Antibody compositions with fc mutations and site-specific conjugation properties

Abstract

To provide antibody compositions with Fc mutations and site-specific conjugation properties.SOLUTION: Disclosed herein is an antibody that binds to Her2, EGFR, Trop2, CDH3, or other TAA containing a triple mutation at L234A, L235A and L328C, and a method for preparing such a triple mutated antibody. Consequently, the triple mutated antibody contains a modified effector function through Fc silencing and is capable of site-specific conjugation at L328C to form an antibody-drug-conjugate (ADC), which can be administered to a patient and can provide a method of treating cancer, immunological and neurological disorders.SELECTED DRAWING: Figure 8

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 973,475, filed Oct. 4, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Submission of Sequence Listing The content of the following paper copy submission is hereby incorporated by reference in its entirety: Paper copy of the sequence listing recorded on Oct. 2, 2020.

[0003] Statement of Rights to Inventions Made Under Federally Sponsored Research Not applicable.

[0004] Field of the Invention The invention described herein relates to antibodies engineered to include multiple functional properties including Fc silencing and site - specific conjugation, antigen - binding fragments thereof, antibody - drug conjugates (ADCs), and antibody - boron conjugates (ABCs). The invention further relates to prognostic, prophylactic, and therapeutic methods and compositions useful for the treatment of cancer and immunological and neurological disorders.

Background Art

[0005] Background of the Invention Cancer is the second most common cause of death worldwide after coronary artery disease. Millions of people die from cancer every year, and in the United States alone, more than half a million people die from cancer every year, with 1,688,780 new cancer cases diagnosed in 2017 (American Cancer Society). Deaths from heart disease have decreased significantly, while deaths from cancer generally have increased. By 2040, there will be more than 16 million cancer deaths worldwide (source: International Agency for Research on Cancer, 2018), and thus, it is estimated that cancer will overtake heart disease as the leading cause of death unless medical developments change current trends.

[0006] Some cancers stand out as having high mortality rates. In particular, lung cancer (18.4% of all cancer deaths), breast cancer (6.6% of all cancer deaths), colorectal cancer (9.2% of all cancer deaths), liver cancer (8.2% of all cancer deaths), and stomach cancer (8.2% of all cancer deaths) represent the leading causes of cancer death in both men and women of all ages worldwide (GLOBOCAN 2018). These and virtually all other cancers share the common lethal feature that they metastasize to sites distant from their primary tumors, with few exceptions. Furthermore, even cancer patients who initially survive their primary cancer generally experience a dramatic change in their lives. Many cancer patients experience intense anxiety that stems from the recognition of the possibility of recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Additionally, many cancer patients experience a recurrence of their disease.

[0007] Cancer treatment has improved over the past several decades and survival rates have increased, but the heterogeneity of cancer still requires new treatment strategies that utilize multiple treatment modalities. This is particularly true for the treatment of solid tumors (e.g., glioblastoma, head and neck squamous cell carcinoma, and lung adenocarcinoma) in anatomically critical sites that are sometimes limited to standard radiotherapy and / or chemotherapy. Nevertheless, the adverse effects of these therapies are chemo-resistance and radio-resistance that promote local-regional recurrence, distant metastasis, and second primary tumors, in addition to severe side effects that reduce the quality of life of patients.

[0008] In the fight against cancer and other medical conditions, the therapeutic utility of monoclonal antibodies (mAbs) (G. KOHLER and C. MILSTEIN, Nature 256:495-497 (1975)) is being realized. mAbs are currently approved as therapies in transplantation, cancer, infectious diseases, cardiovascular diseases and inflammation. Different isotypes have different effector functions. Such functional differences are reflected in the different three-dimensional structures of the various immunoglobulin isotypes (P.M. ALZARI et al., Annual Rev. Immunol., 6:555-580 (1988)).

[0009] In general, antibodies act by several mechanisms, most of which involve other arms of the immune system. Antibodies can simply block molecular interactions or activate the classical complement pathway (known as complement-dependent cytotoxicity or CDC) by the interaction of C1q on the C1 complex with clustered antibodies. Importantly, antibodies also act as a link between antibody-mediated and cell-mediated immune responses through the involvement of Fc receptors.

[0010] The Fc engineering approach has been used to determine the key interaction sites of the Fc domain with Fc gamma receptors and C1q, and then to mutate these positions to reduce or abolish binding in order to modulate effector functions and improve therapeutic properties such as reduction of toxicity. See HEZAREH et al., J. Virol. 75(24):12161-8 (Dec. 2001) and OGANESYAN et al., Acta Crystallographica. D. Biol Crystallogr, 64:(Pt.6):700-4 (Jun 2008).

[0011] Furthermore, antibody-drug conjugates (ADCs) are a new class of targeted therapeutics with an improved therapeutic index compared to conventional chemotherapy. Drugs and linkers have become the focus of ADC development, in addition to (monoclonal) antibodies (mAbs) and target selection. However, recently, the importance of conjugate homogeneity has been investigated. By applying site-specific conjugation techniques that utilize engineered surface-exposed cysteine residues on antibodies, the pharmacological profile of ADCs can be improved, which are then conjugated to linker drugs, and as a result, it has been reported that site-specific conjugate ADCs with a defined drug-to-antibody ratio (DAR) can be obtained. Compared to heterogeneous mixtures produced using conventional lysine and cysteine conjugation methodologies, site-specifically conjugated ADCs generally showed at least equivalent in vivo efficacy, improved PK, and an expanded therapeutic window.

[0012] The prior art discloses several approaches for obtaining site-specific conjugation and the resulting ADCs. See, for example, International Publication No. WO 2006 / 034488 (Genentech), Sutherland et al., Blood 122(8):1455-1463 (2013), International Publication No. WO 2014 / 124316 (Novartis), and U.S. Patent Application Publication No. 2017 / 0080103 (Synthon Biopharmaceuticals).

[0013] In all of the prior art methods disclosed to date, the focus has been on positions on the surface / solvent-exposed, positions showing high thiol reactivity, and positions in the specific constant regions of monoclonal antibodies for conjugating linker drugs for the purpose of improving homogeneity and pharmacokinetic properties.

[0014] The above-described conventional lysine and cysteine conjugation methods have led to FDA-approved antibody-drug conjugates, which are being used to construct the majority of the numerous ADCs in current preclinical and clinical trials. However, there is still a need for new conjugation strategies aimed at (further) improving the physicochemical, pharmacokinetic, pharmacological, and / or toxicological properties of ADCs in order to obtain ADCs with acceptable antigen-binding properties, in vivo efficacy, therapeutic index, and / or stability.

[0015] From the above, it is readily apparent to those skilled in the art that new treatment paradigms are needed in the treatment of cancer and immunological diseases. By using the latest antibody engineering techniques and new conjugation methodologies, new classes of antibodies can be achieved with the overall goals of more effective treatment, reduction of side effects, and reduction of production costs.

[0016] In view of the current deficiencies known in the art, it is an object of the present invention to provide novel and improved antibodies and binding ligands, as well as methods for treating cancer(s), immune disorders, and other diseases, by utilizing antibodies engineered with triple mutations that reduce antibody effector function and contain site-specific conjugation points.

Prior Art Documents

Patent Documents

[0017]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0018]

Non-Patent Document 1

[0019] The present invention provides antibodies, antigen-binding fragments, antibody-drug conjugates (ADCs), antibody-immunomodifying conjugates, and antibody-boron conjugates (ABCs) that bind to proteins including but not limited to Her2, EGFR, Trop2, CDH3, and polypeptide fragments of proteins including but not limited to Her2, EGFR, Trop2, CDH3, and. In some embodiments, the present invention includes fully human antibodies conjugated to a therapeutic agent. In some embodiments, the present invention includes fully human antibodies conjugated to a borylated compound. In some embodiments, the antibody is engineered to reduce effector function by Fc silencing. In some embodiments, the antibody contains a site-specific mutation that can be conjugated to a drug moiety. In some embodiments, the antibody contains a triple mutation in which the Fc is silenced and a site-specific mutation is inserted for conjugation to a drug moiety. In a further embodiment, the triple mutation includes L234A, L235A, L328C.

[0020] The present invention further provides various immunogenic or therapeutic compositions such as antibodies, antibody-drug conjugates, and strategies for treating cancers that express other tumor-associated antigens (TAAs) such as Her2, EGFR, Trop2, CDH3, and those listed in Table IV.

[0021] In another embodiment, the present disclosure teaches a method for synthesizing a triple mutant antibody.

[0022] In another embodiment, the present disclosure teaches a method for synthesizing a triple mutant antibody and conjugating a drug moiety at a site-specific position thereon.

[0023] In another embodiment, the present disclosure teaches a method for synthesizing a single mutant antibody.

[0024] In another embodiment, the present disclosure teaches a method for synthesizing a single mutant antibody and conjugating a drug moiety at a site-specific position thereon.

[0025] In another embodiment, the present disclosure teaches a method for treating human cancer(s), immunological disorders, and other diseases. In certain embodiments, for example, the following are provided: (Item 1) An antibody composition comprising a triple mutation, wherein the triple mutation comprises an L234A modification, an L235A modification, and an L328C modification, and the triple mutation modifies Fcγ receptor binding and antibody effector function. (Item 2) The antibody according to Item 1, wherein the antibody comprises an EGFR antibody. (Item 3) The antibody according to Item 1, wherein the antibody comprises a Her2 antibody. (Item 4) The antibody according to Item 1, wherein the antibody comprises a Trop2 antibody. (Item 5) The antibody according to Item 1, wherein the antibody comprises a CDH3 antibody. (Item 6) The antibody according to item 1, wherein the antibody comprises a tumor-associated antigen (TAA) antibody. (Item 7) The antibody according to item 6, wherein the TAA is as shown in Table IV. (Item 8) An antibody-drug conjugate (ADC) comprising: (i) An antibody composition comprising a triple mutation, wherein the triple mutation comprises L234A modification, L235A modification and L328C modification, and the triple mutation modifies the antibody effector function; (ii) A linker; (iii) A drug unit, wherein the drug unit is specifically conjugated at site L328C. (Item 8) The ADC according to item 8, wherein the antibody composition comprises an EGFR antibody. (Item 9) The ADC according to item 8, wherein the antibody composition comprises a Her2 antibody. (Item 10) The ADC according to item 8, wherein the antibody composition comprises a Trop2 antibody. (Item 11) The antibody according to item 8, wherein the antibody comprises a CDH3 antibody. (Item 12) The ADC according to item 8, wherein the antibody composition comprises a tumor-associated antigen (TAA) antibody. (Item 13) The antibody according to item 12, wherein the TAA is as shown in Table IV. (Item 14) The ADC according to item 8, further comprising a stretcher unit. (Item 15) The ADC according to item 8, further comprising a spacer unit. (Item 16) The ADC according to item 8, further comprising an amino acid unit. (Item 17) A manufactured product comprising the antibody according to item 1. (Item 18) A manufactured product comprising the ADC according to item 8. (Item 19) A pharmaceutical composition comprising an ADC according to item 8 in a therapeutically effective amount and a pharmaceutically acceptable excipient. (Item 20) A pharmaceutical composition comprising an antibody according to item 1 in a therapeutically effective amount and a pharmaceutically acceptable excipient. (Item 21) An antibody-boron-conjugate (ABC), comprising: (i) An antibody composition comprising a triple mutation, wherein the triple mutation comprises L234A modification, L235A modification and L328C modification, and the triple mutation modifies the antibody effector function; (ii) A linker; (iii) A drug unit, wherein the drug unit comprises a borylated composition and the drug unit is specifically conjugated at site L328C. (Item 22) The ABC according to item 21, wherein the antibody composition comprises an EGFR antibody. (Item 23) The ABC according to item 21, wherein the antibody composition comprises a Her2 antibody. (Item 24) The ABC according to item 21, wherein the antibody composition comprises a Trop2 antibody. (Item 25) The ABC according to item 21, wherein the antibody composition comprises a CDH3 antibody. (Item 26) The ABC according to item 21, wherein the antibody composition comprises a tumor-associated antigen (TAA) antibody. (Item 27) The antibody according to item 26, wherein the TAA is shown in Table IV. (Item 28) The ABC according to item 21, further comprising a stretcher unit. (Item 29) The ABC according to item 21, further comprising a spacer unit. (Item 30) The ABC according to item 21, further comprising an amino acid unit. (Item 31) A pharmaceutical composition comprising a therapeutically effective amount of ABC described in Item 21 and a pharmaceutically acceptable excipient. (Item 32) A method of treating cancer in an individual, (i) comprising administering to the individual a therapeutically effective amount of ABC described in Item 21, wherein the cancer comprises cells expressing the cancer shown in Table I.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0087] Detailed Description of the Invention Summary of Sections I.) Definitions II.) Antibodies III.) Fc Mutations that Modify Effector Functions IV.) Antibody-Drug-Conjugates V.) Site-Specific Conjugation Formats for ADCs VI.) Linker Units VII.) Stretcher Units VIII.) Amino Acid Units IX.) Spacer Units X.) Drug Units XI.) Methods for Determining the Cytotoxic Effects of ADCs XII.) Treatment of cancers (multiple) expressing Her2, EGFR, Trop2, CDH3, and TAA XIII.) Combination therapy XIV.) Kit / manufactured product

[0088] I.) Definitions Unless otherwise defined, all technical terms, notations, and other scientific or specialized terms used herein shall, unless the context clearly indicates otherwise, be intended to have the meaning commonly understood by those of ordinary skill in the art to which the present invention pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and including such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0089] When a trade name is used herein, reference to the trade name shall, unless otherwise indicated by the context, also refer to the product formulation, generic pharmaceuticals, and pharmaceutical active ingredient(s) of the trade name product.

[0090] The terms "advanced cancer", "locally advanced cancer", "advanced disease", and "locally advanced disease" mean cancer that has spread through the associated tissue capsule, and include stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients with clinically localized (organ-confined) cancer.

[0091] The term "substituted" means that a particular group or moiety has one or more substituents. The term "unsubstituted" means that a particular group has no substituents. The term "optionally substituted" means that a particular group is unsubstituted or is substituted by one or more substituents. When the term "substituted" is used to describe a structural system, substitution is meant to occur at any valence-allowable position on the system.

