition as described above and performing imaging on the patient to detect the amount of detectable molecules bound to the amyloid deposits, (b) treating the patient with a therapy intended to remove the amyloid deposits, (c) administering to the patient a composition as described above, (d) performing imaging on the patient to detect the amount of detectable molecules bound to the amyloid deposits, and (e) comparing the amount of detectable molecules detected in step (d) with the amount of detectable molecules detected in step (a).

In step (b) of the above method, the patient may be treated with an antibody selected from humanized or chimeric 11-1F4 antibody or an antigen-binding fragment thereof and antibody CAEL-101. In the above method, the composition may be any of the above compositions. In another embodiment, the method of detection may be PET. In another embodiment, when the method of detection is PET, the detectable label is selected from ¹²⁴ I and ⁸⁹ Zr. In another embodiment of the above method, the antibody is selected from humanized or chimeric 11-1F4 or an antigen-binding fragment thereof and CAEL-101. In another embodiment of the above method, the amyloid deposits occur in the heart.

In another embodiment, the present invention provides a method for determining the efficacy of a treatment for removing amyloid deposits in a patient, the method comprising the steps of: (a) treating the patient with a therapeutically effective dose of a humanized or chimeric 11-F4 antibody or antibody fragment thereof; (b) administering to the patient a diagnostically effective amount of one of the compositions described above; and (c) measuring by imaging the amount of a molecule detectable from the diagnostic composition in a lymph node of the patient, where the greater the amount of detectable molecule detected in the lymph node, the more effective the treatment. In one aspect of this embodiment, the amyloid deposits occur in the heart.

In some embodiments of the invention, the chimeric 11-1F4 antibody is CAEL-101, and the antibody is produced by CHO cells deposited with the ATCC under ACC number PTA-125146.

In some embodiments, the primary amyloidosis consists of λ light chain fibrillar aggregate deposits, in other embodiments, the primary amyloidosis consists of κ light chain fibrillar aggregate deposits, and in yet other embodiments, the primary amyloidosis consists of κ and λ light chain fibrillar aggregate deposits.

The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present invention.

FIG. 1 outlines the strategy used to clone mouse _VH and _VK genes from hybridoma cell lines. 1 is a listing of the DNA and amino acid sequences of the murine 11-1F4 antibody _VH region gene (SEQ ID NO:39 and SEQ ID NO:35, respectively). 1 is a listing of the DNA and amino acid sequences of the mouse 11-1F4 antibody V _K region gene (SEQ ID NO:40 and SEQ ID NO:36, respectively). 1 is a map of the immunoglobulin kappa light chain expression vector pKN100. It consists of a pSV2 vector fragment and has the SV40 early and incomplete SV40 late promoters, the SV40 origin of replication and the Co1E1 origin of replication. It also has the ampicillin resistance gene and the neo gene. The incomplete SV40 late promoter drives the neo gene. It also has the HCMVi promoter, a multiple cloning site (including BamHI and HindIII restriction sites) for inserting immunoglobulin variable region genes, and the cDNA of the human kappa constant region gene terminated by the spaC2 termination signal sequence ("Arnie") in the same orientation as the kappa light chain expression cassette. This is a map of the immunoglobulin gamma 1 heavy chain expression vector pG1D200. It consists of a pSV2dhfr vector fragment, and has the SV40 early and incomplete SV40 late promoters, the SV40 origin of replication, and the Co1E1 origin of replication. It also has the ampicillin resistance gene and the dhfr gene. The incomplete SV40 late promoter drives the dhfr gene. As a result, expression is low. This allows the use of relatively low levels of methotrexate to select for multigene/high expression level clones. It also has the HCMVi promoter fragment, multiple cloning sites, the cDNA of the human gamma 1 constant region gene (intron (-)), followed by the spaC2 termination signal sequence ("Arnie"). 1 is a listing of the DNA and amino acid sequences of the modified murine 11-1F4 antibody V _K region gene (SEQ ID NO:42 and SEQ ID NO:47, respectively) and the sequences of oligonucleotide primers used to modify the V _K gene (SEQ ID NO:41 and SEQ ID NO:43, respectively). 1 is a listing of the DNA and amino acid sequences of the modified murine 11-1F4 antibody _VH region gene (SEQ ID NO:45 and SEQ ID NO:48, respectively) and the sequences of oligonucleotide primers used to modify the _VH gene (SEQ ID NO:44 and SEQ ID NO:46, respectively). 1 is a graph showing the results of an amyloid fibril binding ELISA assay. Cos cell supernatants containing chimeric 11-1F4 antibody were tested separately on the same ELISA plate along with purified mouse 11-1F4 antibody. Absorbance was read at OD405. New sv=pG1KD200-11-1F4. New cotransfection=11-1F4VHpG1D200+11-1F4VK.pKN100. This is a PET image of a mouse transplanted with human cardiac amyloidoma, which was injected with [ ¹²⁴ I]CAEL-101, and imaged 4 days after injection.

According to the present invention, compositions are provided that include humanized antibodies, chimeric antibodies (e.g., mouse-human antibodies) or antigen-binding fragments thereof, each linked to a detectable molecule, which are useful for detecting the presence, location and amount of amyloid deposits in vivo. These compositions allow for detection, localization and/or quantification of amyloid deposits with little or no human anti-mouse antibody (HAMA) reaction. The present invention provides compositions that include at least one antibody or antibody fragment linked to a detectable marker and a pharma- ceutically acceptable carrier, and methods for detecting the location and amount of amyloid deposits by administering to a patient an effective amount of the marker-linked antibody or antibody fragment effective for detecting amyloid deposits when present, and detecting the presence, amount and/or location of the detectable marker bound to the amyloid deposits by diagnostic imaging.

Further in accordance with the present invention, a method is provided for monitoring or determining the effectiveness of a treatment for an amyloid deposition disease. A method for monitoring disease progression in a patient diagnosed with an amyloid deposition disease and having amyloid deposits comprises:
(a) administering to a patient a diagnostically effective amount of a humanized or chimeric 11-1F4 antibody, or antigen-binding fragment thereof, linked to a detectable molecule, and detecting the amount of the detectable molecule bound to amyloid deposits;
(b) treating the patient with a therapy designed to remove amyloid deposits, such as a humanized or chimeric 11-1F4 antibody or antigen-binding fragment thereof;
(c) administering to the patient a diagnostically effective amount of a humanized or chimeric 11-1F4 antibody, or antigen-binding fragment thereof, linked to a detectable molecule, and detecting the amount of the detectable molecule bound to amyloid deposits;
(d) comparing the amount of detectable molecule detected in step (c) with the amount of detectable molecule detected in step (a);
Includes.

In this embodiment, the invention aims to measure the degree of change in the amount of amyloid deposits (if present) in a patient by administering to the patient an antibody or antibody fragment linked to a marker as described herein both before and after a treatment intended to remove amyloid deposits, and measuring the difference between the size and extent of amyloid deposits detected after treatment and the size and extent measured before treatment.

Further provided is a method of detecting amyloid deposits in a subject in vivo, the method comprising: (a) administering to the subject a composition comprising one or more of the antibodies or antigen-binding fragments thereof described herein linked to a detectable molecule; and (b) detecting by imaging a detectable agent linked to the antibody or antigen-binding fragment thereof bound to the amyloid deposit.

In the above-described method, the step of detection (step (b)) may be performed using PET (positron emission tomography), SPECT (single photon emission computed tomography), MRI, fluorescence imaging or other suitable imaging method. PET is the preferred imaging method. The use of PET to image tumors and other deposits using detectable molecules is well known. The preparation of this imaging agent and its use in PET is described, for example, in Baillet et al. (Int. J. Mol. Sci. 2017, 18, 57), Boerman et al. (J Nucl Med, 2011; 52: 1171-1172), and Mayer et al. (J Nucl Med, 2017; 58: 538-546), the entire contents of which are incorporated herein by reference. Although these references are primarily directed to the detection of cancerous tumors, the materials and methods described therein are similarly useful for detecting amyloid deposits.

The in vivo imaging step in the above-described methods may be whole-body imaging for diagnostic purposes, or quantitative and localized imaging of specific sites, such as the heart, kidneys, spleen, nervous system or gastrointestinal system, to assess disease progression or patient response to a therapeutic regimen. The detection step in the above-described methods may be any imaging technique, including, but not ^limited to, positron emission tomography (PET) where the detectable agent is an isotope such as ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶⁴ Cu, ⁶² Cu, ¹²⁴ I, 76 Br, ⁸² Rb, ⁸⁹ Zr or ⁶⁸ Ga, single photon emission computed tomography (SPECT) where the detectable agent is a radiotracer such as ^99m Tc, ¹¹¹ In, ¹²³ I, ²⁰¹ Tl or ¹³³ Xe depending on the particular application, and magnetic resonance imaging (MRI) where the detectable agent is, for example, gadolinium, iron oxide nanoparticles and carbon-coated iron-cobalt nanoparticles. In the examples described herein, the antibodies are labeled with ¹²⁴ I or ⁸⁹ Zr.