[0092] The term "antibody" is used in the broadest sense unless otherwise specified. Thus, an "antibody" can be naturally occurring or artificial such as monoclonal antibodies produced by conventional hybridoma or transgenic mouse technology. Her2, EGFR, Trop2, CDH3, and / or TAA antibodies include monoclonal and polyclonal antibodies, as well as fragments thereof that contain the antigen-binding domain of these antibodies and / or one or more complementarity-determining regions. As used herein, the term "antibody" specifically binds to Her2, EGFR, Trop2, CDH3, and / or any TAA, and / or exhibits the desired biological activity, and refers to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and any form of antibody or fragment thereof that specifically encompasses antibody fragments as long as they specifically bind to Her2, EGFR, Trop2, CDH3 and / or exhibit the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one embodiment, the term "antibody" encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that together form a specific binding site for a target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. Antibodies useful in the present methods and compositions can be produced in cell culture, in phage, in yeast, or in various animals including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Thus, in one embodiment, the antibodies of the present invention are mammalian antibodies. Phage technology can be used to isolate initial antibodies or generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art.For example, recombinant antibodies can be produced by transfecting a host cell with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence that expresses at least one VL and at least one VH region in the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shepard et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, latest edition). The antibodies of the present invention can be modified by recombinant means to enhance the effectiveness of the antibody in mediating the desired function. Accordingly, it is within the scope of the present invention that the antibody can be modified by substitution using recombinant means. Typically, the substitution is a conservative substitution. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, International Publication No. 9958572, and Angal et al., Mol. Immunol. 30: 105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Often, antibodies are labeled by covalently or non-covalently connecting a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known and widely reported in both scientific and patent literature. These antibodies can be screened for binding to normal or defective FLT3.For example, see ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies having the desired biological activity can be identified using the following in vitro assays, including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and the following in vivo assays such as inhibition of tumor growth. The antibodies provided herein may also be useful in diagnostic applications. As capture antibodies or non-neutralizing antibodies, they can be screened for the ability to bind specifically to an antigen without inhibiting antigen receptor binding or biological activity. As neutralizing antibodies, the antibodies can be useful in competitive binding assays. They can also be used to quantify Her2, EGFR, Trop2 and / or CDH3 or its receptor.

[0093] As used herein, the term "antigen-binding fragment" or "antibody fragment" (or simply "antibody portion") of an antibody refers to one or more fragments of a Her2, EGFR, Trop2, CDH3, and / or TAA antibody that retain the ability to specifically bind to an antigen (e.g., Her2, EGFR, Trop2, CDH3 and / or variant). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody are: (i) a Fab fragment, which is a monovalent fragment consisting of the V L 、V H 、C L and C H1 domains; (ii) an F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the V H and C H1 domains; (iv) an Fv fragment consisting of the V L and V H domains of a single arm of the antibody; (v) VH dAb fragments consisting of domains (WARD et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining regions (CDRs). Further, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but they can be linked by a synthetic linker that enables the V L and V H regions to pair and form a monovalent molecule known as a single-chain Fv (scFv) as a single protein chain. See, for example, BIRD et al., (1988) Science 242:423-426; and HUSTON et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0094] As used herein, the term "Fc" refers to a region that includes the hinge region, CH2 and / or CH3 domains.

[0095] As used herein, any form of "antigen" can be used to generate antibodies specific for Her2, EGFR, Trop2, CDH3, and / or any TAA of the present invention. Thus, the immunizing antigen may be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunizing antigen can be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein). The antigen can be produced in genetically modified cells. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" in the context of an antigen refers, as appropriate, to the minimum number of amino acids or nucleic acids necessary to constitute the immunogenic epitope of the antigen of interest. Without limitation, any gene vector suitable for transformation of the cell of interest can be used, including but not limited to adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids. In one embodiment, the antibodies of the methods and compositions herein specifically bind to at least a portion of the extracellular domain of the target of interest.

[0096] The antibodies or antigen-binding fragments thereof provided herein can constitute or be part of a "bioactive agent". As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds to an antigen and / or enhances or mediates a desired biological effect to enhance a cell-killing toxin. In one embodiment, the binding fragments useful in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that can bind to a desired antigen epitope and directly or indirectly exert a biological effect. Direct effects include, but are not limited to, modulation, stimulation and / or inhibition of growth signals, modulation, stimulation and / or inhibition of anti-apoptosis signals, modulation, stimulation and / or inhibition of apoptosis or necrosis signals, modulation, stimulation and / or inhibition of the ADCC cascade, and modulation, stimulation and / or inhibition of the CDC cascade and / or Fc silencing.

[0097] As used herein with respect to antigen binding, the term "specifically binds to" a protein means that an antigen-binding protein binds to a target and to distinct domains or distinct amino acid sequences within the target, but does not bind, or binds only slightly, to other (e.g., unrelated) proteins. However, this term does not exclude the fact that an antibody or its binding fragment may be cross-reactive with molecules that are closely related. The antibodies and fragments thereof described herein, and antibody-drug conjugates containing them, can specifically bind to Her2, EGFR, Trop2, CDH3, and / or TAAs disclosed herein with an affinity that is at least 2, 5, 10, 50, 100, or 1000 times higher than binding to closely related molecules.

[0098] "Bispecific" antibodies are also useful in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigen epitopes. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitopes are derived from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using co-expression of two immunoglobulin heavy chain / light chain pairs. See, e.g., Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkages. See, e.g., Brennan et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993); Gruber et al., J. Immunol. 152:5368 (1994).

[0099] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which part of the heavy and / or light chain is identical or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion(s) of the chain(s) is derived from an antibody of another species or belonging to another antibody class or subclass, as long as they specifically bind to the target antigen and / or exhibit the desired biological activity, and is identical or homologous to the corresponding sequences in fragments of such antibodies (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0100] As used herein, the terms “cancer,” “neoplasm,” and “tumor” are used interchangeably and refer to cells that have undergone malignant transformation that is pathological to the host organism, either in the singular or plural form. Primary cancer cells (i.e., cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells used herein includes not only primary cancer cells but also any cells derived from the ancestors of cancer cells. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically present as solid tumors, a “clinically detectable” tumor is one that is detectable based on a tumor mass. For example, it is detectable by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and / or due to the expression of one or more cancer-specific antigens in a sample obtained from the patient. The tumor may be a hematopoietic tumor, such as a tumor of blood cells, and means a liquid tumor. Specific examples of clinical conditions based on such tumors include myelomas such as chronic myelogenous leukemia, acute myelogenous leukemia, multiple myeloma, and lymphomas.

[0101] The term “therapeutic agent” refers to all agents that provide a therapeutic benefit and / or are therapeutically effective as defined herein. A therapeutic agent can, for example, reverse, remit, alleviate, inhibit, or limit the progression of, or reduce the severity of, a disease, disorder, or condition, or affect, improve, or remit one or more symptoms of a disease such as cancer. Such agents can be cytotoxic or cytostatic. The term includes, but is not limited to, chemotherapeutic agents, anti-neoplastic agents, and “drug unit” agents as defined herein.

[0102] The term “anti-neoplastic agent” refers to all agents that provide a therapeutic benefit and / or are therapeutically effective as defined herein in the treatment of neoplasms or cancers.

[0103] The term "chemotherapeutic agent" refers to all chemical compounds that are effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents such as nitrogen mustard, ethyleneimine compounds, and alkyl sulfonates; antimetabolites such as folic acid, purine, or pyrimidine antagonists; mitotic inhibitors such as antitubulin agents like vinca alkaloids, auristatins, and derivatives of podophyllotoxin; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication such as DNA minor groove binders; and growth factor receptor antagonists. Further, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.

[0104] The terms "complementary determining region" and "CDR" are known in the art to refer to discontinuous sequences of amino acids within the antibody variable regions that confer antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3) and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3).

[0105] The exact amino acid sequence boundaries of a given CDR can be readily determined using any of several well-known schemes, including those described in Kabat et al., (1991), ‘‘Sequences of Proteins of Immunological Interest,’’ 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (the ‘‘Kabat’’ numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the ‘‘Chothia’’ numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), ‘‘Antibody-antigen interactions: Contact analysis and binding site topography,’’ J. Mol. Biol. 262, 732-745. ‘‘(Contact’’ numbering scheme), Lefranc M.P. et al., ‘‘IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,’’ Dev Comp Immunol, 2003 January;27(1):55-77 (the ‘‘IMGT’’ numbering scheme), and Honegger A. and Plückthun A., ‘‘Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,’’ J Mol Biol, 2001 Jun. 8;309(3):657-70 (the AHo numbering scheme).

[0106] The boundaries of a given CDR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, and insertions are provided by inserted letters, such as "30a", and deletions appear in some antibodies. The two schemes place specific insertions and deletions ("indels") at different positions, resulting in different numberings. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0107] Accordingly, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof, such as a variable region, and the individual CDRs of an antibody or region thereof (e.g., "CDR-H1, CDR-H2) are to be understood to encompass complementary determining regions as defined by any of the known schemes described in the present specification above. In some cases, a scheme for the identification of a particular one or more CDRs, e.g., CDRs defined by the Kabat, Chothia, or Contact methods, is specified.

[0108] As used herein, the term "conservative substitution" is known to those of skill in the art and generally refers to substitutions of amino acids and / or amino acid sequences that can be made without changing the biological activity of the resulting molecule. Those of skill in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, e.g., WATSON et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made according to those shown in Tables II and III. For example, such changes include substituting any one of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; substituting glutamic acid (E) with aspartic acid (D) and vice versa; substituting asparagine (N) with glutamine (Q) and vice versa; and substituting threonine (T) with serine (S) and vice versa. Depending on the environment of a particular amino acid and its role in the three-dimensional structure of the protein, other substitutions can be considered conservative. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). The relatively hydrophobic methionine (M) is often exchanged with leucine and isoleucine and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable at positions where the important feature of the amino acid residue is its charge and the different pKs of these two amino acid residues are not important. Still other changes can be considered "conservative" in a particular environment (see, e.g., Table III herein; pages 13-15 ‘‘Biochemistry’’ 2nd ED. Lubert Stryer ed (Stanford University); HENIKOFF et al., PNAS 1992 Vol 89 10915-10919; LEI et al., J Biol Chem 1995 May 19; 270(20):11882-6).) Other substitutions are also tolerated and can be determined empirically or according to known conservative substitutions.

[0109] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity and function of cells and / or causes cell destruction. This term is intended to include radioisotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof). Examples of cytotoxic agents include auristatin, auroomycin, maytansinoid, ricin, ricin A chain, combrestatin, duocarmycin, dostaratin, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracinedione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogelin, restrictocin, phenomycin, enomycin, curcin, crotonin, calicheamicin, Sapaonaria officinalis inhibitor and glucocorticoid and other chemotherapeutic agents and At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 or 213 、P 32 radioisotopes such as, and Lu 177 radioisotopes of Lu containing Lu are included, but not limited to these.

[0110] Antibodies comprising the antibody of the present invention may also be conjugated to any of the above cytotoxic agents and also to an anti-cancer prodrug activating enzyme capable of converting a prodrug into its active form.

[0111] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable domain (V H -V L ) bound to a light chain variable domain (V L ) in the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain to create two antigen-binding sites. Diabodies are well described, for example, in European Patent No. 404,097, International Publication No. 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

[0112] The term "homologue" refers to a molecule that shows homology to another molecule, for example, by having the same or similar sequence of chemical residues at corresponding positions.

[0113] The term "identical" or "sequence identity" refers to the degree of identity between two nucleic acid sequences or between two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

[0114] The "percent identity" between two arrays is a function of the number of identical positions shared by the arrays (i.e., % identity = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap. Comparison of arrays and determination of the percent identity between two arrays can be achieved using a mathematical algorithm, as described below. The percent identity between two nucleotide sequences can be determined using the GAP program of the GCG software package using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide sequences or between amino acid sequences can also be determined using the algorithm of Meyers et al., Comput. Appi. Biosci., 4:11-17 (1988), incorporated in the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman et al., J. Mol. Biol. 48:444-453 (1970), incorporated in the GAP program of the GCG software package, using the Blossum62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0115] By way of example, a polynucleotide sequence may be identical to a reference polynucleotide sequence that is 100% identical to a reference sequence, or may contain up to a certain integer number of nucleotide changes compared to the reference sequence, such as at least 50, 60, 70, 75, 80, 85, 90, 95, 98 or 99% identity. Such changes are selected from at least one nucleotide deletion, substitution (including transitions and transversions), or insertion, and the changes may occur at the 5' or 3' end positions of the reference nucleotide sequence, or anywhere between those end positions, and may be individually between nucleotides in the reference sequence, or may be scattered among 1 or more consecutive bases within the reference sequence. The number of nucleotide changes is determined by multiplying the total number of nucleotides in the reference polynucleotide sequence as described herein by the numerical percentage of each percent identity (divided by 100), and subtracting that product from the total number of nucleotides in the reference polynucleotide sequence, or, n n ≤ x n -(x ny ) as determined by the formula, where n n is the number of nucleotide changes, x n is the total number of nucleotides in the reference polynucleotide sequence described herein (see the nucleic acid sequences in the "Sequence Listing" for exemplary reference polynucleotide sequences), y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, and is the symbol for the multiplication operator, and any non-integer product of x n and y is x nRound down to the nearest integer before subtracting it therefrom. Similarly, a polypeptide sequence may be identical to a polypeptide reference sequence described herein, i.e., 100% identical, or may contain amino acid changes up to a certain integer as compared to the reference sequence such that the % identity is less than 100%, e.g., at least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identical. Such changes are selected from the group consisting of at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, and said changes may occur at the amino-terminal position or carboxy-terminal position of the reference polypeptide sequence, or anywhere between those terminal positions, and may occur individually between amino acids in the reference sequence or may be scattered among any of 1 or more consecutive groups within the reference sequence. The number of amino acid changes for a given % identity is determined by multiplying the total number of amino acids in the polypeptide sequence encoded by the polypeptide reference sequence by the numerical percentage of each percent identity (divided by 100), and then subtracting that product from the total number of amino acids in the polypeptide reference sequence described herein or: n a ≦x a -(x ay ) as determined by the formula, wherein n a is the number of amino acid changes, x a is the total number of amino acids in the reference polypeptide sequence, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, and is the symbol for the multiplication operator, wherein any non-integer product of x a and y is rounded down to the nearest integer before subtracting it from x a . Percent identity can be determined over the length of the sequence. As defined herein, the term "more than 75% identical" includes identities greater than 75%, 80%, 85%, 95% and 99%, as well as all discrete values and discrete sub-ranges within this range.