The detectable molecules described herein (sometimes referred to herein as "labels," "markers," or "agents") may be linked (sometimes referred to herein as "conjugated") to an antibody or fragment thereof via any suitable method known in the art. For example, but not limited to, a detectable agent may be linked to an antibody or antibody fragment via a covalent bond or ionic interaction. Linkage may be achieved via chemical cross-linking reactions or via fusion using recombinant DNA methodology combined with any peptide expression system, such as bacterial, yeast, or mammalian cell-based systems. Methods for linking an antibody or fragment thereof to a detectable agent are well known to those of skill in the art, as set forth in the references cited above.

Further provided herein is a method for determining an appropriate dosage of a humanized or chimeric antibody described herein for treatment based on the affinity of a diagnostic composition for amyloid deposits as determined by uptake of the diagnostic composition by the patient's amyloid deposits. That is, the dosage for the patient can be determined based on the detected uptake of the labeled antibody or antibody fragment. A patient exhibiting high uptake of the labeled antibody or antibody fragment may require a lower amount of the therapeutic antibody or antibody fragment than a patient exhibiting low uptake of the labeled antibody or antibody fragment. Thus, the present invention provides a method for determining an appropriate dosage of a humanized or chimeric antibody described herein for treatment. The method includes administering to a patient a labeled antibody or antibody fragment described herein, determining uptake of the labeled antibody or antibody fragment by the amyloid deposits, and administering a dose or series of doses of the chimeric or humanized antibody or antibody fragment based on the detected amount of uptake.

Further provided herein is a method for determining the efficacy of a therapy for removing amyloid deposits using a chimeric or humanized antibody or antibody fragment described herein. The method includes the steps of (a) treating a patient with a therapeutically effective dose of a therapy intended to remove amyloid deposits, such as a chimeric or humanized antibody or antibody fragment described herein; (b) administering to the patient a diagnostically effective amount of a diagnostic composition described herein; and (c) measuring the amount of a molecule detectable from the diagnostic composition in a lymph node of the patient, where the greater the amount of detectable molecule detected in the lymph node, the more effective the therapy.

[Definition]
It is to be understood that the method is not limited to the specific embodiments described herein, but may vary. It is also to be understood that the terminology used herein is used to describe only the specific embodiments, and is not intended to be limiting. The scope of the technology is limited only by the appended claims.

Certain terms used herein may have the following defined meanings. As used in this specification and claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a single cell as well as a plurality of cells, including mixtures thereof.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, means excluding other elements that are of essential importance to the composition or method. "Consisting of" means excluding more than trace elements of other components in the compositions and substantial method steps defined in the claims. Embodiments defined by each of these transitional terms are within the scope of the present invention. Thus, it is intended that the methods and compositions may include additional steps and ingredients (comprising), or may include non-essential steps and compositions (consisting essentially of), or may consist only of the method steps or compositions described herein (consisting of).

As used herein, "about" means plus or minus 10% from the value.

As used herein, "optional" means that the event or circumstance described below may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

As used herein, the terms "individual," "patient," or "subject" may refer to an individual organism, a vertebrate, a mammal (e.g., a cow, a dog, a cat, or a horse), or a human. In a preferred embodiment, the individual, patient, or subject is a human.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to an amyloid fibril is substantially free of antibodies that do not bind to an amyloid fibril). However, an isolated antibody that specifically binds to an epitope of an amyloid light chain fibril (e.g., κ and/or λ fibril) may have cross-reactivity to other proteins, such as amyloid A fibrils. However, the antibody preferably always binds to human amyloid light chain fibrils. Also, an isolated antibody is typically substantially free of other cellular material and/or chemicals.

As used herein, the term "diagnostically effective amount" refers to an amount of an antibody or antibody fragment linked to a detectable marker that, when administered to a patient or subject, is capable of detecting, localizing, and/or quantifying amyloid deposits in the patient or subject in vivo by imaging, if such deposits are present. It is emphasized that a diagnostically effective amount is not necessarily effective for detecting amyloid deposits, even if the amount would be considered a diagnostically effective amount by one of skill in the art. A diagnostically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the condition of the subject, such as the type and stage of amyloidosis at the time of initiation of diagnosis, as well as other factors.

As used herein, the term "humanized antibody" refers to an antibody having the CDRs of an antibody derived from a mammal other than a human, and the framework region (FR) and constant region of a humanized antibody. Humanized antibodies are useful as elements in diagnostic compositions according to the present invention due to the reduced antigenicity of the humanized antibody in the human body.

As used herein, the term "antibody fragment" refers to a portion of an antibody that is designed to delete part of the structure of an intact antibody while still containing the CDRs of the antibody. The design of antibody fragments is well known in the art and is described (for example) by Holliger et al. in Nature Biotechnology: 23(9), 1126-1135 (2005), the entire contents of which are incorporated herein by reference. As used herein, "antigen-binding fragment" refers to an antibody fragment that binds to amyloid deposits.

As used herein, the term "pharmaceutically-acceptable carrier" refers to a material for mixing with a pharmaceutical or therapeutic compound (e.g., a chimeric antibody linked to a detectable molecule) for administration to a patient, e.g., as described in Ansel's Pharmaceutical Dosage Forms and Delivery Systems, 10th Edition (2014).

As used herein, the terms "detectable molecule," "detectable marker," "detectable agent," and "detectable label" refer to a substance that may be detected in vivo by diagnostic imaging techniques, including, but not limited to, PET, SPECT, and MRI.

As used herein, the term "diagnostic imaging" or "imaging" refers to a method of imaging detectable molecules in a patient or subject in vivo to determine the presence, location and/or amount of tissue or material (such as amyloid deposits) in a patient or subject having the detectable molecule localized by linking it to an antibody or antibody fragment that binds to the tissue or material to be detected.

[Anti-AL antibody]
[[Mouse Antigen-Fibrin Antibody]]
Recent animal studies have shown that administration of the murine 11-1F4 antibody and other murine anti-human light chain specific antibodies directed against epitopes common to the β-pleated sheet structure present in AL fibrils results in complete degradation of human ALκ and ALλ amyloid deposits. Some of these murine antibodies are described in U.S. Patent No. 8,105,594 (the "'594 Patent"), the entire contents of which are incorporated herein by reference.

Mouse antibodies are generally not suitable for administration to other animal species (such as humans) because the recipient species recognizes the mouse antibody as an antigen and produces antibodies against it. Antigenicity when an antibody from one species is injected into another species is usually caused by a portion of the constant domain. Such an antigenic reaction inhibits or prevents the desired therapeutic effect of the mouse antibody. In humans, this antigenic reaction is called human anti-mouse antibody (HAMA). The antibodies described in the '594 patent can be highly immunogenic in humans through a human anti-mouse antibody (HAMA) response. Since the HAMA reaction usually results in rapid clearance of the mouse antibody from the human recipient, HAMA greatly limits the benefits that mouse antibodies can bring to human therapy and diagnosis. As such, these mouse antibodies are not suitable for administration to patients to detect amyloid fibril deposits in the patient. Thus, the present invention provides a composition for detecting and/or monitoring amyloid deposition disease that is unlikely to produce an immunogenic HAMA reaction in the patient after administration.

[[Humanized and chimeric antigen-fiber antibodies]]
The present invention provides compositions comprising humanized and chimeric antibodies or antigen-binding fragments thereof linked to detectable molecules. The compositions described herein are useful for imaging the location and amount of amyloid deposits in a subject. Typically, an antibody consists of four polypeptides, including two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. Typically, each heavy chain has two N-terminal variable (V _H ) regions and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain has one N-terminal variable (V _L or V _K ) region and one C-terminal constant (CL) region. Each variable domain of the light and heavy chains of an antibody also contains three segments called complementarity determining regions ("CDRs") or hypervariable regions. Each CDR in the light chain, together with the corresponding CDR in the adjacent heavy chain, forms the antigen-binding site of the antibody. The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody, while the constant regions provide structural support and regulate the immune response initiated by antigen binding.

Chimeric antibodies incorporate the variable regions of a non-humanized antibody into the constant region of a humanized antibody. For example, a chimeric 11-1F4 antibody may be produced by expressing mouse variable regions in the Fc region of a humanized antibody, such as human IgG1.