[0116] In one embodiment, the antibodies provided herein are "human antibodies." As used herein, the term "human antibody" refers to an antibody in which essentially the entire sequences of the light and heavy chain sequences, including the complementarity determining regions (CDRs), are derived from human genes. In one embodiment, human monoclonal antibodies are prepared by the trioma technique, human B-cell technology (see, e.g., KOZBOR et al., Immunol. Today 4:72 (1983), EBV transformation technique (see, e.g., COLE et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)), or using phage display (see, e.g., MARKS et al., J. Mol. Biol. 222:581 (1991)). In a specific embodiment, human antibodies are produced in transgenic mice. Techniques for making such partial to fully human antibodies are known in the art and any such technique can be used. According to a particularly preferred embodiment, the fully human antibody sequences are prepared in transgenic mice engineered to express human heavy and light chain antibody genes. Exemplary descriptions of preparing transgenic mice for making human antibodies can be found in International Publication No. WO 02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix) and its progeny. B cells from the transgenic mice that produce the desired antibody can then be fused to generate a hybridoma cell line for continuous production of the antibody. See, e.g., U.S. Patent Nos. 5,569,825, 5,625,126, 5,633,425, 5,661,016, and 5,545,806, as well as JAKOBOVITS, Adv. Drug Del. Rev. 31:33-42 (1998), GREEN et al., J. Exp. Med. 188:483-95 (1998).

[0117] As used herein, the term "humanized antibody" refers to a form of antibody that includes sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody includes substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being of human immunoglobulin sequence. A humanized antibody also optionally includes at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., CABILLY, U.S. Patent No. 4,816,567, QUEEN et al., (1989) Proc. Nat’l Acad. Sci. USA 86:10029-10033, and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996).

[0118] As used herein, the terms "inhibit" or "inhibition of" mean to reduce by a measurable amount or to prevent completely.

[0119] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0120] The terms "metastatic cancer" and "metastatic disease" mean cancer that has metastasized to regional lymph nodes or distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system.

[0121] As used herein, the term "modified" refers to the presence of a change to a natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide. Such changes or modifications can be obtained by post-synthetic modification of a natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide, or by co-translation, or by post-translational modification of a natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide.

[0122] "Molecular recognition" means a chemical event in which a host molecule can form a complex with a second molecule (i.e., a guest). This process occurs by non-covalent chemical bonds including, but not limited to, hydrogen bonds, hydrophobic interactions, and ionic interactions.

[0123] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a multitude of antibodies directed against (or specific for) different epitopes. In one embodiment, a polyclonal antibody comprises a plurality of monoclonal antibodies having different epitope specificities, affinities, or avidities within a single antigen that contains multiple antigenic epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). These monoclonal antibodies typically bind with a Kd of at least about 1 μM, more generally at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, as determined by ELISA.

[0124] What is referred to as "non-natural amino acid" or otherwise "nnAA" refers to an amino acid that is not one of the 20 (twenty) common amino acids or pyrrolidine or selenocysteine. Other terms that may be used synonymously with the term nnAA are "non-naturally encoded amino acid", "unnatural amino acid", "amino acid that occurs non-naturally". Furthermore, the term nnAA includes, but is not limited to, amino acids that do not occur naturally and can be obtained synthetically or by modification of non-natural amino acids.

[0125] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, isotonicity adjusters, wetting agents, preservatives and the like.

[0126] "Pharmaceutically acceptable" refers to a composition that is non-toxic, inert and / or physiologically compatible with humans or other mammals.

[0127] The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout this specification, the standard three-letter (see Table II.) or one-letter notation for amino acids is used. In the art, this term is often used interchangeably with "peptide" or "protein".

[0128] As used herein, the term "single-chain Fv" or "scFv" or "single-chain" antibody refers to an antibody fragment that comprises the V H and V L domains of the antibody, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide comprises the V H domain and the V LIt further includes a polypeptide linker between the domains. For a review of sFv, see PLUCKTHUN, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0129] As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the selective binding of an antibody to a target antigen epitope. An antibody can be tested for binding specificity by comparing its binding to the appropriate antigen to its binding to irrelevant antigens or antigen mixtures under a given set of conditions. An antibody is considered specific if it binds at least 2, 5, 7, preferably 10-fold more to the appropriate antigen than to irrelevant antigens or antigen mixtures. In one embodiment, a specific antibody is an antibody that binds only to Her2, EGFR, Trop2, CDH3 antigens and does not bind to irrelevant antigens. In another embodiment, a specific antibody is an antibody that binds to human Her2, EGFR, Trop2, CDH3-4 antigens but does not bind to non-human Her2, EGFR, Trop2, CDH3 antigens that have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid homology to the Her2, EGFR, Trop2, CDH3 antigens. In another embodiment, a specific antibody is an antibody that binds to human Her2, EGFR, Trop2, CDH3 antigens but does not bind to non-human Her2, EGFR, Trop2, CDH3 antigens that have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent identity to the amino acid sequences of the Her2, EGFR, Trop2, CDH3 antigens. In another embodiment, a specific antibody is an antibody that binds to human Her2, EGFR, Trop2, CDH3 antigens and binds to mouse Her2, EGFR, Trop2, CDH3 antigens, but has a higher degree of binding to human antigens. In another embodiment, a specific antibody is an antibody that binds to human Her2, EGFR, Trop2, CDH3 antigens and binds to primate Her2, EGFR, Trop2, CDH3 antigens, but has a higher degree of binding to human antigens.In another embodiment, the specific antibody binds to human Her2, EGFR, Trop2, CDH3 antigens and any non-human Her2, EGFR, Trop2, CDH3 antigens, but has a higher degree of binding to human antigens or any combination thereof.

[0130] As used herein, "treating" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as prolongation of survival, reduction in morbidity, and / or reduction of side effects that are by-products of alternative therapies. As will be readily understood in the art, complete eradication of the disease is preferred but not a requirement for the act of treatment.

[0131] The term "variant" refers to a molecule that exhibits a variation from a described type or standard, such as a protein having one or more different amino acid residues at a corresponding position of a specifically described protein (e.g., Her2, EGFR, Trop2, CDH3 protein). Analogs are examples of variant proteins. Splice isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

[0132] II.) Antibodies Another aspect of the invention provides an antibody that binds to Her2, EGFR, Trop2, CDH3, and other TAAs disclosed herein. In one embodiment, an antibody that binds to Her2, EGFR, Trop2, CDH3 and other TAA-related proteins.

[0133] As is known in the art, the Her2, EGFR, Trop2, CDH3 and other TAA antibodies of the present invention are particularly useful in cancer (e.g., see Table I) for prognostic assays, imaging, diagnostic and treatment methodologies. In one embodiment, the Her2, EGFR, Trop2, CDH3 and other TAA binding assays disclosed herein for use in the detection of cancer, for example in immunoassays. Similarly, such antibodies are useful in the treatment and / or prognosis of cancer (e.g., the cancers shown in Table I) to the extent that Her2, EGFR, Trop2, CDH3 and / or other TAAs are expressed or overexpressed in these and other cancers, for example, when combined with a therapeutic agent, in an ADC. Further, intracellular expression antibodies (e.g., single-chain antibodies) are therapeutically useful in the treatment of cancers in which the expression of Her2, EGFR, Trop2, CDH3, and other targets is involved.

[0134] A variety of methods for the preparation of antibodies, specifically monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)) using Her2, EGFR, Trop2, CDH3, and other TAA-related proteins, peptides, or fragments. Further, fusion proteins of Her2, EGFR, Trop2, CDH3, and fusion proteins of other TAAs such as Her2, EGFR, Trop2, CDH3 GST fusion proteins can also be used. In certain embodiments, GST fusion proteins containing all or most of the amino acid sequences of Her2, EGFR, Trop2, CDH3 are produced and then used as an immunogen for generating suitable antibodies. In another embodiment, Her2, EGFR, Trop2, CDH3, and other TAA-related proteins are synthesized and used as an immunogen.

[0135] Furthermore, using naked DNA immunization techniques known in the art (regardless of the presence or absence of purified Her2, EGFR, and other TAA-related proteins or Her2, EGFR, Trop2, CDH3, and other TAA-expressing cells), an immune response to the encoded immunogen is generated (for a review, see DONNELLY et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0136] Preferred methods for the generation of Her2, EGFR, Trop2, CDH3, and other TAA antibodies are further illustrated by the examples provided herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers, such as BSA, KLH, or another carrier protein, are also well known in the art. In some situations, direct conjugation using, for example, carbodiimide reagents is used. In other examples, linking reagents, such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of Her2, EGFR, Trop2, CDH3, and other TAA immunogens is often carried out by injection using appropriate adjuvants over an appropriate period, as understood in the art. During the immunization schedule, the titer of the antibody can be obtained to determine the validity of antibody formation.

[0137] Her2, EGFR, Trop2, CDH3, and other TAA monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete the desired monoclonal antibody are prepared using the standard hybridoma technique of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibody are screened by immunoassays where the antigen is Her2, EGFR, Trop2, CDH3, and other TAA-related proteins. Once an appropriate immortalized cell culture is identified, the cells can be expanded and the antibody produced from either in vitro culture or ascites fluid.

[0138] The antibodies or fragments of the present invention can also be produced by recombinant means. Regions that specifically bind to desired regions of Her2, EGFR, Trop2, CDH3, and other TAA proteins can also be produced in relation to chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human Her2, EGFR, Trop2, CDH3, and other TAA antibodies can also be produced and are preferred for use in therapeutic settings. Methods for humanizing mouse and other non-human antibodies by substituting one or more non-human antibody CDRs for the corresponding human antibody sequences are well known (see, for example, JONES et al., 1986, Nature 321:522-525; RIECHMANN et al., 1988, Nature 332:323-327; VERHOEYEN et al., 1988, Science 239:1534-1536). See also CARTER et al., 1993, Proc. Natl. Acad. Sci. USA 89:4285 and SIMS et al., 1993, J. Immunol. 151:2296.

[0139] In one embodiment, the human monoclonal antibodies of the invention can be prepared using VelocImmune mice in which genomic sequences having endogenous murine variable segments at the immunoglobulin heavy chain (VH, DH, and JH segments) and / or kappa light chain (VK and JK) loci have been wholly or partially replaced with human genomic sequences (Regeneron, Tarrytown, N.Y.) having germline variable segments of the human immunoglobulin heavy chain (VH, DH, and JH) and / or kappa light chain (VK and JK) loci that have not been rearranged. See, for example, U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

[0140] Furthermore, the human antibodies of the invention can be generated using HuMAb mice (Medarex, Inc.) that contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain immunoglobulin sequences (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., LONBERG et al., (1994) Nature 368(6474):856-859).

[0141] In another embodiment, the fully human antibodies of the invention can be produced using mice having human immunoglobulin sequences on transgenes and transchromosomes, such as mice having human heavy chain transgenes and human light chain transchromosomes. Such mice, referred to herein as "KM mice", are described in TOMIZUKA et al., (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication No. WO 02 / 43478 to TOMIZUKA et al.

[0142] The human monoclonal antibodies of the present invention can also be prepared using a phage display method for screening a library of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well established in the art. For example, U.S. Patent No. 5,223,409 to LADNER et al., U.S. Patent No. 5,403,484, and U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908 to DOWER et al., No. 5,580,717, U.S. Patent No. 5,969,108 and No. 6,172,197 to MCCAFFERTY et al., and U.S. Patent No. 5,885,793 to GRIFFITHS et al., No. 6,521,404, No. 6,544,731, No. 6,555,313, No. 6,582,915 and No. 6,593,081.

[0143] The human monoclonal antibodies of the present invention can also be prepared using SCID mice reconstituted with human immune cells so that a human antibody response can occur upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to WILSON et al.

[0144] Furthermore, the human antibodies of the present invention can be produced using techniques that employ transgenic mice that have been inactivated for antibody production and engineered at the human heavy and light chain loci, called Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.). An exemplary description of preparing transgenic mice that produce human antibodies can be found in U.S. Patent No. 6,657,103. See also U.S. Patent Nos. 5,569,825, 5,625,126, 5,633,425, 5,661,016 and 5,545,806, as well as MENDEZ et al., Nature Genetics, 15:146-156 (1998); KELLERMAN, S.A. and GREEN, L.L., Curr. Opin. Biotechnol 13, 593-597 (2002).

[0145] Any of the above production methods results in an antibody having a certain ability to bind to Her2, EGFR, Trop2, CDH3, and other TAAs, or a homolog or fragment or polypeptide sequence having 85, 90, 91, 92, 93, 94, 95, 96, 9, 98, or 99% sequence identity to Her2, EGFR, Trop2, CDH3, and other TAAs.

[0146] The binding affinity (K D ) of antibodies, their binding fragments, and antibody-drug conjugates containing them to Her2, EGFR, Trop2, CDH3, and other TAAs can be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, K D can be between 5 - 10 nM, or 1 - 2 nM. K D can be from 1 micromolar concentration to 500 micromolar concentration or from 500 micromolar concentration to 1 nM.

[0147] The binding affinity of an antigen-binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd / Ka). The binding affinity can be measured by BIACORE, for example, by capturing the test antibody on a sensor surface coated with protein A and flowing Her2, EGFR, Trop2, CDH3, and other TAAs over this surface. Alternatively, the binding affinity can be measured by FORTEBIO, for example, by capturing the test antibody receptor on a needle coated with protein A and flowing Her2, EGFR, Trop2, CDH3, and other TAAs over this surface. Those skilled in the art can identify other suitable assays known in the art for measuring binding affinity.

[0148] The engineered antibodies of the present invention include V H and / or V LThose that have been modified with respect to the framework residues therein are included (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "revert mutate" one or more framework residues to their corresponding germline sequences. More specifically, an antibody that has undergone somatic mutations may contain framework residues that are different from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configurations, the somatic mutations can be "reverted mutated" to the germline sequences by, for example, site-directed mutagenesis or PCR-mediated mutagenesis (e.g., a "revert mutation" from leucine to methionine). Such "revert mutated" antibodies are also intended to be encompassed by the present invention.

[0149] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by CARR et al.

[0150] In addition to, or instead of, the modifications made within the framework region or the CDR region, the antibodies of the present invention can typically be engineered to include modifications within the Fc region in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Furthermore, the Her2, EGFR, Trop2, CDH3, and other TAA MAbs of the present invention may be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody), or may be modified to alter their glycosylation, where again the modification may be to alter one or more functional properties of the MAb. Each of these embodiments will be described in further detail below. In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues within the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues within the hinge region of CH1 is altered, e.g., to facilitate the construction of the light and heavy chains, or to increase or decrease the stability of the Her2, EGFR, Trop2, CDH3, and other TAA MAbs.

[0151] In another embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the Her2, EGFR, Trop2, CDH3, and other TAA MAbs. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody impairs staphylococcal protein A (SpA) binding as compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0152] In another embodiment, Her2, EGFR, Trop2, CDH3, and other TAA MAbs are modified to increase their biological half-life. Various approaches are possible. For example, mutations can be introduced as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to PRESTA, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope derived from two loops of the CH2 domain of the Fc region of IgG.

[0153] In yet another embodiment, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the effector function(s) of Her2, EGFR, Trop2, CDH3, and other TAA MAbs. For example, one or more amino acids selected from amino acid-specific residues can be replaced with different amino acid residues such that the antibody has an altered affinity for an effector ligand while retaining the antigen-binding ability of the parental antibody. Effector ligands whose affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in both U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0154] In a preferred embodiment, Her2, EGFR, Trop2, CDH3, and other TAA Mabs contain a triple substitution at the following positions: L234A, L235A, L328C.

[0155] In a preferred embodiment, Her2 Mab contains a triple substitution at the following positions: L234A, L235A, L328C.

[0156] In a preferred embodiment, EGFR Mab contains a triple substitution at the following positions: L234A, L235A, L328C.

[0157] In a preferred embodiment, the Trop2 Mab contains triple substitutions at the following positions: L234A, L235A, L328C.

[0158] In a preferred embodiment, the CDH3 Mab contains triple substitutions at the following positions: L234A, L235A, L328C.

[0159] In another preferred embodiment, the TAA Mab contains triple substitutions at the following positions: L234A, L235A, L328C.

[0160] In another embodiment, Her2, EGFR, Trop2, CDH3, and other TAA Mabs contain single substitutions at the position: L328C.

[0161] In another embodiment, the Her2 Mab contains a single substitution at the position: L328C.

[0162] In another embodiment, the EGFR Mab contains a single substitution at the position: L328C.

[0163] In another embodiment, the Trop2 Mab contains a single substitution at the position: L328C.

[0164] In another embodiment, the CDH3 Mab contains a single substitution at the position: L328C.

[0165] In another embodiment, the TAA Mab of the present disclosure contains a single substitution at the position: L328C.

[0166] In another embodiment, Her2, EGFR, Trop2, CDH3, and other TAA Mabs contain double substitutions at the positions: L234A, L235A.

[0167] In another embodiment, the Her2 Mab contains double substitutions at the positions: L234A, L235A.

[0168] In another embodiment, the EGFR Mab contains double replacements at the following positions: L234A, L235A.

[0169] In another embodiment, the Trop2 Mab contains double replacements at the following positions: L234A, L235A.

[0170] In another embodiment, the CDH3 Mab contains double replacements at the following positions: L234A, L235A.

[0171] In another embodiment, the TAA Mab of the present disclosure contains double replacements at the following positions: L234A, L235A.

[0172] The reactivity of Her2, EGFR, Trop2, CDH3, and other TAA antibodies can be established by many well-known means, including Western blot, immunoprecipitation, ELISA, and FACS analysis, using Her2, EGFR, Trop2, CDH3, and other TAA-related proteins, Her2, EGFR, Trop2, CDH3, and other TAA-expressing cells or their extracts as appropriate. Her2, EGFR, Trop2, CDH3, and other TAA antibodies or their fragments can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents, or enzymes.

[0173] III.) Fc mutations that modify effector function The Fc region of an antibody (i.e., the end of the heavy chain of the antibody spanning domains CH2, CH3, and part of the hinge region) has limited variability and is involved in performing the physiological role of the antibody. The effector functions resulting from the Fc region of an antibody vary depending on the class and subclass of the antibody and include the binding of the antibody to specific Fc receptors ( "FcR") via the Fc region on the cell, which causes various biological responses.

[0174] These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that can mediate some intracellular signaling events. These receptors are expressed on various immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. The formation of the Fc / FcγR complex recruits these effector cells to the site of the bound antigen and typically results in important subsequent immune responses such as intracellular signaling events and the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies can destroy target cells. The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the bound antibody on the target cell and subsequently cause lysis of the target cell is called antibody-dependent cell-mediated cytotoxicity (ADCC) (see RAVETCH et al., Annu. Rev. Immunol. 19 (2001) 275-290). The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the bound antibody on the target cell and subsequently cause phagocytosis of the target cell is called antibody-dependent cell-mediated phagocytosis (ADCP). Furthermore, overlapping sites on the Fc region of the molecule also control the activation of a complement-mediated cell-independent cytotoxic function known as complement-dependent cytotoxicity (CDC).

[0175] Furthermore, the complement inflammatory cascade is part of the innate immune response and is important for an individual's ability to prevent infection. Another important Fc ligand is the complement protein C1q. Binding of Fc to C1q mediates a process called complement-dependent cytotoxicity (CDC). C1q can bind to six antibodies, but binding to two IgGs is sufficient to activate the complement cascade. C1q forms a complex with the C1r and C1s serine proteases to form the C1 complex of the complement pathway.

[0176] In many situations, the binding and stimulation of effector functions mediated by the Fc region of immunoglobulins are highly beneficial. However, in certain instances, it may be more advantageous to reduce or eliminate effector functions. This is particularly true for antibodies designed to deliver drugs (e.g., toxins and isotopes) to target cells where Fc / FcγR-mediated effector functions carry healthy immune cells near a lethal payload, resulting in depletion of normal lymphoid tissue along with the target cells (see HUTCHINS et al., PNAS USA 92 (1995) 11980-11984). In these cases, the use of antibodies that mobilize few complement cells or effector cells would be highly beneficial (see also U.S. Patent No. 6,194,551, U.S. Patent No. 5,885,573, and PCT Publication WO 04 / 029207).

[0177] In other examples, for instance, when the goal is to block the interaction between a widely expressed receptor and its cognate ligand, it would be advantageous to reduce or eliminate all antibody effector functions to reduce unwanted toxicity. Further, if a therapeutic antibody exhibits indiscriminate binding across multiple human tissues, it would be prudent to limit effector function targeting to a diverse set of tissues to limit toxicity. Finally, a reduced affinity of an antibody for the FcγRII receptor in particular is advantageous for antibodies that induce platelet activation and aggregation via FcγRII receptor binding, which can be a significant side effect of such antibodies. See TAM et al., Antibodies 6:12 (2017). See also WEBER et al., Pharm Res 35:169 (2018).

[0178] There are certain subclasses of human immunoglobulins that lack specific effector functions, but there are no known naturally occurring immunoglobulins that lack all effector functions. Another approach would be to manipulate or mutate important residues in the Fc region that is responsible for effector functions. See SCHLOTHAUER et al., Protein Eng. Design, and Selection, vol. 29, no. 10 pp457-466 (2016) and WANG et al., Protein Cell, 9(1) pp63-73 (2018). In fact, several research groups have tried such attempts. See, for example, PCT Publication WO 2009 / 100309 (Medimmune), WO 2006 / 076594 (Xencor), WO 1999 / 58572 (Univ. Cambridge), US Patent Application Publication No. 2006 / 0134709 (Macrogenics), WO 2006 / 047350 (Xencor), WO 2006 / 053301 (Xencor), US Patent No. 6,737,056 (Genentech), US Patent Application Publication No. 5,624,821 (Scotgen Pharmaceuticals), US Patent Application Publication No. 2010 / 0166740 (Roche), and US Patent No. 8,969,526 (Roche Glycart AG).

[0179] Binding of IgG to activating and inhibitory Fcγ receptors or to the first component of complement (C1q) depends on residues located in the hinge region and CH2 domain. Two regions of the CH2 domain are important for binding of FcγR and complement C1q and have unique sequences. Substitution of human IgG1 and IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduced ADCC and CDC (see ARMOUR et al., Eur. J. Immunol. 29(8) (1999) 2613-2624; SHIELDS et al., J. Biol. Chem. 276(9) (2001) 6591-6604).

[0180] Furthermore, IDUSOGIE et al., J. Immunol. 166 (2000) 2571-2575 mapped the C1q binding site of RITUXAN and showed that Pro329Ala reduced the ability of rituximab to bind C1q and activate complement. Additionally, substitution of Pro329 with Ala has been reported to lead to decreased binding to the FcγRI, FcγRII, and FcγRIIIA receptors (see SHIELDS et al., J. Biol. Chem. 276(9)(2001)6591-6604). However, this mutation has also been described as showing wild-type-like binding to FcγRI and FcγRII and only a slight decrease in binding to the FcγRIIIA receptor (European Patent No. 1,068,241, Genentech).

[0181] Furthermore, OGANESYAN et al., Acta Cristallographica D64 (2008) 700-704 introduced the triple mutation L234F / L235E / P331S into the lower hinge and C2H domains and showed a decrease in the binding activity of the human IgG1 molecule to the human C1q receptor, FcγRI, FcγRII, and FcγRIIIA.

[0182] In view of the foregoing background, the invention herein relates to a method for producing a polypeptide comprising an Fc variant in which the Fc is silenced and / or inhibited. The "parent", "starting", "non-variant" or "wild-type" polypeptide is prepared using techniques available in the art for generating polypeptides or antibodies comprising an Fc region. In a preferred embodiment of the invention, the parent polypeptide is an antibody, and exemplary methods for generating antibodies are described in more detail in the present disclosure. In a further preferred embodiment, the wild-type polypeptide is an antibody that binds to Her2, EGFR, Trop2, CDH3, and / or any TAA of the present disclosure (see Table IV).

[0183] One embodiment of the present invention encompasses a polypeptide comprising an Fc region of an antibody, which includes an addition, substitution, or deletion of at least one amino acid residue to the Fc region, resulting in a decrease or removal of affinity for at least one Fc receptor. The Fc region interacts with several receptors or ligands including, but not limited to, Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIIA), complement protein C1q, and other molecules such as protein A and G. As described in the present disclosure, these interactions are essential for various effector functions and downstream signaling events including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0184] Accordingly, in certain embodiments, a variant of the present invention has the same amino acid sequence as a polypeptide comprising an Fc variant of the present invention, but has a decreased or removed affinity for Fc receptors responsible for effector functions as compared to a polypeptide (also referred to herein as a "wild-type polypeptide") that does not include an addition, substitution, and deletion of at least one amino acid residue to the Fc region. In certain embodiments, a polypeptide comprising an Fc variant of the present invention includes at least one or more of the following characteristics: decreased or removed effector (ADCC and / or CDC and / or ADCP) function, decreased or removed binding to Fc receptors, decreased or removed binding to C1q, or decreased or removed toxicity. More specifically, embodiments of the present invention provide anti-Her2, anti-EGFR, anti-Trop2, anti-CDH3, and anti-TAA (tumor-associated antigen (Table IV)) antibodies with decreased affinity for Fcγ receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB) and / or complement protein C1q.

[0185] In one embodiment, the present invention includes Her2, EGFR, Trop2, CDH3, and TAA MAbs that include a triple mutation.

[0186] In one embodiment, the antibody of the present invention comprises an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue at position 328, and the numbering system for the constant region is the EU index numbering system as shown in Kabat et al., NIH Publication 91(1991)3242, National Technical Information Service, Springfield, Va.

[0187] In one embodiment, the antibody of the present invention comprises an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue at position 234, and the numbering system for the constant region is the EU index numbering system as shown in Kabat et al., NIH Publication 91(1991)3242, National Technical Information Service, Springfield, Va.

[0188] In one embodiment, the antibody of the present invention comprises an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue at position 235, and the numbering system for the constant region is the EU index numbering system as shown in Kabat et al., NIH Publication 91(1991)3242, National Technical Information Service, Springfield, Va.

[0189] In certain aspects of the present invention, the polypeptide comprising the Fc variant comprises an antibody. In yet another aspect of the present invention, the polypeptide comprising the Fc variant comprises a human IgG1, IgG2, IgG3, or IgG4 Fc region. In still further aspects of the present invention, the variant is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0190] In a further specific embodiment, the polypeptide described above comprises a human IgG1 region.

[0191] In a specific embodiment, the polypeptide of the present invention comprises an Fc variant of a wild-type human Fc polypeptide, said variant comprising an amino acid substitution at position L328C, and the numbering of the residues of the IgG Fc region is according to the EU index numbering as in the case of Kabat. In yet another embodiment, said variant comprises at least one additional amino acid substitution. In yet another embodiment, said variant comprises at least one additional amino acid substitution. In yet another embodiment, said variant comprises three (3) amino acid substitutions at L234A, L235A, and L328C.

[0192] In a specific embodiment, the anti-Her2 MAb of the present invention comprises an Fc variant of a wild-type human Fc Her2 Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0193] In a specific embodiment, the anti-EGFR MAb of the present invention comprises an Fc variant of a wild-type human Fc EGFR Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0194] In a specific embodiment, the anti-Trop2 MAb of the present invention comprises an Fc variant of a wild-type human Fc Trop2 Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0195] In a specific embodiment, the anti-CDH3 MAb of the present invention comprises an Fc variant of a wild-type human Fc CDH3 Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0196] In a specific embodiment, the TAA MAb of the present invention comprises an Fc variant of a wild-type human Fc TAA Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0197] In one aspect, the polypeptide comprising the Fc variant does not affect target binding as compared to the unmodified antibody.