Humanized versions of non-human (e.g., murine) antibodies can be obtained that contain minimal sequences derived from non-human immunoglobulin. Generally, humanized antibodies may have one or more variable domains. The variable regions are derived from a non-human immunoglobulin, and the framework regions (FR) correspond to human immunoglobulin sequences. Thus, in some embodiments, a humanized anti-AL antibody has a humanized antibody framework region. Such antibodies can be prepared by known techniques.

The murine 11-1F4 monoclonal antibody is an anti-AL antibody produced by SP2/0 hybridoma cells deposited at Dr. Alan Solomon, MD (University of Tennessee Medical Science Center, Knoxville, Tennessee, USA). The hybridoma cell line is available from the American Type Culture Collection (ATCC deposit PTA-105). Table 1 below shows the _VK region (SEQ ID NO:36) and _VH region (SEQ ID NO:35) of the 11-1F4 antibody. Table 2 shows the CDR sequences of the heavy and light chains.

Using known human antibody sequences, the genes for the _VH and _VK regions described above can be cloned to generate the chimeric 11-1F4 antibody. The chimeric 11-1F4 antibody binds to an epitope expressed by the beta-pleated sheet structure of amyloid, similar to its mouse counterpart. Surprisingly, as Example 6 below shows, this chimeric antibody binds to AL amyloid fibrils with higher affinity than the 11-1F4 mouse antibody from which it is derived.

Known human antibody sequences can also be used to generate humanized versions of the antibody by cloning the genes of the CDR regions. Like the chimeric 11-1F4 antibody, the humanized version can have a higher binding affinity for amyloid fibrils than the corresponding mouse antibody.

Those skilled in the art will appreciate that the humanized and chimeric antibodies described herein can utilize any type of human constant region and/or framework region. For example, the humanized and chimeric antibodies described herein may have human IgG (including IgG1, IgG2, IgG3 and IgG4), IgA, IgE, IgH, or IgM constant regions and/or framework regions. In a preferred embodiment, the humanized or chimeric 11-1F4 antibodies described herein have a human IgG1 constant region.

In some embodiments, the antibodies described herein may contain one or more substitutions, insertions, or deletions, so long as the antibody maintains its ability to bind to amyloid fibrils (e.g., κ and/or λ light chain fibrils). For example, in some embodiments, the chimeric 11-1F4 antibodies of the invention may have heavy and light chains that have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity compared to the corresponding heavy and light chain sequences described herein, so long as the antibody maintains its ability to bind to amyloid fibrils. In some embodiments, the humanized 11-1F4 antibodies of the invention may have CDRs that are about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the corresponding CDR sequences described herein, so long as the antibody maintains the ability to bind to amyloid fibrils.

[Abbreviation]
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), copy DNA (cDNA), polymerase chain reaction (PCR), minutes (min), seconds (sec), Tris-borate buffer (TBE).

Amino acids are represented by their IUPAC abbreviations as follows: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Nucleotides are similarly represented as follows: adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), adenine or guanine (R), cytosine or thymine (Y), guanine or cytosine (S), adenine or thymine (W), guanine or thymine (K), adenine or cytosine (M), cytosine or guanine or thymine (B), adenine or guanine or thymine (D), adenine or cytosine or thymine (H), adenine or cytosine or guanine (V), and any base (N).

Humanized or chimeric antibodies
To produce the chimeric antibodies of the present invention, the murine 11-1F4 monoclonal antibody heavy and kappa light chain variable region genes described in U.S. Pat. No. 8,105,594 were PCR modified to facilitate expression of the chimeric 11-1F4 antibody in mammalian cells. Detailed sequence analysis of the modified variable region genes was performed. The modified variable region genes were cloned into appropriate mammalian expression vectors to generate constructs 11-1F4VHpG1D200 and 11-1F4VK.pKN100. A single super vector construct pG1KD200-11-1F4 was generated from the 11-1F4VHpG1D200 and 11-1F4VK.pKN100 constructs by EcoRI restriction enzyme digestion and ligation. Finally, the chimeric 11-1F4 antibody was transiently expressed in COS cells by both co-transfection and single super vector transfection. For convenience, COS cells were chosen for co-transfection or transfection, although one skilled in the art would recognize that other mammalian cell lines could be used. The binding ability of the chimeric 11-1F4 antibody to amyloid fibrils was characterized by direct binding ELISA. Unexpectedly and beneficially, the chimeric 11-1F4 antibody bound to amyloid fibrils with higher affinity than the murine 11-1F4 antibody.

Typically, an antibody consists of four polypeptides, including two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. Typically, each heavy chain has one N-terminal variable ( _VH ) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain has one N-terminal variable ( _VL or _VK ) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody.

The antibodies useful in the compositions and methods of the invention may be chimeric mouse-human monoclonal antibodies having a V _K region of SEQ ID NO: 47 and a V _H region of SEQ ID NO: 48, or humanized monoclonal antibodies comprising the CDR sequences of SEQ ID NOs: 49-54. These antibodies bind to an epitope expressed by the β-pleated sheet structure of amyloid fibrils. Moreover, surprisingly, these antibodies bind to this epitope with a higher affinity than the 11-1F4 mouse antibody from which they are derived, having a V _K region of SEQ ID NO: 36 and a V _H region of SEQ ID NO: 35. The present invention includes a method of detecting amyloid deposits in a human patient. The method includes the step of administering to the patient a diagnostically effective dose of one of the above-mentioned antibodies linked to a detectable molecule in a pharma-ceutically acceptable carrier. The antibody compositions may be administered by any conventional route of administration, although parenteral administration (such as intravenous) is preferred. Pharmaceutically acceptable carriers are well known in the art and an appropriate one can be selected by one skilled in the medical arts.

Chimeric antibodies and antigen-binding fragments thereof useful in the compositions and methods described and claimed herein (as well as methods of making chimeric antibodies) are described in commonly owned Patent Cooperation Treaty Application PCT/US18/399805, filed June 28, 2018 (attorney docket no. 8441-0004WO, which has priority from U.S. Patent Application No. 62/526,835, filed June 29, 2017), and commonly owned Patent Cooperation Treaty Application PCT/US2018/043374, filed July 24, 2018 (attorney docket no. 8441-0009WO), the entire contents of which are incorporated herein by reference. Materials useful for making the antibodies include 11-1F4VK.pKN100 and 11-F4VH.pKN100, which are shown in Figures 5 and 6, respectively. pG1D200, and the superconstruct pG.1KD20011-1F4 made from the two vector constructs described above. Other useful materials include the modified murine 11-1F4 antibody V _K region gene (SEQ ID NO:42) and the modified 11-1F4 antibody V _H region gene (SEQ ID NO:45) and the respective primers SEQ ID NOs:41, 43, 44 and 46. The antibody may be produced by co-transfection of the vector constructs 11-1F4VK.pKN100 and 11-F4VH.pG1D200 or the superconstruct pG.1KD20011-1F4 into a suitable mammalian host cell, such as a COS (Chinese Hamster Ovary) cell.

Methods for making, testing and using humanized or chimeric 11-1F4 antibodies are described in further detail in the Examples section below.
[Diagnostic Method]
Diagnostic compositions suitable for the methods described herein can include a humanized or chimeric 11-1F4 antibody, humanized antibody or antigen-binding antibody fragment described herein, each linked to a detectable marker, and a pharma- ceutically acceptable carrier or diluent.

The composition may be formulated for intravenous, subcutaneous, intraperitoneal or intramuscular administration, although intravenous administration is preferred.

Pharmacologically acceptable carriers for various dosage forms are known in the art. For example, solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents for liquid formulations are known. In some embodiments, the pharmaceutical composition includes additional components such as one or more preservatives, antioxidants, stabilizers, etc.

Sterile injectable solutions can be prepared by incorporating the required amount of marker-linked antibody or antibody fragment in an appropriate solvent with one or more combinations of ingredients as required above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the diagnostic composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
Diagnostic and Monitoring Methods
In general, amyloidosis is caused by the accumulation of abnormal proteins called amyloids. Amyloids are produced in the bone marrow and can be deposited in any tissue or organ. The specific cause depends on the type of amyloidosis.

There are several types of amyloidosis, or amyloid disease, including AL amyloidosis, AA amyloidosis, and hereditary amyloidosis.

AL amyloidosis (immunoglobulin light chain amyloidosis) is the most common type and affects the heart, kidneys, skin, nerves, and liver. Formerly known as primary amyloidosis, AL amyloidosis occurs when the bone marrow produces abnormal antibodies that it cannot break down. The antibodies are deposited in various tissues as amyloid plaques and prevent the normal functioning of the tissue or organ.