[0198] In one aspect, the polypeptide comprising the Fc variant of the invention exhibits inhibition of FcγRI binding as compared to an unmodified antibody.

[0199] In one aspect, the polypeptide comprising the Fc variant of the invention exhibits inhibition of FcγRII binding as compared to an unmodified antibody.

[0200] In one aspect, the polypeptide comprising the Fc variant of the invention exhibits inhibition of FcγRIIIa binding as compared to an unmodified antibody.

[0201] In one aspect, the polypeptide comprising the Fc variant of the invention does not substantially affect FcRn binding as compared to an unmodified antibody.

[0202] As will be appreciated by those skilled in the art, the surprising recognition discovered by the present invention is that the need for the triple mutation, specifically the addition of amino acid substitution(s) at L234A and L235A, was found to be due to the following observation(s). First, L328C by itself only shows a partial reduction of Fc effector function. By combining additional mutations, a greater degree of Fc silencing can be achieved. L234 and L235 were selected because these residues are located near the hinge region and can partially reduce FcγR binding when mutated to alanine. The combination of L234A and L235A with L328C showed almost complete inhibition of FcγR interaction for all isotypes tested.

[0203] As observed, despite complete suppression of FcγR binding, equivalent binding activity was observed when compared to the wild-type counterpart, so the triple mutation (L328C in combination with L234A / L235A) did not affect target antigen binding. The stability and expression of the antibody also remained equivalent to the wild-type antibody. Thus, the triple mutation enables complete inhibition of Fc effector function without compromising target specificity, antibody quality and yield.

[0204] Furthermore, as described below, the specific triple mutation can promote efficient site-specific conjugation.

[0205] IV.) Antibody-Drug Conjugate In another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody conjugated to a therapeutic agent. The therapeutic agent can be a cytotoxic agent, a cytostatic agent, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radiolabeled conjugate). In another aspect, the present invention further provides a method of using the ADC. In one aspect, the ADC comprises any of the above Her2, EGFR, Trop2, CDH3, and other TAA Mab covalently attached or attached via an oxime bond to a cytotoxic agent or a detectable agent.

[0206] In a further embodiment, the ADC comprises Her2, EGFR, Trop2, CDH3, and other TAA Mab further comprising a triple substitution at the following positions: L234A, L235A, L328C.

[0207] In a preferred embodiment, the ADC comprises Her2, EGFR, Trop2, CDH3, and other TAA Mab further comprising a triple substitution at the following positions: L234A, L235A, L328C, and further comprising site-specific conjugation at L328C.

[0208] In the background, in the treatment of cancer, the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) enables the targeted delivery of the drug moiety to the tumor and its intracellular accumulation. The systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity not only to the tumor cells to be eliminated but also to normal cells (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986): 603-05; Thorpe, (1985) ‘‘Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,’’ in Monoclonal Antibodies’84: Biological and Clinical Applications, A. Pinchera et al. (ed.), pp. 475-506). Thereby, minimal toxicity and maximal efficacy are sought. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (MANDLER et al., (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; MANDLER et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; MANDLER et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (European Patent No. 1391213; LIU et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (LODE et al (1998) Cancer Res. 58:2928; HINMAN et al (1993) Cancer Res. 53:3336-3342). Toxins can affect their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0209] Examples of antibody-drug conjugates are ADCETRIS (brentuximab vedotin, Seattle Genetics), ZEVALIN® (ibritumomab tiuxetan, Biogen / Idec), MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), KADCYLA® (ado-trastuzumab emtansine, Genentech), BESPONSA® (inotuzumab ozogamicin, Pfizer / Wyeth), POLIVY (polatuzumab vedotin, Genentech / Roche), canzumab mertansine (Immunogen, Inc.) and MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.).

[0210] Furthermore, although not limited thereto, therapeutic agents including chemotherapeutic agents useful for the generation of ADCs are described herein. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene. See, for example, International Publication No. WO 93 / 21232, published Oct. 28, 1993. A variety of radionuclides are available for the production of radiolabeled antibodies. By way of example, 212 Bi, 131 I, 131 In, 90 Y and 186Examples include Re. Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al. (1987) Science, 238:1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radio nucleotides to antibodies (International Publication No. WO 94 / 11026). Other antitumor agents that can be conjugated to the antibodies of the present invention include BCNU, streptozocin, vincristine, and 5-fluorouracil, the family of agents collectively known as the LL-E33288 complex described in U.S. Patent Nos. 5,053,394 and 5,770,710, and esperamicin (U.S. Patent No. 5,877,296).

[0211] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene. See, for example, International Publication No. 93 / 21232 (published October 28, 1993).

[0212] The present invention further contemplates an ADC formed between an antibody and a compound having nuclease activity (e.g., ribonuclease or DNA endonuclease, such as deoxyribonuclease; DNase).

[0213] For the selective destruction of tumors, the antibody may contain a radioactive atom. Various radioisotopes are available for the production of radiolabeled antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 88 , Sm 53 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu. When the conjugate is used for detection, the conjugate may contain a radioactive atom for scintigraphy studies, such as tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0214] Radioactive labels or other labels can be incorporated into the conjugate by known methods. For example, the peptide may be biosynthesized or synthesized by chemical amino acid synthesis using a suitable amino acid precursor containing, for example, fluorine-19 instead of hydrogen. tc 99m or I 123 、Re 186 、Re 188 and In 111 Labels such as can be attached via cysteine residues in the peptide. Yttrium-90 can be attached via lysine residues. Iodine-123 can be incorporated using the IODOGEN method (FRAKER et al., (1978) Biochem. Biophys. Res. Commun. 80:49-57). "Monoclonal Antibodies in Immunoscintigraphy" (CHATAL, CRC Press 1989) describes other methods in detail.

[0215] The present invention provides, inter alia, antibody-drug conjugate compounds for the targeted delivery of therapeutic agents. The inventors have discovered that antibody-drug conjugate compounds have potent cytotoxic activity and / or cell growth inhibitory activity against cells expressing Her2, EGFR, Trop2, CDH3 and other TAAs.

[0216] Antibody-drug conjugate compounds comprise an antibody unit covalently bound to at least one drug unit. The drug unit can be covalently bound to the antibody unit directly or via a linker unit (-LU-). Furthermore, the drug unit is conjugated at the site-specific position of L328C.

[0217] In some embodiments, the antibody-drug conjugate compound has the following formula: L-(LU-D) p (I) or a pharmaceutically acceptable salt or solvate thereof; wherein, ·L is an antibody unit, such as Her2, EGFR, Trop2, CDH3, or another TAA MAb of the present invention, and the MAb contains triple mutations at the following positions: L234A, L235A, and L328C. ·(LU-D) is a linker unit-drug unit moiety, wherein ·LU- is a linker unit. ·-D is a drug unit having cytostatic or cytotoxic activity against target cells. ·p ranges from 1 to 20 or alternatively from 1 to 50. Furthermore, the drug unit moiety is conjugated at the site-specific position on the MAb at L328C.

[0218] In some embodiments, the antibody-drug conjugate compound has the following formula: L-(A a -W w -Y y -D) p (II) or a pharmaceutically acceptable salt or solvate thereof; wherein ·L is an antibody unit, such as Her2, EGFR, Trop2, CDH3, or another TAA MAb, and the MAb contains triple mutations at the following positions: L234A, L235A, and L328C. ·-A a -W w -Y y - is a linker unit (LU), wherein ·-A- is a stretcher unit. ·a is 0 or 1 or 2 or 3. ·Each -W- is independently an amino acid unit. ·w is an integer in the range of 0 to 12. ·-Y- is a self-immolative spacer unit. ·y is 0, 1, or 2. ·-D is a drug unit having cytostatic or cytotoxic activity against target cells. ·p is an integer from 1 to 20, or alternatively an integer from 1 to 50. Furthermore, the drug unit moiety is conjugated at the site-specific position on the MAb with L328C.

[0219] In the case of a composition comprising multiple antibodies, the drug loading is represented by p, the average number of drug molecules per antibody. The drug loading can range from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in the preparation of the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the antibody-drug-conjugate with respect to p can also be determined. In some examples, the isolation, purification, and characterization of a homogeneous antibody-drug-conjugate where p is a specific value from other antibody-drug-conjugates with different drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis. In an exemplary embodiment, p is from 2 to 8.

[0220] The generation of the antibody-drug conjugate compound can be achieved by any technique known to those skilled in the art. Briefly, the antibody-drug conjugate compound comprises, as the antibody unit, Her2, EGFR, Trop2, CDH3, or another TAA MAb containing triple mutations at the following positions: L234A, L235A, and L328C, a drug, and optionally a linker that binds the drug and a binder. In a preferred embodiment, the drug unit moiety is conjugated at the site-specific position of L328C.

[0221] Several different reactions are available for covalently attaching a drug and / or linker to a binder. This is often accomplished by reaction of the binder, e.g., amino groups of lysine, free carboxylic acid groups of glutamic acid and aspartic acid, sulfhydryl groups of cysteine, and various moieties of aromatic amino acids, with amino acid residues of an antibody molecule. One of the most commonly used non-specific methods of covalent bonding is the carbodiimide reaction that links the carboxy (or amino) group of a compound to the amino (or carboxy) group of an antibody. Additionally, bifunctional agents such as dialdehydes or imido esters have been used to link the amino group of a compound to the amino group of an antibody molecule. Also available for attachment of a drug to a binder is the Schiff base reaction. This method involves periodate oxidation of a drug containing a glycol or hydroxy group, thus forming an aldehyde, which is then reacted with the binder. The attachment occurs via formation of a Schiff base with an amino group of the binder. Isothiocyanates can also be used as coupling agents for covalently attaching a drug to a binder. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0222] In certain embodiments, an intermediate that is a precursor of a linker is reacted with a drug under suitable conditions. In certain embodiments, the reactive groups are used on the drug and / or the intermediate. The product of the reaction between the drug and the intermediate or derivatized drug is then reacted with Her2, EGFR, Trop2, CDH3, or other TAA MAb under suitable conditions.

[0223] V.) Site-Specific Conjugation Formats for ADCs As will be appreciated by those skilled in the art, the ability to optimize payload placement and conjugate composition in the context of an ADC is a beneficial endeavor. See ABHIJIIT et al., Bioprocess Int., Mab Upstream Processing (Oct 2014).

[0224] Generally speaking, bioconjugation strategies involve covalently linking a protein or peptide (biologic) to a small molecule, carbohydrate, oligonucleotide, synthetic polymer, or another protein / peptide. This approach can be extremely important for generating differentiation in the highly competitive biologic market and driving product development. These strategies were the basis for the development of highly successful conjugate vaccines such as Prevnar 13, Menactra, Menomune, and HibTITER. Four (4) of them were created by conjugating bacterial polysaccharides to immunogenic carrier proteins. Similarly, the application of bioconjugation to half-life extending polymer carriers (such as polyethylene glycol (PEG)) has created drugs currently on the market (e.g., certolizumab (Cimzia), pegfilgrastim (Neulasta) and pegvisomant (Somavert)) that have longer durations of action than their unconjugated counterparts and dosing regimens that facilitate patient compliance.

[0225] As is known in the art, the choice of linker and conjugation chemistry for making first-generation ADCs was dictated by the limitations of working with proteins. Thus, the linker was functionalized with reactive groups designed to specifically react with nucleophilic amino acid side chains accessible on the surface belonging to natural amino acids such as cysteine (thiol) or lysine (amine).

[0226] However, when antibodies are treated with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), their disulfide bonds can be cleaved to expose free thiols, which can then be readily conjugated to a maleimide-containing linker. Up to four interchain disulfide bonds can be reduced, thereby exposing up to eight reactive thiol groups for conjugation. The conditions developed for thiol chemical conjugation result in either complete or partial reduction of the disulfide bonds, and conjugates made using this method can contain either 0, 2, 4, 6, or 8 drugs per antibody molecule.

[0227] It is important to note that beyond the number of drugs per antibody molecule, another level of heterogeneity occurs at the conjugation site. Thus, ADCs with a specific drug-to-antibody ratio (DAR) generated by cysteine conjugation are still a heterogeneous mixture of conjugates with different sites of conjugation. However, since there are only eight sites available for cysteine conjugation (compared to a maximum of 80 available for lysine-directed chemistry), it is not an overstatement to say that the cysteine conjugation approach provides greater control over the conjugation site and facilitates better characterization. The controlled reduction-alkylation strategy has been successfully used to manufacture the approved ADCETRIS (Seattle Genetics, Bothell, WA) product, along with several other ADCs currently in clinical trials.

[0228] The researchers studied the in vivo effects of ADCs targeting CD30+ tumor cells with 2, 4, and 8 monomethyl auristatin E (MMAE) toxins per antibody molecule and demonstrated that the drug loading stoichiometry significantly affects the pharmacokinetics (PK), efficacy, and toxicity of the drug. Hamblett et al. found that in their system, ADCs with 4 drugs per antibody were more potent than ADCs with 2 drugs but had equivalent efficacy and better tolerability than ADCs with 8 drugs / antibody. The results generally showed that ADCs with higher drug loadings had greater clearance, higher efficacy, and increased toxicity. This implied that each ADC has an optimal drug loading that has the right balance of efficacy and toxicity.

[0229] Their groundbreaking research established the concept that the drug-to-antibody ratio (DAR) is an important design parameter for ADCs. Chemical conjugation to native cysteine or lysine residues has been shown to be suboptimal as they produce heterogeneous ADC mixtures. Heterogeneity results from differences in DAR and conjugation sites, leading to subpopulations of ADCs with low potency, high toxicity, and different dispositions and PK characteristics. Furthermore, for such non-selective conjugation methods, the analytical characterization and control of batch-to-batch variability during manufacturing remain a major challenge. To overcome these limitations, the concept of site-specific conjugation has evolved, which aims to first produce homogeneous ADCs and control the DAR and conjugation sites.