AA amyloidosis, previously known as secondary amyloidosis, commonly affects the kidneys, but may also affect the gastrointestinal tract, liver, or heart. It often occurs in association with chronic infectious or inflammatory diseases, such as rheumatoid arthritis or inflammatory bowel disease.

Hereditary amyloidosis (familial amyloidosis) is a genetic disorder that usually affects the liver, nerves, heart and/or kidneys. Many different types of genetic abnormalities present at birth are associated with an increased risk of amyloid disease or hereditary amyloidosis. The type and location of the amyloid genetic abnormality influences the risk of certain complications, the age at which symptoms first appear and the progression of the disease over time.

When amyloid diseases affect the heart, they can cause many types of complications. Amyloid deposits, or plaques, reduce the heart's ability to fill with blood between heartbeats. Less blood is pumped with each beat, causing shortness of breath. Amyloid deposits and plaques in or around the heart can also cause arrhythmias and organ damage, such as congestive heart failure.

When amyloid disease affects the kidneys, it impairs the kidneys' filtering ability, leading to increased leakage of protein from the blood into the urine (i.e., proteinuria). This in turn reduces the kidneys' ability to remove waste products from the body, which can eventually lead to kidney failure.

Provided herein is a method for detecting, localizing, and/or quantifying amyloid deposits in a patient suffering from or suspected of suffering from an amyloid deposition disease, such as primary (AL) amyloidosis, comprising administering to a patient (e.g., a human patient) a humanized or chimeric 11-1F4 antibody or antigen-binding fragment thereof linked to a detectable marker and a pharma- tically acceptable carrier in an amount effective to detect, localize, and/or quantify amyloid deposits, if present, and then detecting detectable molecules bound to amyloid deposits in the patient by imaging to determine the extent, location, and/or amount of amyloid deposits, if present.

Also provided herein is a method for monitoring disease progression in a patient diagnosed with an amyloid deposition disease and having amyloid deposits, the method comprising:
(a) administering to a patient a diagnostically effective amount of a humanized or chimeric 11-1F4 antibody, or antigen-binding fragment thereof, linked to a detectable molecule, and performing imaging on the patient to detect the amount of the detectable molecule bound to amyloid deposits;
(b) treating the patient with a therapy designed to remove amyloid deposits, such as a humanized or chimeric 11-1F4 antibody or antigen-binding fragment thereof;
(c) administering to the patient a diagnostically effective amount of a humanized or chimeric 11-1F4 antibody, or antigen-binding fragment thereof, linked to a detectable molecule and performing imaging on the patient to detect the amount of the detectable molecule bound to amyloid deposits;
(d) comparing the amount of the detectable molecule detected in step (c) with the amount of the detectable molecule detected in step (a).

The above-described monitoring methods can be performed before any treatment is initiated or can be used during a treatment regimen. That is, step (a) above can determine a baseline amount of amyloid deposits in a patient prior to any treatment, or step (a) above can be performed after one or more courses of treatment to determine the effectiveness of a subsequent course of treatment.

In some embodiments, the amyloid deposition disease (e.g., primary amyloidosis) involves at least one organ or tissue selected from the group consisting of the heart, kidney, liver, lungs, gastrointestinal tract, nervous system, musculoskeletal system, soft tissue, and skin.

In some embodiments, the methods described herein include treating a patient suffering from relapsed or refractory ALA. In some embodiments, the patient may have κ ALA. In some embodiments, the patient may have λ ALA.

Exemplary diagnostic amounts may vary depending on the size and health of the individual being treated. In some embodiments, a diagnostically effective amount of radiolabeled humanized or chimeric 11-1F4 antibody for PET is about 1-10 mCi. However, in some situations and for other imaging modalities, the dosage may be higher or lower. One skilled in the art of imaging will know how to select an appropriate amount of labeled antibody or antibody fragment, as described (for example) in the references cited above.

The following examples are provided to illustrate the invention. However, it should be noted that the invention is not limited to the specific conditions or details described in these examples. All publications referenced herein are incorporated herein by reference.

PCR cloning and DNA sequencing of mouse 11-1F4 antibody.
The mouse 11-1F4 monoclonal antibody heavy and light chain variable region genes were PCR cloned, and detailed sequence analysis was performed on all isolated variable region genes (pseudo and functional). Detailed DNA and amino acid sequences of the mouse 11-1F4 antibody heavy and light chain variable region genes were obtained.

[material]
Media components and all other tissue culture materials were obtained from Life Technologies (UK). RNA solution kit was obtained from Stratagene (USA) and first strand cDNA synthesis kit was purchased from Pharmacia (UK). All components and equipment for PCR reaction including AmpliTaq® DNA polymerase were purchased from PerkinElmer (USA). TOPO TA Cloning® kit was obtained from Invitrogen (USA). Agarose (UltraPure™) was obtained from Life Technologies (UK). ABI PRISM® Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit Premix Cycle Sequencing Kit and ABI PRISM® 310 Sequencing Machine were both purchased from PE Applied Biosystems (USA). All other molecular biology products were obtained from New England Biolabs (USA) and Promega (USA).

[method]
FIG. 1 is a diagram outlining the strategy used for PCR cloning of mouse V _H and V _K genes from a hybridoma cell line producing mouse monoclonal antibody 11-1F4.

Two clones (B2C4 and B2D6) of the SP2/0 hybridoma cell line producing the α-human light chain monoclonal antibody 11-1F4 were kindly provided by Dr. Alan Solomon, MD (University of Tennessee Medical Science Center, Knoxville, TN, USA). The hybridoma cell line is available from the American Type Culture Collection (ATCC deposit PTA-105). The cell line was cultured using DMEM medium supplemented with 20% (v/v) FBS, penicillin/streptomycin and L-glutamine. The cells were cultured until a total viable cell count of ¹⁰⁸ cells was reached.

Cells were harvested separately from each clone as follows: Mouse hybridoma cell lines were grown in suspension in the appropriate culture medium to sufficient quantities to provide a total viable cell count of approximately ¹⁰⁸ cells. Culture supernatants were harvested and hybridoma cells were pelleted in a benchtop centrifuge (250g, 5 min). Cells were gently resuspended in 20 ml of PBS and a 100 μl aliquot was taken for viable cell count. An aliquot of cells was pelleted again and 200 μl of PBS and 200 μl of trypan blue were added to the 100 μl of cells and mixed gently. 10 μl of this mixture was pipetted onto a disposable cell counting slide and the number of white blood cells in nine small squares was counted under a microscope. Blue cells (i.e., dead cells) were not counted. The counting process was repeated, the results averaged, and the average result multiplied by 9× ¹⁰⁵ to obtain the viable cell count of cells in 20 ml of PBS. We harvested enough cells and resuspended them in 10 ml of solution D for RNA isolation (using the Stratagene RNA isolation kit, see below).

Total RNA was then isolated from cells of each clone individually using a Stratagene RNA isolation kit according to the manufacturer's instructions. 1 ml of 2 M sodium acetate (pH 4.0) was added to the sample and the contents of the tube were thoroughly mixed by repeatedly inverting the tube. 10.0 ml of phenol (pH 5.3-5.7) was added to the tube and the contents were thoroughly mixed again by inversion. 2.0 ml of chloroform-isoamyl alcohol mixture was added to the mixture, the tube was capped and shaken vigorously for 10 seconds, and the tube was incubated in ice for 15 minutes. The sample was transferred to a 50 ml thick-walled round-bottom centrifuge tube pre-chilled on ice and the tube was spun in a centrifuge at 10,000 x g for 20 minutes at 4°C. After centrifugation, two phases were visible in the tube. The upper aqueous phase contained RNA, and the lower phenol phase and intermediate phase contained DNA and protein. The upper aqueous phase containing RNA was transferred to a new centrifuge tube and the lower phenol phase was discarded. An equal volume of isopropanol was added to the aqueous phase, the contents mixed by inversion, and the tube was then incubated at -20°C for 1 hour to precipitate the RNA. The tube was spun in a centrifuge at 10,000 xg for 20 minutes at 4°C. After centrifugation, the pellet at the bottom of the tube containing the RNA was removed and the supernatant discarded. The pellet was dissolved in 3.0 ml of solution D, 3.0 ml of isopropanol was added to the tube, and the contents mixed well. The tube was incubated at -20°C for 1 hour, then spun again in a centrifuge at 10,000 xg for 10 minutes at 4°C, and the supernatant removed from the tube and discarded. (Note: Up until this point, the RNA had been protected from ribonucleases by the presence of guanidine isothiocyanate, but is now no longer protected.) The pellet was washed with 75% (v/v) ethanol (DEPC-treated water (25%)) and the pellet dried under vacuum for 2-3 minutes. The RNA pellet was resuspended in 0.5-2 ml of DEPC-treated water.

The Not I-d(T) ¹⁸ primer provided in the Amersham Pharmacia Biotech First Strand cDNA Synthesis Kit was used to generate single stranded DNA copies of the 11-1F4 hybridoma mRNA according to the manufacturer's instructions. One reaction was performed for each of the two RNA samples isolated as described below. The components used were bulk first strand cDNA reaction mix, cloning FPLCpure™ mouse reverse transcriptase, RNAguard™, BSA, dATP, dCTP, dGTP and dTTP, 200 mM DIT in water, Not I-d(T) ¹⁸ primer: 5'-d[AACTGGAAGAATTCGCGGCCGCAGGAA ₁₈ ]-3', and DEPC-treated water.

Approximately 5 μg of total RNA in 20 μl DEPC water was heated at 65° C. for 10 minutes and then cooled on ice. The bulk first strand cDNA reaction mixture was gently pipetted to obtain a uniform suspension and the reaction was set up in a 0.5 ml microcentrifuge tube as follows: 20 μl denatured RNA solution, 11 μl bulk first strand cDNA reaction mixture, 1 μl Not I-d(T) ¹⁸ primer and 1 μl DTT solution for a total volume of 33 μl. The reaction materials were pipetted and mixed gently and incubated at 37° C. for 1 hour.

The mouse heavy and kappa light chain variable region genes ( _VH and _VK genes, respectively) were then PCR amplified from ssDNA templates using the method described by Jones and Bendig (Bio/Technology, 9:88).

Separate PCR reactions were _set up for each of the degenerate leader sequence specific primers (MHV1-MHV12 for _VH and MKV1-MKV11 for _VK ) using the appropriate constant region primers (equimolar mixture of MHC1-MHC3 for VH and _MKC for VK). Tables 1 and 2 detail the primers used to amplify the _VH and _VK region genes, respectively. In total, 12 heavy chain reactions and 11 kappa light chain reactions were performed. Template cDNA was amplified in all cases using AmpliTaq® DNA polymerase as described below.

The completed cDNA first strand synthesis reaction was heated to 90°C for 5 minutes to denature the RNA-cDNA duplex and inactivate the reverse transcriptase, and cooled on ice. Eleven GeneAmp™ PCR reaction tubes were labeled MKV1-11. For each tube, a 100 μl reaction mixture was prepared, with each reaction mixture containing 69.3 μl sterile water, 10 μl 10× PCR buffer II, 6 μl 25 mM MgCl ₂ , 2 μl each of a 10 mM stock solution of dNTPs, 2.5 μl 10 mM MKC primer, 2.5 μl one of the 10 mM MKV primers, and 1 μl of the RNA-cDNA template mixture. 0.7 μl of AmpliTaq® DNA polymerase was then added to each of the tubes, and the completed reaction mixture was overlaid with 50 μl of mineral oil.

A similar series of reaction mixtures was prepared as described above to PCR clone mouse heavy chain variable region genes, except this time 12 reaction tubes were labeled and one of the 12 MHV primers and the appropriate MHC primer were added to each. That is, for example, to PCR amplify the variable domain gene of the mouse γ1 heavy chain, the MHC G1 primer was used.

The reaction tubes were loaded into a DNA thermal cycler and cycled 25 times at 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min (after an initial melt at 94°C for 1.5 min). The last cycle was followed by a final extension step at 72°C for 10 min before cooling to 4°C. A 30 s ramp time was allowed between each step of the cycle, except between the annealing (50°C) and extension (72°C) steps, where an extended ramp time of 2.5 min was used. A 10 μl aliquot from each PCR reaction was run on a 1% (w/v) agarose/1×TBE buffer gel containing 0.5 μg/ml ethidium bromide to determine which leader primer produced a PCR product. The size of the positive PCR clones was approximately 420 bp to 500 bp.

The PCR amplification steps described above were repeated two more times to select for PCR reactions that appeared to amplify full-length variable domain genes. A 6 μl aliquot of each potential PCR product was directly cloned into the pCR™II vector provided by the TA Cloning® kit as described in the manufacturer's instructions. Aliquots of 10.0% (v/v), 1.0% (v/v) and 0.1% (v/v) of transformed E. coli cells were pipetted onto individual 90 mm diameter LB agar plates containing 50 μg/ml ampicillin, overlaid with 25 μl of X-Gal stock solution and 40 μl of IPTG stock solution, and incubated overnight at 37°C. Positive colonies were identified by PCR screening.

A 5 μl aliquot from each PCR reaction was run on a 1% agarose/TBE (pH 8.8) gel to determine those that produced a PCR product of the correct size (approximately 450 bp). These putative positive PCR products thus identified were directly cloned into the pCR2.1 vector provided by the TA Cloning® kit and transformed into TOP10 competent cells as described in the manufacturer's protocol. Colonies containing plasmids with the correct size insert were identified by PCR screening colonies using the 1212 and 1233 oligonucleotide primers (Table 3) according to the method of Gussow and Clackson (Nucleic Acids Res., 17:4000). These putative positive clones thus identified were sequenced using an ABI PRISM 310 Genetic Analyzer and an ABI PRISM BigDye™ Terminator to sequence the double-stranded plasmid DNA. Three positive clones each of the _VH and _VK genes from the B2C4 hybridoma cell line clones were sequenced, as were four positive clones of the _VK gene and six of the _VH gene from the B2D6 hybridoma cell line clones.

The results of 12 PCR reactions performed on each hybridoma clone (B2C4 and BCD6) to amplify the mouse 11-1F4 antibody heavy chain variable region gene are shown in Table 4(a).

The degenerate leader sequence primer MHV7, when combined with a mixture of MHCGI-3 constant region primers (Table 1), produced a PCR product of approximately 600 bp from template cDNA from both the B2C4 and B2D6 hybridoma cell lines. This band was larger than the expected size for the average _VH gene (450 bp) and was not investigated further. On the other hand, the degenerate leader sequence primer MHV6, when combined with a mixture of MHCGI-3 constant region primers (Table 1), produced a PCR product of the expected size for the _VH gene (450 bp) from template cDNA from both the B2C4 and B2D6 hybridoma cell lines.

Table 4 shows the results of PCR amplification performed to clone the mouse 11-1F4 monoclonal antibody heavy chain variable region gene (a) and light chain variable region gene (b) from SP2/0 hybridoma cell lines B2C4 and B2D6. In the third column, the actual PCR results are recorded. When bands were observed for a particular combination of primers, the base pair size (bp) was recorded in the appropriate space.

Sequence analysis of three clones from the B2C4-derived PCR product and five clones from the B2D6-derived PCR product revealed a single heavy chain variable region sequence (Figure 2).

The cloning strategy used (amplification of the entire variable region gene by using primers flanking this region, i.e. specific for the leader and constant region sequences) allowed the identification of the complete FR1 sequence. All eight clones sequenced had identical sequences in this region (Figure 2).

The results of 11 PCR reactions performed on each hybridoma clone (B2C4 and BCD6) to amplify the mouse 11-1F4 antibody κ light chain variable region gene are shown in Table 4(b).

The degenerate leader sequence primer MKV6, in combination with the MKC constant region primers (Table 2), produced a PCR product of approximately 200 bp from template cDNA derived only from the B2C4 hybridoma cell line, and because this band was much smaller than the expected size for the _VK gene (450 bp), it was not investigated further.

The degenerate leader sequence primer MKV2, in combination with the MKC constant region primer (Table 2), produced PCR products smaller than the expected band size of 450 bp (as viewed on an agarose gel) from template cDNA from both the B2C4 and B2D6 hybridoma cell lines. Furthermore, previous V _K cloning showed that the MKV2 primers amplify a known kappa light chain pseudogene. Therefore, to confirm that the products were pseudogenes and not the mouse 11-1F4 antibody V _K gene, sequence analysis of one clone from each PCR product was performed. This sequence analysis revealed that the PCR clones were indeed pseudogenes.

Finally, the degenerate leader sequence primer MKV1, in combination with the MKC constant region primers (Table 1), produced a PCR product of approximately the expected size (450 bp) for the _VK gene from template cDNA derived from both the B2C4 and B2D6 hybridoma cell lines.

Sequence analysis of three clones of PCR products derived from B2C4 and four clones of PCR products derived from B2D6 revealed a single kappa light chain variable region sequence that could not be identified as a pseudogene.

Here, the 11-1F4 antibody heavy chain variable region gene was cloned from hybridoma mRNA (using constant region-specific and leader sequence-specific primers) and sequenced.