[0230] Several site-specific conjugation techniques have been demonstrated to optimize payload disposition using various technologies (see, for example, ThioMab (Genentech), Seattle Genetics, and Ambrx, Inc.).

[0231] Studies on the reported ThioMab have revealed another fundamental concept. The homogeneity of the ADC is not only key to improving biophysical and therapeutic properties, but also the actual site of conjugation on the antibody backbone has a major impact on the in vivo behavior of the ADC molecule. SHEN et al. generated multiple homogeneous TDC conjugates with a HER2-targeted antibody using the MMAE payload, where the engineered cysteine for conjugation was located in either the light chain (LC), heavy chain (HC) or Fc region of the antibody. All conjugates showed comparable in vitro potency, but the authors reported significant differences in their in vivo efficacy and PK properties.

[0232] The LC conjugate showed the greatest efficacy when studied in a mouse xenograft model where the HC conjugate was intermediate and the Fc conjugate had little or no activity. Mouse PK studies showed a similar trend that the LC conjugate exhibited the greatest stability and lowest clearance, followed by the HC conjugate, and the Fc conjugate ADC was removed the fastest and provided the lowest ADC exposure. These results were due to differences in the local microenvironment and accessibility to the solvent contributing to different stabilities of the linker systems at different sites.

[0233] Several additional site-specific bioconjugation methods have been reported that aim to deliver homogeneous ADCs compared to first-generation lysine and cysteine conjugations, but only a subset of these technologies offer greater versatility in finding the optimal conjugation site for a given antibody-payload combination. These include conjugation with non-natural amino acids (nnAAs) introduced by genetic code modification (Ambrx, La Jolla, CA; Sutro Biopharma, South San Francisco, CA; Allozyne, acquired by Medimmune, Seattle, WA). Also, transglutaminase (TG)-mediated conjugation to engineered glutamine tags (Pfizer, New York, NY). Also, conjugation with aldehyde-tagged antibodies generated by co-expressed formylglycine-generating enzyme (FGE) (Redwood Bioscience, acquired by Catalent Pharma Solutions, Emeryville, CA).

[0234] Based on the above, studies using technologies that offer flexible site-specific conjugation options have firmly established that the site of conjugation significantly affects the pharmacological properties of ADCs. Therefore, it should be considered an important parameter in product design, and the ability to find the optimal site for a particular payload-antibody combination can be important for the success of product or product(s) development. See SCHUMACHER et al., Clin. Immunol, 36(Suppl 1):S100-S107 (2016); DEONARAIN et al., Expert Opin. Drug Discov, 10(5) (2015); ZHOU, Biomedicines, 5:64 (2017); PANOWSKI et al., mAbs 6:1 pp 34-45 (Jan / Feb 2014); PCT Patent Publication WO 2018 / 20081 and US Patent Publication 2017 / 0080103.

[0235] In one embodiment, the site-specific conjugation method by converting Leu-328 to Cys in the Fc domain of the monoclonal antibody enables controlled conjugation without affecting target binding. Furthermore, it has been shown that neither the expression level nor the stability of the antibody is impaired by the introduction of L328C.

[0236] In a further embodiment, the site-specific conjugation method by converting Leu-328 to Cys in the Fc domain of the Her2 antibody enables controlled conjugation without affecting target binding. Furthermore, it has been shown that neither the expression level nor the stability of the antibody is impaired by the introduction of L328C.

[0237] In a further embodiment, the site-specific conjugation method by converting Leu-328 to Cys in the Fc domain of the EGFR antibody enables controlled conjugation without affecting target binding. Furthermore, it has been shown that neither the expression level nor the stability of the antibody is impaired by the introduction of L328C.

[0238] In a further embodiment, the site-specific conjugation method by converting Leu-328 to Cys in the Fc domain of the Trop2 antibody enables controlled conjugation without affecting target binding. Furthermore, it can be shown that neither the expression level nor the stability of the antibody is impaired by the introduction of L328C.

[0239] In a further embodiment, the site-specific conjugation method by converting Leu-328 to Cys in the Fc domain of the CDH3 antibody enables controlled conjugation without affecting target binding. Furthermore, it has been shown that neither the expression level nor the stability of the antibody is impaired by the introduction of L328C.

[0240] In a further embodiment, the site-specific conjugation technique by conversion of Leu-328 to Cys in the Fc domain of a TAA antibody (such as the TAAs listed in Table IV) enables controlled conjugation without affecting target binding. Furthermore, it has been shown that neither the expression level nor the stability of the antibody is impaired by the introduction of L328C.

[0241] The site-specific conjugation technique mediated by L328C was selected to enable more uniform drug products and improved conjugation efficiency. Various treatment modalities, including antibody-drug conjugates (ADCs), can benefit from site-specific conjugation as it can prevent the formation of heterogeneous mixtures that can have a negative impact on in vivo efficacy and the therapeutic index. Similarly, the binding of boron-containing entities to specifically defined sites on antibody molecules can improve the efficacy of boron neutron capture therapy (BNCT), a non-invasive treatment modality for treating cancer.

[0242] The conversion of Leu-328 to Cys in the Fc domain of monoclonal antibodies enables controlled conjugation without affecting target binding. Furthermore, neither the expression level nor the stability of the antibody is impaired by the introduction of L328C. Quality assessment by size exclusion chromatography shows a major peak exceeding 99% comparable to the wild-type counterpart. Thus, the tendency to aggregate, which is often associated with the introduction of unpaired cysteines, is not observed for L328C. The formation of a uniform product with 100% conjugation efficiency mediated by L328C implies a simpler manufacturing process compared to the complex and inefficient production processes required for non-specifically conjugated counterparts. The defined uniform composition mediated by L328C conjugation has a much lower therapeutic burden, a simpler manufacturing process, and enables the rapid discovery and development of ADCs and antibody-boron conjugates for the application of BNCT.

[0243] Furthermore, L328C also exhibits the additional advantage of reducing Fc effector function. Safety liabilities associated with infusion reactions caused by therapeutic monoclonal antibodies and their interactions with FcγRs have been reported. The ability to partially silence Fc effector function without compromising antibody stability is an attractive feature of L328C. Potential mitigation strategies for an improved clinical safety profile can be further developed for L328C-mediated conjugation or, as a naked antibody, in possible combinations with other variables that have previously been shown to reduce Fc effector function.

[0244] VI.) Linker unit Typically, an antibody-drug conjugate compound includes a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In still other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

[0245] In a preferred embodiment, the linker is conjugated at the site-specific position L328C.

[0246] In some embodiments, the linker is cleavable by a cleavage factor present in the intracellular environment (e.g., within a lysosome or endosome or caveola). The linker can be a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, for example, lysosomal or endosomal proteases. The linker can also be cleaved by a cleavage factor present in the extracellular environment (e.g., in the vicinity of the cell membrane or tissue space). The linker can be a peptidyl linker that is cleaved by an extracellular peptidase or protease enzyme (including but not limited to members of the cathepsin family of enzymes or matrix metalloproteinases).

[0247] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, the pH-sensitive linker is hydrolysable under acidic conditions. For example, acid-labile linkers that are hydrolysable in lysosomes (e.g., oximes, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, orthoesters, acetals, ketals, etc.) can be used. (See, for example, U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; DUBOWCHIK AND WALKER, 1999, Pharm. Therapeutics 83:67-123; NEVILLE et al., 1989, Biol. Chem. 264:14653-14661.)

[0248] In yet other embodiments, the linker is cleavable under reducing conditions known in the art. (See, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; WAWRZYNCZAK et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. VOGEL ed., Oxford U.Press, 1987, U.S. Pat. No. 4,880,935).) The linker can also be cleaved under reducing conditions found intracellularly (or extracellularly). For example, in a preferred embodiment, the specific linker N-O bond can be formally reduced and cleaved to effect cleavage of the linker.

[0249] In yet other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation. (See PCT Publication No. WO 2012 / 166560 (Ambrx, Inc.), which is incorporated herein by reference in its entirety.)

[0250] In other non-mutually exclusive embodiments, the linker promotes endocytosis as known in the art.

[0251] A variety of exemplary linkers that can be used with the compositions and methods of the present invention are described in WO 2004 / 010957, US 2006 / 0074008, US 20050238649, and US 2006 / 0024317 (each incorporated herein by reference in its entirety for all purposes).

[0252] VII.) Stretcher unit The stretcher unit (A), when present, can link the antibody unit to the amino acid unit (-W-), when present, to the spacer unit (-Y-), when present, or to the drug unit (-D). Useful functional groups that can be present on Her2, EGFR, Trop2, CDH3, or other TAA Mab either naturally or via chemical manipulation include, but are not limited to, keto, aldehyde, sulfhydryl, amino, hydroxyl, anomeric hydroxyl groups of carbohydrates, and carboxyl. Suitable functional groups are keto, aldehyde, sulfhydryl, and amino. In one example, the keto group is on a non-natural amino acid (nnAA) incorporated into the Mab of the present invention. In a further example, the aldehyde group is on an nnAA incorporated into the Mab of the present invention. In another example, the sulfhydryl group can be generated by reduction of the intramolecular disulfide bond of Her2, EGFR, Trop2, CDH3, or other TAA Mab. In another embodiment, the sulfhydryl group can be generated by reaction of the amino group of the lysine moiety of Her2, EGFR, Trop2, CDH3, or other TAA Mab with 2-iminothiolane (Traut reagent) or other sulfhydryl generating reagent. In certain embodiments, Her2, EGFR, Trop2, CDH3, or other TAA Mab is a recombinant antibody and is engineered to carry one or more lysines. In certain other embodiments, the recombinant Her2, EGFR, Trop2, CDH3, or other TAA Mab is engineered to have additional sulfhydryl groups, such as additional cysteines.

[0253] In preferred embodiments, Her2, EGFR, Trop2, CDH3 or other TAA Mab contains Fc modification at L234A, L235A, further contains another modification at L328C, and further contains site-specific conjugation at L328C.

[0254] In preferred embodiments, the stretcher unit is located at L328C.

[0255] VIII.) Amino acid unit The amino acid unit (-W-), when present, connects the stretcher unit to the spacer unit if a spacer unit is present, connects the stretcher unit to the drug moiety if no spacer unit is present, and connects the antibody unit to the drug unit if neither a stretcher unit nor a spacer unit is present.

[0256] In certain embodiments, the amino acid unit can include natural amino acids. In other embodiments, the amino acid unit can include non-natural amino acids.

[0257] In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes including cancer or tumor-related proteases to release the drug unit (-D), and in one embodiment, is protonated in vivo upon release to provide the drug (D).

[0258] In preferred embodiments, the amino acid unit is located at L328C.

[0259] IX.) Spacer unit The spacer unit (-Y-), when present and when an amino acid unit is present, links the amino acid unit to the drug unit. Alternatively, the spacer unit links the stretcher unit to the drug unit when no amino acid unit is present. The spacer unit also links the drug unit to the antibody unit when neither an amino acid unit nor a stretcher unit is present. The spacer unit is of two general types: non-self-sacrificing or self-sacrificing. Examples of possible spacers of the present invention are known in the art. See TOKI et al., 2002, J. Org. Chem. 67:1866-1872 and Nature Biotechnology 21(7):778-784).

[0260] Other examples of self-sacrificing spacers include, but are not limited to, 2-aminoimidazole-5-methanol derivatives (HAY et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and aromatic compounds electronically similar to PAB groups such as ortho or para-aminobenzyl acetals. Substituted and unsubstituted 4-aminobutyric acid amides (RODRIGUES et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (STORM et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amides (AMSBERRY et al., 1990, J. Org. Chem. 55:5867), etc., spacers that undergo cyclization upon amide bond hydrolysis can be used. The elimination of amine-containing drugs substituted at the a-position of glycine (KINGSBURY et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-sacrificing spacer.

[0261] In a preferred embodiment, the amino acid unit is located at L328C.

[0262] X.) Drug unit The drug unit (D) can be any therapeutic agent. For example, the drug unit can be a cytotoxic agent, a cytostatic agent, or an immunomodulatory (e.g., immunosuppressive) agent or a chemotherapeutic agent. D is a drug unit (moiety) having atoms capable of forming a bond with a spacer unit (if present), an amino acid unit (if present), a stretcher unit (if present), or an antibody unit. In some embodiments, the drug unit D has a nitrogen atom capable of forming a bond with a spacer unit (if used). As used herein, the terms "drug unit" and "drug moiety" are synonymous and are used interchangeably.

[0263] XI.) Method for Determining the Cytotoxic Effect of an ADC Methods for determining whether a drug or antibody-drug conjugate exerts a cytostatic and / or cytotoxic effect on cells are known. Generally, the cytotoxic or cytostatic activity of an ADC can be measured by exposing mammalian cells expressing the target protein of the antibody drug conjugate in cell culture medium, culturing the cells for about 6 hours to about 5 days, and measuring cell viability. Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the antibody drug conjugate.

[0264] To determine whether an ADC exerts a cytostatic effect, a thymidine incorporation assay can be used. For example, cancer cells expressing the target antigen can be cultured at a density of 5,000 cells per well in a 96-well plate for 72 hours and exposed to 0.5 μCi of 3 H-thymidine during the last 8 hours of the 72-hour period. Incorporation of 3 H-thymidine into the cells of the culture is measured in the presence and absence of the ADC.

[0265] To determine cell injury, necrosis or apoptosis (programmed cell death) can be measured. Necrosis typically involves an increase in plasma membrane permeability, cell swelling, and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. Determination of any of these effects on cancer cells indicates that the ADC is useful for the treatment of cancer.

[0266] Cell viability can be measured by determining the uptake of dyes such as neutral red, trypan blue, or ALAMAR™ Blue (e.g., PAGE et al., 1993, Intl. J. Oncology 3:473-476) in the cells. In such assays, the cells are incubated in a medium containing the dye, the cells are washed, and the remaining dye, which reflects the cellular uptake of the dye, is measured spectrophotometrically. Cytotoxicity can also be measured using the protein-binding dye sulforhodamine B (SRB) (SKEHAN et al., 1990, J. Natl. Cancer Inst. 82:1107-12).