This sequence was translated to give the TVSS peptide sequence. Analysis of 122 rearranged human _VH genes recorded in the Kabat database (Kabat et al., Sequences of Proteins of Immunological Interest) revealed that 84% of these sequences had the TVSS peptide sequence. It was therefore concluded that the isolated _VH gene was the correct 11-1F4 antibody gene sequence.

The variable region kappa light chain gene of the murine 11-1F4 antibody was also successfully cloned and sequenced, as was the non-functional _VK pseudogene first identified by Carroll et al. (Molecular Immunology (1988) 25:991). This sequence arises from an aberrant mRNA transcript present in all canonical fusion partners derived from the original MOPC-21 tumor (including SP2/0). As a result of the aberrant mRNA, the invariant cysteine at position 23 is replaced by a tyrosine residue, the VJ joint is out of frame, and a stop codon occurs at position 105.

It is common for lymphoid and hybridoma cells to synthesize two or more rearranged light immunoglobulin mRNAs. These mRNAs are usually non-productive due to the presence of stop codons or frameshifts that are not normally found in functional _VK genes. These false messengers often cause great problems when cloning immunoglobulin genes from hybridomas, because they are excellent substrates for V region PCR, despite the fact that they do not code for functional polypeptides.

The 11-1F4 antibody _VK gene sequence was identified after detailed sequence analysis of seven separate PCR clones isolated from two different PCR products, resulting in SEQ ID NO: 36. Since all sequences were identical, this was accepted as the correct 11-1F4 antibody kappa light chain variable region sequence.

The cloned V _H and V _K region genes were used to generate the chimeric mouse-human 11-1F4 monoclonal antibody, which was then analyzed to confirm specific binding to AL fibrils.

[Construction of chimeric mouse-human 11-1F4 (c11-1F4) antibody]
To allow for transient expression of the 11-1F4 _VH and _VK variable region genes described above in mammalian cells as part of a chimeric mouse-human antibody, it was necessary to modify the 5' and 3' ends using specifically designed PCR primers (Table 5). The 11-1F4 _VK gene was PCR modified using oligonucleotide primers F39836 and F39837, and the 11-1F4 _VH gene was PCR modified using primers F39835 and F58933. The reverse (BAK) primers F39836 and F39835 introduced a HindIII restriction site, a Kozak translation initiation site, and an immunoglobulin leader sequence at the 5' end of the _VK and _VH genes, respectively. The forward (FOR) oligonucleotide primer F39837 introduced a splice donor site and a BamHI restriction site at the 3' end of the _VK gene. The forward (FOR) oligonucleotide primer F58933 added the first 22 base pairs of the gamma-1 CH ₁ gene, including an ApaI restriction site, to the 3' end of the V _H gene.

The Kozak consensus sequence is essential for efficient translation of the variable region sequence (Kozak, J Mol Bio, 196:947). It defines the correct AUG codon at which the ribosome begins translation, with the most critical base being an adenine (or slightly more preferably a guanine) at position -3 upstream of the AUG start.

Immunoglobulin leader sequences allow for easy harvesting and purification to ensure that the expressed antibody is secreted into the medium. The leader sequences used in this example were the _VK and _VH leader sequences of mouse 11-1F4, which were cloned from hybridoma cDNA during the _VH and _VK cloning steps.

The splice donor sequence is important for attaching the light chain variable region correctly in frame to the appropriate constant region and splicing out the 130 bp _VK :C _K intron. The heavy chain variable region was attached directly to its appropriate constant region gene via an Apal site, eliminating the need for a splice donor site.

The subcloning restriction sites HindIII and BamHI, and HindIII and Apal, respectively, bracket the modified _VK and _VH variable region genes, and various unique restriction sites are used to ensure directional subcloning into an appropriate mammalian expression vector.

First, the 11-1F4 light chain variable region gene was carefully analyzed to identify undesirable splice donor sites, splice acceptor sites and Kozak sequences (see Table 6). Both the heavy and light chain variable region genes were analyzed for the presence of extraneous subcloning restriction sites that would later prevent subcloning and/or expression of a functional whole antibody. None were found.

For each variable region gene, a separate PCR reaction was set up as follows: Plasmids 11-1F4V _H . pCR2.1 and 11-1F4V _K . pCR2.1 described above were used as templates. 100 μl reaction mixture was prepared in each PCR tube. Each mixture contained up to 41 μl sterile water, 10 μl 10×PCR buffer I, 8 μl 10 mM stock solution of dNTPs, 1 μl 10 mM 5′ forward primer, 1 μl 10 mM 3′ reverse primer and 1 μl of 1/10 diluted template DNA. Finally, after adding 0.5 μl AmpliTaq® DNA polymerase (2.5 units), the completed reaction mixture was overlaid with 50 μl mineral oil. The reaction tubes were loaded into a DNA thermal cycler and cycled 25 times (after an initial melt at 94°C for 1 min) at 94°C for 30 s, 68°C for 30 s, and 72°C for 50 s. The last cycle was followed by a final extension step at 72°C for 7 min and cooling to 4°C. A 10 μl aliquot from each PCR reaction tube was run on a 1.2% (w/v) agarose/1×TBE buffer gel containing 0.5 μg/ml ethidium bromide to determine the size and presence of PCR products. The size of the positive PCR clones was approximately 420 bp. The putative positive PCR products thus identified were directly cloned into the pCR2.1 vector provided by the Topo TA Cloning® kit and transformed into TOP10 competent cells as described in the manufacturer's protocol. Colonies were PCR screened using 1212 and 1233 oligonucleotide primers (Table 3) according to the method of Gussow and Clackson to identify colonies containing plasmids with the correct size insert. The putative positive clones described above were subjected to double-stranded plasmid DNA sequencing using an ABI PRISM 310 Genetic Analyzer and ABI PRISM BigDye™ Terminators. Two positive clones of the _VH and _VK genes cloned into Topo TA were sequenced.

Clones containing the correctly modified 11-1F4 _{V H} and 11-1F4 V _K genes were identified, and the modified V genes from these clones were subcloned into the respective expression vectors to facilitate the expression of the chimeric heavy and kappa light chains in mammalian cells. The modified 11-1F4 V _K gene was subcloned as a HindIII-BamHI fragment into the expression vector pKN100 (Figure 4), which contains the human kappa constant region gene (allotype Km (3 Ala153, Ser191)). The modified 11-1F4 V _H gene was also subcloned as a HindIII-ApaI fragment into the expression vector pG1D200 (Figure 5), which contains the human gamma 1 constant region gene (allotype: G1m (-1 Glu377, Met38I, -2 Ala462, 3 Arg222, Ser229)). The kappa and gamma 1 constant region allotypes used are both commonly found in the Caucasian population. The ligated expression constructs 11-1F4VK.pKN100 and 11-1F4VH.pG1D200 were then used to transform DH5α competent cells and positive clones were identified by the PCR screening method described above using the original PCR modified primers (Table 4). Expression vectors are readily available.

[Construction of a single supervector for transient expression of chimeric 11-1F4 in COS cells]
A single super vector expressing both immunoglobulin chains of the chimeric 11-1F4 antibody was constructed as follows: the 11-1F4 kappa light chain expression cassette (containing the HCMVi promoter, the 11-1F4 kappa light chain variable region gene and the kappa light chain constant region gene) was restriction enzyme digested (EcoRI at positions 1 and 2490) from the 11-1F4VK.pKN100 construct (Figure 4) and then ligated into the 11-1F4VHpG1D200 construct via a unique EcoRI (position 4297, Figure 5). This ligation created the super vector construct pG1KD200-11-1F4, which contains both the heavy and kappa light chains of the 11-1F4 chimeric antibody.

Transient expression of chimeric γ1/κ.11-1F4 whole antibody in COS cells.
The chimeric 11-1F4 antibody was transiently expressed in COS cells from the European Collection of Cell Cultures (ECACC) in two ways:
(i) By co-transfection of 10 μg each of the vector constructs 11-1F4VK.pKN100 and 11-1F4VH.pG1D200. Co-transfections were performed in duplicate.
(ii) by transfection of 13 μg of the single supervector construct pG1KD200-11-1F4. Supervector transfections were performed five times.