[0267] Alternatively, tetrazolium salts such as MTT are used in quantitative colorimetric assays for the survival and proliferation of mammalian cells by detecting living cells but not dead cells (e.g., MOSMANN, 1983, J. Immunol. Methods 65:55-63).

[0268] Apoptosis can be quantified, for example, by measuring DNA fragmentation.

[0269] Commercially available photometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0270] Apoptosis can also be determined by measuring the morphological changes of cells. For example, similar to necrosis, the loss of plasma membrane integrity can be determined by measuring the uptake of specific dyes (e.g., fluorescent dyes, such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described in Duke and Cohen, Current Protocols in Immunology (COLIGAN et al., eds., 1992, pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide or propidium iodide), and the cells are observed for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage.

[0271] The presence of apoptotic cells can be measured in both the adherent and "floating" compartments of the culture. For example, both compartments can be collected by removing the supernatant, trypsinizing the adherent cells, combining the preparations after a centrifugation wash step (e.g., 2000 rpm for 10 minutes), and detecting apoptosis (e.g., by measuring DNA fragmentation). (See, for example, PIAZZA et al., 1995, Cancer Research 55:3110-16.)

[0272] In vivo, the effects of Her2, EGFR, Trop2, CDH3, or other TAA MAb therapeutic compositions can be evaluated in appropriate animal models. For example, xenogeneic cancer models can be used in which cancer explants or passaged xenograft tissues are introduced into immunocompromised animals such as nude mice or SCID mice (KLEIN et al., 1997, Nature Medicine 3:402-408). For example, PCT Patent Application International Publication No. 98 / 16628 and U.S. Patent No. 6,107,540 describe various xenograft models of human prostate cancer that can recapitulate the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late-stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression, or metastasis, etc.

[0273] In vivo assays for evaluating the promotion of apoptosis are useful for the evaluation of therapeutic compositions. In one embodiment, xenografts from mice having tumors treated with the therapeutic composition may be examined for the presence of apoptotic foci and compared to mice having untreated control xenografts. The degree to which apoptotic foci are found in the tumors of treated mice provides an indication of the therapeutic efficacy of the composition.

[0274] The therapeutic compositions used in the practice of the methods described above can be formulated into pharmaceutical compositions containing carriers suitable for the desired delivery method. Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solution, bacteriostatic water, etc. (see generally Remington’s Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

[0275] The therapeutic agent can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, topical, etc. Preferred formulations for intravenous injection contain the therapeutic composition in bacteriostatic water for injection, sterile non-preserved water solutions, and / or are diluted in polyvinyl chloride or polyethylene bags containing 0.9% sterile sodium chloride for injection, USP. Therapeutic protein preparations can be lyophilized, stored as sterile powders, preferably under vacuum, and then reconstituted with bacteriostatic water (e.g., containing benzyl alcohol preservative) or sterile water prior to injection.

[0276] The dosages and administration protocols for treating cancer using the methods described above vary depending on the method and the target cancer and generally depend on many other factors understood in the art.

[0277] In one embodiment, the pharmaceutical composition of the invention can comprise two or more of the ADCs of the invention by modification of Her2, EGFR, Trop2, CDH3, or other TAA MAb. For example, the invention includes a pharmaceutical composition comprising an ADC of the invention, wherein the Her2, EGFR, Trop2, CDH3, or other TAA MAb is an antibody in which the C-terminal lysine has been partially or completely removed, an antibody having an N-terminal post-translational modification, an antibody lacking the heavy chain C-terminal lysine and having an N-terminal post-translational modification, and / or an antibody having a heavy chain C-terminal lysine and no N-terminal post-translational modification.

[0278] In a preferred embodiment, the Her2, EGFR, Trop2, CDH3, or other TAA MAb is a triple mutation at L234A, L235A, and L328C, where L328C is the position for site-specific conjugation.

[0279] XII.) Treatment of cancers (s) expressing Her2, EGFR, Trop2, CDH3 and TAA Although typically expressed in a limited set of tissues or cells, the identification of Her2, EGFR, Trop2, CDH3, or other TAAs as proteins (plural) that are also expressed in cancers such as those listed in Table I opens up numerous therapeutic approaches for the treatment of such cancers.

[0280] Notably, targeted anti-tumor therapies have been useful even when the targeted protein is expressed in normal tissues or cells, and even in normal organ tissues necessary for life support. Organs necessary for life support are organs necessary for maintaining life, such as the heart or colon. Organs not necessary for life support are organs that can be removed, and as a result, the individual can still survive. Examples of organs not necessary for life support are the ovaries, breasts, and prostate.

[0281] Expression of the target protein in normal tissues (and further, normal tissues necessary for life support) does not lose the usefulness of the targeting agent for the protein as a therapeutic agent for a specific tumor in which the protein is also overexpressed. For example, expression in organs necessary for life support is not harmful per se. Furthermore, organs considered non-essential, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some organs necessary for life support are not affected by normal organ expression due to immune privilege. Immune privileged organs are organs that are protected from the blood by the blood-organ barrier and thus inaccessible to immunotherapy. Examples of immune privileged organs are the brain and testis.

[0282] Accordingly, therapeutic approaches that inhibit the activity of Her2, EGFR, Trop2, CDH3, or other TAA proteins are useful for patients suffering from cancers that express Her2, EGFR, Trop2, CDH3, or other TAAs (such as, for example, the cancers shown in Table I). These therapeutic approaches are generally classified into three classes. The first class modulates Her2, EGFR, Trop2, CDH3, or other TAA function when it relates to tumor cell growth, resulting in inhibition or delay of tumor cell growth, or induction of its killing. The second class includes various methods for inhibiting the binding or association of Her2, EGFR, Trop2, CDH3, or other TAA proteins with their binding partners or with other proteins. The third class includes various methods for inhibiting the transcription of Her2, EGFR, Trop2, CDH3, or other TAA genes or the translation of Her2, EGFR, Trop2, CDH3, or other TAA mRNAs.

[0283] Accordingly, cancer patients can be evaluated for the presence and level of Her2, EGFR, Trop2, CDH3, or other TAA expression, preferably using immunohistochemical evaluation of tumor tissue, quantitative Her2, EGFR, Trop2, CDH3, or other TAA imaging, or other techniques that reliably demonstrate the presence and extent of Her2, EGFR, Trop2, CDH3, or other TAA expression. If applicable, immunohistochemical analysis of a tumor biopsy or surgical specimen is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

[0284] XIII.) Combination Therapy In one embodiment, there is a synergistic effect when a tumor, including a human tumor, is treated with a chemotherapeutic agent or radiation or a combination thereof in combination with a Her2, EGFR, Trop2, CDH3, or other TAA ADC. In other words, the inhibition of tumor growth by Her2, EGFR, Trop2, CDH3 or other TAA ADC is enhanced more than expected when combined with a chemotherapeutic agent or radiation or a combination thereof. The synergistic effect can be shown, for example, by a greater inhibition of tumor growth by the combination treatment than expected from the additive effect of treatment with only Her2, EGFR, Trop2, CDH3, or other TAA ADC, or treatment with Her2, EGFR, Trop2, CDH3, or other TAA ADC and a chemotherapeutic agent or radiation. Preferably, the synergistic effect is demonstrated by remission of a cancer for which remission is not expected from either treatment with Her2, EGFR, Trop2, CDH3, or other TAA ADC, or treatment with an additive combination of Her2, EGFR, Trop2, CDH3, or other TAA ADC and a chemotherapeutic agent or radiation.

[0285] A method of inhibiting the growth of tumor cells using Her2, EGFR, Trop2, CDH3, or other TAA ADC, and a combination of chemotherapy or radiation, or both, includes administering Her2, EGFR, Trop2, CDH3, or other TAA ADC before, during, or after the initiation of chemotherapy or radiation therapy, and any combination thereof (i.e., before and during chemotherapy and / or radiation therapy, before and after, during and after, or before, during, and after). For example, Her2, EGFR, Trop2, CDH3, or other TAA ADC is typically administered for 1 to 60 days, preferably 3 to 40 days, more preferably 5 to 12 days before initiating radiation therapy and / or chemotherapy. However, depending on the treatment protocol and the specific patient needs, the method is implemented to provide the most effective treatment and ultimately extend the patient's lifespan.

[0286] Administration of chemotherapeutic agents can be achieved by various methods including systemic administration via parenteral and enteral routes. In one embodiment, Her2, EGFR, Trop2, CDH3, or other TAA ADCs and chemotherapeutic agents are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol and combinations thereof.

[0287] A radiation source used in combination with Her2, EGFR, Trop2, CDH3, or other TAA ADCs can be either external or internal to the patient being treated. When the radiation source is external to the patient, the treatment is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT). In one embodiment, the radiation therapy is boron neutron capture therapy. In one embodiment, the radiation is proton boron fusion therapy.

[0288] The above treatment regimen can be further combined with additional cancer treatment agents and / or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful for treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies described in WO 2005 / 092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

[0289] When a mammal is subjected to further chemotherapy, the above chemotherapy agent can be used. Additionally, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogens can be used. For example, antihormonal agents such as antiestrogen agents such as Nolvadex (tamoxifen) or antiandrogen agents such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) can be used.

[0290] The above treatment approach can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The treatment approach of the present invention can enable the use of low-dose chemotherapy (or other therapies) and / or low-frequency administrations, which is advantageous for all patients, particularly those who are not sufficiently tolerant of the toxicity of chemotherapy agents.

[0291] XIV.) Kit / Manufactured Product For use in the laboratories, prognoses, prophylaxis, diagnosis and therapeutic applications described herein, the kit is within the scope of the present invention. Such a kit can comprise a transport container, package, or container compartmentalized to receive one or more containers such as vials, tubes, etc., each of the container(s) comprising one of the separate elements used in the method, together with a label or insert containing instructions for use such as the uses described herein. For example, the container(s) can comprise Her2, EGFR, Trop2, CDH3 or other TAA Mab of the present disclosure or several Her2, EGFR, Trop2, CDH3 or other TAA Mab. The kit can comprise a container containing a drug unit. The kit can comprise all or part of Her2, EGFR, Trop2, CDH3, or other TAA ADC, and / or a diagnostic assay for detecting cancer and / or other immunological disorders.

[0292] The kits of the present invention typically comprise the above-described containers, as well as buffers, diluents, filters, needles, syringes; transport containers, packages, containers, vials and / or tube labels listing the contents and / or instructions for use, and one or more other containers associated therewith, including the materials desirable from a commercial and user perspective.

[0293] Labels can be present on or with the container to indicate that the composition is for use in a particular therapeutic or non-therapeutic use, such as prognostic, prophylactic, diagnostic or laboratory use, and can also indicate instructions for use in either in vivo or in vitro, such as those described herein. The instructions and / or other information can also be included in an insert(s) or label(s) included on or with the kit. The label can be on the container or associated with the container. The label can be on the container if the letters, numbers or other characters forming the label are molded or etched into the container itself, and the label can be present within the receptacle or shipping container holding the container, for example, associated with the container as an enclosure. The label can indicate that the composition is used for diagnosing, treating, preventing or prognosticating a condition such as cancer or other immunological disorders.

[0294] The terms "kit" and "manufactured article" can be used synonymously.

[0295] In another embodiment of the invention, a manufactured article(s) containing a composition such as a Her2, EGFR, Trop2, CDH3, or other TAA ADC of the present disclosure. The manufactured article typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from various materials such as glass, metal or plastic. The container can hold one or several Her2, EGFR, Trop2, CDH3, or other TAA ADC, and / or one or more therapeutic doses of Her2, EGFR, Trop2, CDH3, or other TAA ADC.

[0296] The container can alternatively hold a composition effective for treatment, diagnosis, prognosis or prevention of a condition and can have a sterile access port (e.g., the container can be a vial having a stopper pierceable by an intravenous solution bag or a hypodermic needle). The active agent in the composition can be a Her2, EGFR, Trop2, CDH3, or other TAA Mab or ADC of the present disclosure.

[0297] The product can further include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and / or dextrose solution. It can further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and package inserts with instructions for indications and / or use.

[0298] Exemplary embodiments Among the provided embodiments are the following: 1) An antibody composition comprising a triple mutation, wherein the triple mutation comprises L234A modification, L235A modification and L328C modification, and the triple mutation modifies Fcγ receptor binding and antibody effector function.

[0299] 2) The antibody according to claim 1, wherein the antibody comprises an EGFR antibody.

[0300] 3) The antibody according to claim 1, wherein the antibody comprises a Her2 antibody.

[0301] 4) The antibody according to claim 1, wherein the antibody comprises a Trop2 antibody.

[0302] 5) The antibody according to claim 1, wherein the antibody comprises a CDH3 antibody.

[0303] 6) The antibody according to claim 1, wherein the antibody comprises a GPNMB antibody.

[0304] 7) The antibody according to claim 1, wherein the antibody comprises a DLL3 antibody.

[0305] 8) The antibody according to claim 1, wherein the antibody comprises an ENPP3 antibody.

[0306] 9) The antibody according to claim 1, wherein the antibody comprises a SLITRK6 antibody.

[0307] 10) The antibody according to claim 1, wherein the antibody comprises a CA9 antibody.

[0308] 11) The antibody according to claim 1, wherein the antibody comprises a PSMA antibody.

[0309] 12) The antibody according to claim 1, wherein the antibody comprises a CDH6 antibody.

[0310] 13) The antibody according to claim 1, wherein the antibody comprises a glypican 3 antibody.

[0311] 14) The antibody according to claim 1, wherein the antibody comprises an EDNRB antibody.

[0312] 15) The antibody according to claim 1, wherein the antibody comprises a nectin-4 antibody.

[0313] 16) The antibody according to claim 1, wherein the antibody comprises an SLC34A2 antibody.

[0314] 17) The antibody according to claim 1, wherein the antibody comprises a Her3 antibody.

[0315] 18) The antibody according to claim 1, wherein the antibody comprises an NRP1 antibody.

[0316] 19) The antibody according to claim 1, wherein the antibody comprises a tumor-associated antigen (TAA) antibody.

[0317] 20) The Her2 antibody according to claim 3, further comprising an antibody heavy chain comprising SEQ ID NO: 2.