The transfection method was as follows: COS cell lines were grown to confluence in 150 cm2 flasks in DMEM supplemented with 10% (v/v) FCS, 580 μg/ml L-glutamine, and 50 units/ml penicillin/50 μg/ml streptomycin (hereafter "medium"). Cells were trypsinized, spun down in a ^benchtop centrifuge (250 g for 5 min), then resuspended in 6 ml of medium before being split evenly into three 150 ^cm2 flasks each containing 25 ml of fresh pre-warmed medium. Cells were incubated overnight at 37°C in 5% _CO2 and harvested the following day while exponentially growing. Each flask contained approximately 1 x ¹⁰⁷ cells. Cells were trypsinized again, pelleted as before, washed with 20 ml of PBS, and then resuspended in enough PBS to give a cell concentration of 1 x ¹⁰⁷ cells/ml. 700 μl of washed COS cells were pipetted into a Gene Pulser® cuvette, to which 1 μl of both heavy and kappa light chain expression vector DNA (10 μg each) or 13 μg of supervector construct was then added. A Bio-Rad Gene Pulser® instrument was used to deliver a 1900V, 25 μF capacitance pulse to the mixture. The pulse was repeated for each experimental transfection and for the "no DNA" control (COS cells were electroporated in the absence of DNA). A positive control of previously expressed antibody was also performed to test the efficiency of COS cells.

COS cells were allowed to recover for 10 min at room temperature and then gently pipetted into a 10 cm diameter tissue culture dish containing 8 ml prewarmed DMEM supplemented with 10% (v/v) γ-globulin-free FBS, 580 μg/ml L-glutamine and 50 units/ml penicillin/50 μg/ml streptomycin and incubated for 72 h at 37° C. with 5% CO ₂ before COS cell supernatants were harvested for analysis. After 72 h incubation, the medium was collected, spun to remove cell debris and analyzed by ELISA for chimeric antibody production and antigen binding of the c11-1F4 antibody.

Quantification of chimeric γ1/κ11-1F4 antibodies via capture ELISA.
After expression, total IgG molecules present in the COS cell supernatant were quantified using a capture ELISA assay. IgG molecules were captured on a Nunc-Immuno MaxiSorb™ plate via immobilized goat anti-human IgG, Fcγ fragment specific antibody and detected via anti-human kappa light chain peroxidase conjugated antibody. A standard curve was generated by capturing and detecting standard IgG antibodies of known concentrations on the same plate in the same manner as follows. Each well of a 96-well (hole) immunoplate was coated with a 100 μl aliquot of 0.4 μg/ml goat anti-human IgG antibody diluted in PBS and incubated overnight at 4° C. Excess coating solution was removed and the plate was washed three times with 200 μl/well of wash buffer (1×PBS, 0.1% TWEEN). 100 μl of SEC buffer was dispensed into all wells except wells in row B-G of column 2. A 1 μg/ml solution of human IgG1/κ antibody in SEC buffer was prepared as a standard and 200 μl/well was pipetted into wells in rows B and C of column 2. The medium of the transfected cos cells was centrifuged (250 g, 5 min) and the supernatant was saved. A 200 μl aliquot of supernatant from the "no DNA" control (COS cells were transfected in the absence of DNA) was pipetted into wells in row D of column 2 and 200 μl/well aliquots of experimental supernatants were pipetted into wells in rows E, F and G of column 2. The 200 μl aliquots from wells in rows B-G of column 2 were mixed and then 100 μl was transferred from each well to the adjacent well in column 3. This process continued through column 11 with a series of two-fold dilutions of standards, controls and experimental samples, then all were incubated at 37°C for 1 hour and all wells were rinsed six times with 200 μl aliquots of wash buffer. Goat anti-human kappa light chain peroxidase conjugate was diluted 5000-fold in SEC buffer and 100 μl of diluted conjugate was added to each well and the incubation and rinsing steps were repeated. 150 μl of K-BLUE substrate was added to each well and incubated for 10 minutes at 25°C in the dark. 50 μl of RED STOP solution was added to each well to stop the reaction and the optical density was read at 655 nm.

Binding analysis of chimeric 11-1F4 antibody
The chimeric 11-1F4 antibody was tested for binding to amyloid fibrils using a direct binding ELISA assay. Synthetic fibrils were formed from immunoglobulin light chain proteins and used to monitor the reactivity of the antibodies in a solid-phase ELISA-based assay using "low-binding" polystyrene plates (Costar, #3474). Immediately before coating the plates, 250 μg of fibril mass was diluted to 1 ml with coating buffer (0.1% bovine serum albumin in phosphate buffered saline, pH 7.5). The sample was then sonicated for 20 seconds using the ultrasonic probe of a Tekmar Sonic Disruptor set at 40% of maximum power to obtain a solution of short fibrils, each consisting of up to 2-5 protofilaments. This solution was then diluted to 5 ml, mixed thoroughly by vortexing, and aliquoted into the wells of the plate. This process resulted in 50 μl of fibril solution in each well at a concentration of 50 μg/ml. The plates were then placed uncovered in a 37° C. incubator to dry overnight.

The ELISA assay was then performed within 48 hours of preparing the plates as follows: Wells were blocked by adding 100 μl of 1% BSA in PBS and incubated for 1 hour at room temperature on a shaker. The plates were washed 3 times with PBS, 0.05% Tween 20 (v/v). To each well of the plate, 50 μl of a solution of c11-1F4 (3 μg/ml antibody in 0.1% BSA/PBS) was added and the plates were incubated for 1 hour at room temperature on a shaker. The plates were washed 3 times (as before) and detection of bound antibody was performed using a biotinylated goat anti-mouse IgG antibody (Sigma no. B-8774, anti-heavy and light chain).

Sequence analysis of the successfully modified _VH and _VK genes revealed the presence of the correct sequences. The detailed DNA and amino acid sequences of the modified 11-1F4 _VK and _VH genes are shown in Figures 3 and 4. The modified _VK and _VH genes were successfully cloned into mammalian expression vectors pG1D200 and pKN100, respectively, and the resulting 11-1F4VK.pKN100 and 11-1F4VHpG1D200 constructs were used for co-transfection of mammalian cells.

The 11-1F4VK.pKN100 and 11-1F4VHpG1D200 constructs were then used to construct a single supervector (pG1KD200-11-1F4) expressing the chimeric 11-1F4 antibody in mammalian cells. The expression levels of the chimeric 11-1F4 antibody by cotransfection and supervector transfection of COS cells at ECACC were analyzed. The expression level observed from transfection of the pG1KD200-11-1F4 supervector (10326 ng/ml) was 3.7-fold higher than the level observed from the corresponding cotransfection of the 11-1F4VK.pKN100 and 11-1F4VHpG1D200 constructs (2820 ng/ml).

After expression and quantification, the chimeric 11-1F4 antibody was tested for binding to the target antigen (amyloid fibrils kindly provided by NCI) by direct binding ELISA. The results of the binding ELISA are shown in Figure 8. Supernatants from the two best individual pG1KD200-11-1F4 supervector transfections were analyzed in parallel with one supernatant from the corresponding cotransfection.

The results showed that the chimeric 11-1F4 antibody bound to amyloid fibrils with higher affinity than its murine antibody. This result was surprising and unexpected, as chimeric antibodies are typically expected to have binding affinities comparable to the original murine antibody. While not intending to be bound by a particular mechanism, the inventors believe that the net effect of combining the 11-1F4 murine V region with the human γ1/κC region used to generate the chimeric 11-1F4 antibody may have resulted in an antibody with higher affinity.

A sample of CHO cells (identified as CAEL-101) secreting one embodiment of the chimeric 11-1F4 monoclonal antibody used herein (which may be referred to in this embodiment as CAEL-101 or antibody CAEL-101) was deposited with the American Type Culture Collection (ATCC deposit number: PTA-125146) on June 27, 2018 pursuant to the Budapest Treaty.

Imaging human amyloid deposits in mice
Human amyloid extracts from heart (κ1), liver (κ1), spleen (λ1) and kidney (λ6) were kindly provided by Tufts University. Lyophilized human amyloid extracts were suspended in 25 ml of sterile PBS and homogenized for 3 min before centrifugation at 12,000 g for 30 min. 100 mg of the resulting pellet was resuspended in sterile saline. Amyloid extracts were subcutaneously injected into Balb/c mice.

[ ¹²⁴ I] CAEL-101:
cGMP grade CAEL-101 was radiolabeled with ^124I , a positron-emitting nuclide used for PET imaging, using the standard iodogen reaction (Fraker et al., Biochem Biophys Res Commun 80(4):849-57, and Markwell et al., Biochem 17:4807-17). Approximately 5 days after implantation of human amyloid extract to form subcutaneous amyloidomas, mice were injected with 50-200 μCi of [ ^124I ]CAEL-101 and imaging was performed on days 1, 4, and 7 post-injection using an Inveon microPET scanner. Regions of interest were drawn with the PMOD software package and tumor to background (amyloid deposit to background) ratios (T:B) were calculated on days 1 and 4 after tracer injection to obtain the SUVmax of the amyloidoma and contralateral background.

[ ¹²⁴ I]CAEL-101 successfully imaged 100% of mice bearing human amyloid extracts (K1, λ1 and λ6 subtypes from heart, liver, spleen and kidney). It also demonstrated for the first time imaging of amyloidosis from the heart, which was previously reported to be non-functional (Wall et al., Blood. 2010 116:2241) (FIG. 9). Human amyloidomas were visualized both at day 1 and day 4 after tracer injection, with a marked increase in T:B ratio at day 4. T:B ratios at day 4 ranged from 2.1 to 4.2. Heterogeneity of uptake was observed among the various amyloidomas. For example, mice transplanted with K subtypes had significantly better in vivo T:B ratios (4.1±0.20) compared to λ subtypes (2.8±0.46). However, all amyloidomas had a T:B uptake greater than 2.1, which is considered clinically significant.

Thus, the heterogeneity of uptake among various amyloidomas can be used to aid in the use of real-time PET imaging to classify patients for treatment with the chimeric or humanized antibodies described herein and to determine appropriate dosages of antibody therapy. That is, patient dosages can be determined based on the detected uptake of the labeled antibody or antibody fragment. Patients who exhibit strong uptake (affinity) for the labeled antibody or antibody fragment may require a smaller amount of therapeutic antibody or antibody fragment than patients who exhibit weak uptake (affinity) for the labeled antibody or antibody fragment. Thus, the present invention provides a method for determining an appropriate dosage of a humanized or chimeric antibody described herein for treatment. The method includes administering to a patient a labeled antibody or antibody fragment described herein, determining the uptake of the labeled antibody or antibody fragment by amyloid deposits in the patient, and administering to the patient a dose or series of doses of the chimeric or humanized antibody or antibody fragment based on the determined amount of the labeled antibody or antibody fragment.

[ ⁸⁹ Zr] CAEL-101:
Although [ ¹²⁴ I]CAEL-101 showed significant binding to amyloidoma, much of the activity detected was in the thyroid or not targeted (if the thyroid was blocked) due to dehalogenation of ^{the 124} I radiolabel. Therefore, we sought a method to radiolabel with a more stable radionuclide that could be imaged after unbound radiolabel had been cleared from the blood (after 7-10 days). Using two different strategies, we radiolabeled CAEL-101 with 89-zirconium ( ⁸⁹ Zr) to produce [ ⁸⁹ Zr]-CAEL-101. One strategy employs standard NCS linkers that are attached to random lysines on the antibody (Eur J Nucl Mol Imaging. 2010 Feb. 37(2):250-59, and Curr Radiopharm. 2011 Apr. 1;4(2):131-139). This method has been used in many clinical applications, but has been shown to generate multiple radioisomeric forms that could theoretically alter binding and biodistribution between batches. Therefore, applicants have radiolabeled CAEL-101 using a cysteine-targeted site-specific approach using a bifunctional deferoxamine-maleimide crosslinker (Nuclear Medicine and Biology, 37 (2010) 289-297). Approximately 5 days after subcutaneous implantation of human cardiac amyloid extract to form amyloidomas, animals were injected with 50-200 μCi of [ ⁸⁹ Zr]CAEL-101 and images were performed on days 1, 4, 7, 11, and 14 after injection using an Inveon microPET scanner.

Both variants of [ ⁸⁹ Zr]CAEL-101 were successfully used to image mice bearing cardiac amyloid extracts. Human amyloidomas were very well visualized throughout the experimental period (14 days after tracer injection) without any significant degradation of the radiotracer. This is in contrast to [ ¹²⁴ I]CAEL-101, which showed significant degradation at days 4 and 7. This extended imaging of amyloid deposits allows imaging with the labeled antibody after significant clearance of unbound labeled antibody from the blood. This improves the tumor-to-background (amyloid deposits to background) ratio. This plays an important role in imaging diseases in organs with many adjacent blood compartments, such as cardiac amyloid deposits. Amyloidomas were observed even 14 days after tracer injection, indicating the long-term stability of the antibody-bound components in vivo. We were again able to demonstrate imaging of amyloidosis originating from the heart, which was reported to be non-functional in the prior literature. We also observed ipsilateral uptake in lymphatic tissues for the first time. This may be due to the transplantation of amyloidomas into immunodeficient mice, which results in the migration of amyloid in phagocytosed immune cells to the draining nodes. In this model, the presence of human amyloid results in spontaneous amyloid clearance. This new observation may provide the basis for imaging responses to therapy by examining lymph node uptake of PET tracers.

The words "comprise", "comprises", "comprising" and variations of such words used in the description and claims of this specification are not intended to exclude other features, additives, ingredients, integers or steps unless expressly stated. The scope of these words is to be interpreted broadly to have an inclusive rather than exclusive meaning.

The compositions and methods of the present invention have been described herein with reference to examples. However, it will be understood that the present invention is not limited thereto, and that various modifications are possible as known to those skilled in the art without departing from the teachings of the present invention as defined by the appended claims.

Claims (16)

1. A system for detecting the presence, location and/or amount of amyloid deposits in the heart, comprising:
a) an antibody linked to a detectable molecule;
b) an imaging device that detects the presence, location and/or amount of said amyloid deposits;
The antibody of a) comprises a variable heavy chain (VH) having SEQ ID NO: 48 and a variable light chain (VK) having SEQ ID NO: 47;
Here, the system uses the imaging device of b) to detect the presence, location and/or amount of amyloid deposits in the heart by detecting a detectable molecule linked to the antibody of a) bound to the amyloid deposits. The system of claim 1, wherein the antibody is a chimeric mouse-human antibody. The system of claim 1, wherein the imaging device is a positron emission tomography (PET) scanner. The system of claim 3 , wherein the detectable label of the detectable molecule is ¹²⁴ I. The system of claim 3 , wherein the detectable label of the detectable molecule is ⁸⁹ Zr. The system of claim 1, comprising an antibody having a higher affinity than an antibody comprising a VH having SEQ ID NO: 35 and a VK having SEQ ID NO: 36. 1. A system for determining efficacy of removal of amyloid deposits in a patient's heart, comprising:
a) a chimeric antibody for removing amyloid deposits, comprising a variable heavy chain (VH) having SEQ ID NO: 48, a variable light chain (VK) having SEQ ID NO: 47, and a constant region derived from human IgG1;
b) a composition comprising an antibody linked to a detectable molecule;
c) an imaging device for detecting the presence, location and/or amount of said amyloid deposits;
The antibody of b) comprises a variable heavy chain (VH) having SEQ ID NO: 48 and a variable light chain (VK) having SEQ ID NO: 47;
wherein said imaging device is utilized to detect the presence, location and/or amount of said amyloid deposits in the heart by detecting the detectable molecule bound to said detectable molecule-linked antibody of said b) that is bound to amyloid deposits before and after administration of said chimeric antibody of said a).
A system for determining the effect of removing the amyloid deposits in the heart by measuring the reduction in the amount of a detectable molecule linked to the antibody contained in the composition of (b). The system of claim 7, wherein the imaging device is a positron emission tomography (PET) scanner. The system of claim 7 , wherein the detectable label of the detectable molecule is ¹²⁴ I. The system of claim 7 , wherein the detectable label of the detectable molecule is ⁸⁹ Zr. The system of claim 7 , comprising a chimeric antibody having a higher affinity than an antibody comprising a VH having sequence number 35 and a VK having sequence number 36. 1. A system for monitoring disease progression of amyloid deposits in the heart, comprising:
a) a composition comprising an antibody linked to a detectable molecule;
b) an imaging device for detecting the presence, location and/or amount of said amyloid deposits at the first and second or subsequent times;
and c) a comparison means for comparing the presence, location, and/or amount of amyloid deposits detected at the first detection with the presence, location, and/or amount of amyloid deposits detected at a second or subsequent detection. The antibody of a) comprises a variable heavy chain (VH) having SEQ ID NO: 48 and a variable light chain (VK) having SEQ ID NO: 47.
Here, a system for monitoring disease progression of amyloid deposits in the heart by detecting the presence, location and/or amount of amyloid deposits in the heart by utilizing the imaging device of b) to detect a detectable molecule linked to the antibody of a) bound to the amyloid deposits at different time points. The system of claim 12, wherein the imaging device is a positron emission tomography (PET) scanner. The system of claim 12 , wherein the detectable label of the detectable molecule is ¹²⁴ I. The system of claim 12 , wherein the detectable label of the detectable molecule is ⁸⁹ Zr. The system of claim 12, comprising an antibody having a higher affinity than an antibody comprising a VH having SEQ ID NO: 35 and a VK having SEQ ID NO: 36.