[0318] 21) The Her2 antibody according to claim 3, further comprising an antibody heavy chain comprising SEQ ID NO: 3.

[0319] 22) The Her2 antibody according to claim 3, further comprising an antibody heavy chain comprising SEQ ID NO: 4.

[0320] 23) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 7.

[0321] 24) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 8.

[0322] 25) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 9.

[0323] 26) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 12.

[0324] 27) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 13.

[0325] 28) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 14.

[0326] 29) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 17.

[0327] 30) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 18.

[0328] 31) The EGFR antibody according to claim 2, further comprising an antibody heavy chain comprising SEQ ID NO: 19.

[0329] 32) The Trop2 antibody according to claim 4, further comprising an antibody heavy chain comprising SEQ ID NO: 22.

[0330] 33) The Trop2 antibody according to claim 4, further comprising an antibody heavy chain comprising SEQ ID NO: 23.

[0331] 34) The Trop2 antibody according to claim 4, further comprising an antibody heavy chain comprising SEQ ID NO: 24.

[0332] 35) The CDH3 antibody according to claim 5, further comprising an antibody heavy chain comprising SEQ ID NO: 27.

[0333] 36) The CDH3 antibody according to claim 5, further comprising an antibody heavy chain comprising SEQ ID NO: 28.

[0334] 37) The CDH3 antibody according to claim 5, further comprising an antibody heavy chain comprising SEQ ID NO: 29.

[0335] 38) An antibody-drug conjugate (ADC) comprising: (i) an antibody composition comprising a triple mutation, wherein the triple mutation comprises an L234A modification, an L235A modification and an L328C modification, and the triple mutation modifies the antibody effector function; (ii) a linker; (iii) a drug unit, wherein the drug unit is specifically conjugated at site L328C.

[0336] 39) The ADC according to claim 38, wherein the antibody composition comprises an EGFR antibody.

[0337] 40) The ADC according to claim 38, wherein the antibody composition comprises a Her2 antibody.

[0338] 41) The ADC according to claim 38, wherein the antibody composition comprises a Trop2 antibody.

[0339] 42) The antibody according to claim 38, wherein the antibody comprises a CDH3 antibody.

[0340] 43) The ADC according to claim 38, wherein the antibody composition comprises a tumor-associated antigen (TAA) antibody.

[0341] 44) The ADC according to claim 38, wherein the TAA antibody is shown in Table IV.

[0342] 45) The ADC according to claim 38, further comprising a stretcher unit.

[0343] 46) The ADC according to claim 38, further comprising a spacer unit.

[0344] 47) The ADC according to claim 38, further comprising an amino acid unit.

[0345] 48) A manufactured product comprising the antibody according to claim 1.

[0346] 49) A manufactured product comprising the ADC according to claim 38.

[0347] 50) A pharmaceutical composition comprising a therapeutically effective amount of the ADC according to claim 38 and a pharmaceutically acceptable excipient.

[0348] 51) A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to claim 1 and a pharmaceutically acceptable excipient.

[0349] 52) A method of treating cancer in an individual, comprising: (i) administering to the individual a therapeutically effective amount of the ADC according to claim 38, wherein the cancer comprises cells expressing the cancer shown in Table I.

[0350] 53) A method of treating cancer in an individual, comprising: (i) administering to the individual a therapeutically effective amount of the antibody according to claim 1, wherein the cancer comprises cells expressing the cancer shown in Table I.

[0351] 54) A method of treating a disease in an individual, comprising: (i) administering to the individual a therapeutically effective amount of the ADC according to claim 38, wherein the disease comprises cells expressing the cancer shown in Table V.

[0352] 55) A method of treating a disease in an individual, comprising: (i) A method comprising administering to the individual a therapeutically effective amount of the antibody according to claim 1, wherein the disease is a cancer expressed by the cells shown in Table V.

[0353] 56) An antibody-boron conjugate (ABC), comprising: (i) An antibody composition comprising a triple mutation, wherein the triple mutation comprises an L234A modification, an L235A modification and an L328C modification, and the triple mutation modifies the antibody effector function; (ii) A linker; (iii) A drug unit, wherein the drug unit comprises a borylated composition and the drug unit is specifically conjugated at site L328C.

[0354] 57) The ABC according to claim 56, wherein the antibody composition comprises an EGFR antibody.

[0355] 58) The ABC according to claim 56, wherein the antibody composition comprises a Her2 antibody.

[0356] 59) The ABC according to claim 56, wherein the antibody composition comprises a Trop2 antibody.

[0357] 60) The ABC according to claim 56, wherein the antibody composition comprises a CDH3 antibody.

[0358] 61) The antibody according to claim 56, wherein the antibody comprises a GPNMB antibody.

[0359] 62) The antibody according to claim 56, wherein the antibody comprises a DLL3 antibody.

[0360] 63) The antibody according to claim 56, wherein the antibody comprises an ENPP3 antibody.

[0361] 64) The antibody according to claim 56, wherein the antibody comprises a SLITRK6 antibody.

[0362] 65) The antibody according to claim 56, wherein the antibody comprises a CA9 antibody.

[0363] 66) The antibody according to claim 56, wherein the antibody comprises a PSMA antibody.

[0364] 67) The antibody according to claim 56, wherein the antibody comprises a CDH6 antibody.

[0365] 68) The antibody according to claim 56, wherein the antibody comprises a glypican 3 antibody.

[0366] 69) The antibody according to claim 56, wherein the antibody comprises an EDNRB antibody.

[0367] 70) The antibody according to claim 56, wherein the antibody comprises a nectin-4 antibody.

[0368] 71) The antibody according to claim 56, wherein the antibody comprises an SLC34A2 antibody.

[0369] 72) The antibody according to claim 56, wherein the antibody comprises a Her3 antibody.

[0370] 73) The antibody according to claim 56, wherein the antibody comprises an NRP1 antibody.

[0371] 74) The ABC according to claim 56, wherein the antibody composition comprises a tumor-associated antigen (TAA) antibody.

[0372] 75) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 2.

[0373] 76) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 3.

[0374] 77) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 4.

[0375] 78) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 7.

[0376] 79) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 8.

[0377] 80) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 9.

[0378] 81) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 12.

[0379] 82) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 13.

[0380] 83) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 14.

[0381] 84) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 17.

[0382] 85) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 18.

[0383] 86) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 19.

[0384] 87) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 22.

[0385] 88) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 23.

[0386] 89) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 24.

[0387] 90) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 27.

[0388] 91) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 28.

[0389] 92) The ABC according to claim 56, further comprising an antibody heavy chain comprising SEQ ID NO: 29.

[0390] 93) The ABC according to claim 56, further comprising a stretcher unit.

[0391] 94) The ABC according to claim 56, further comprising a spacer unit.

[0392] 95) The ABC according to claim 56, further comprising an amino acid unit.

[0393] 96) A pharmaceutical composition comprising a therapeutically effective amount of the ABC according to claim 56 and a pharmaceutically acceptable excipient.

[0394] 97) A method of treating cancer in an individual, (i) comprising administering to the individual a therapeutically effective amount of the ABC according to claim 56, wherein the cancer comprises cells expressing the cancer shown in Table I.

Example

[0395] Various aspects of the present invention are further illustrated and exemplified by several of the following examples, none of which are intended to limit the scope of the present invention.

[0396] Example 1: A method for generating an antibody. Anti-Her2, anti-EGFR, anti-Trop2 and anti-CDH3 mAbs were generated by obtaining the amino acid sequences and performing codon optimization on the corresponding nucleotide sequences. (See Table VI - Antibody Sequences 1 - 30). Gene fragments were synthesized and cloned into the restriction enzyme sites of the dual HC and LC expression vector cassettes for stable expression. For transient constructs, separate expression vectors for HC and LC were generated and co-transfected at a 1:3 HC to LC ratio. Fc variants were constructed by either site-directed mutagenesis or partial gene fragment synthesis followed by subcloning using techniques known in the art. Stable or transient expression vectors were constructed and purified using an endotoxin-free DNA purification kit prior to transfection. Stably transfected Chinese Hamster Ovary (CHO) cells were subjected to a selection and recovery process for the generation of stable pools expressing the recombinant antibody and Fc variants. For antibody production, a fed-batch production process with a typical culture period of 8 - 12 days was used for the stably transfected pools, and for transient expression, the transfected cells were cultured for 6 - 8 days after transfection and then recovered. Subsequently, protein A affinity purification was performed on the recovered cell culture fluid, and the purified material was buffer exchanged into phosphate-buffered saline (PBS). The quality of the recombinant antibody can be evaluated by size exclusion chromatography, SDS-PAGE and other methods known in the art.

[0397] Example 2: Single and / or triple mutant mAbs do not affect target binding. Target antigen binding was evaluated using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with 0.05 - 0.075 μg of recombinant human soluble extracellular domain (ECD) of Her2 / ErbB2 or EGFR or Trop2 (Sino Biologicals, Inc., Beijing, China) or 0.075 μg of recombinant human CDH3 full length (Abnova, Taiwan). Subsequently, the plates were washed and blocked with ELISA blocking buffer. Then, the plates were incubated at room temperature for 2 (2) hours with test samples (i.e., wild type or Fc mutants of anti-HER2, anti-EGFR, anti-TROP2 and anti-CDH3 mAbs) serially diluted 3-fold in 1×PBS containing 1% BSA and 0.05% Tween®-20, at concentrations ranging from 0.0001 - 6.667 nM for anti-HER2 and anti-EGFR mAbs and from 0.0003 - 20 nM for anti-TROP2 and anti-CDH3 mAbs. After washing to remove unbound antibody, the bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-human Fc specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells. In those wells, color development was proportional to the amount of test sample. The optical density (OD) of the color was measured at 650 nm and used to determine the amount of test sample bound to the target antigen. Detectable binding of the antibodies over the range of test concentrations was analyzed using one-site binding non-linear regression analysis with GraphPad Prism.

[0398] The results show that mAbs with single mutation L328C, double mutations L234A and L235A, and triple mutations L234A, L235A and L328C bind to the target and that the triple mutation does not affect target binding, as in the case of single and double mutations, when compared to the wild type (Figs. 1, 2, 3, 4, 5 and 6).

[0399] Example 3: Inhibition of FcγRI binding is observed for the triple mutant MAb. FcγRI binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with recombinant human soluble ECD of CD64 / FCGRIA (Sino Biological, Inc. Beijing, China) in 1×PBS (pH 7.4) coating buffer. The plates were then washed and blocked with ELISA blocking buffer. The plates were incubated at room temperature for 2 (2) hours with test samples (i.e., trastuzumab, panitumumab, cetuximab, nimotuzumab, sacituzumab, or wild-type or Fc variants of anti-CDH3(5836)) in a concentration range of 0.005 - 80 μg / ml (or 0.002 - 30 μg / ml) that were serially diluted 4-fold in 1×PBS containing 1% BSA and 0.05% Tween®-20. After washing to remove unbound antibody, the bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-human Fc specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells. Color develops in those wells in proportion to the amount of test sample. The optical density (OD) of the color was measured at 650 nm and used to determine the amount of test sample bound to FcγRI. Detectable binding of the antibody over the range of test concentrations was analyzed using one-site binding non-linear regression analysis with GraphPad Prism.

[0400] The results show that substantial inhibition of FcγRI binding was observed for the triple mutant compared to the wild-type, single mutant L328C and double mutants L234A and L235A (Figures 7, 8, 9, 10, 11 and 12).

[0401] Example 4: Inhibition of FcγRIIA (CD32) binding is observed for the triple mutant MAb. The binding of antibodies to the low-affinity FcyRIIA receptor was determined by modified sandwich format ELISA. Briefly, microtiter plates were coated with 100 μl / well of anti-his tag antibody (Novus, NBP1-25939 lot#A4) at 4 μg / ml. The wells were then blocked with PBS containing BSA. Serial dilutions of Fc variants mixed with FcγRIIa at different ratios (CD32) (Sino Biological, Inc. Beijing, China, cat#10374-H08H) were then added and the plates were incubated overnight at 4 °C. After washing with PBS, a 1:3,000 dilution of horseradish peroxidase (HRP)-labeled goat anti-hIgG Fc, Jackson ImmunoResearch) in PBS-T containing 1% BSA was added to detect His tag-captured FcyRIIA and Mab complexes. Results were visualized by the addition of tetramethylbenzidine (TMB) and the optical density (OD) was measured at 650 nm. Detectable binding of antibodies to FcyRIIA over the range of Fc variant test concentrations was represented and data captured by anti-his tag Ab were analyzed by one-site binding non-linear regression analysis using GraphPad Prism software.

[0402] Results show that for all antibodies tested, suppression of FcγRIIA binding was observed for the triple mutant and single mutant L328C compared to the wild type and double mutants L234A and L235A (Figures 13, 14, 15, and 16).

[0403] Example 5: Inhibition of FcγRIIIa binding was observed for the triple mutant MAb. FcγRIIIa binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with recombinant human soluble ECD of hCD16a / FCGRIIIA (Sino Biological, Inc., Beijing, China) in 1×PBS (pH 7.4) coating buffer. The plates were then washed and blocked with ELISA blocking buffer. The plates were incubated for 2 (two) hours at room temperature with test samples (i.e., wild-type or Fc variants of trastuzumab, panitumumab, cetuximab or nimotuzumab) serially diluted 4-fold in 1×PBS containing 1% BSA, 0.05% Tween®-20 in a concentration range of 0.005 - 80 μg / ml. After washing to remove unbound antibody, the bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-human Fc specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells. In those wells, color development was proportional to the amount of test sample. The optical density (OD) of the color was measured at 650 nm and used to determine the amount of test sample bound to FcγRIIa. Detectable binding of antibodies across the range of test concentrations was analyzed using one-site binding non-linear regression analysis with GraphPad Prism.

[0404] The results show that substantial inhibition of FcγRIIIa binding was observed for the triple mutant and single mutant L328C compared to wild-type and double mutants L234A and L235A for all antibodies tested (Figures 17, 18, 19, 20, 21, and 22).

[0405] Example 6: Inhibition of FcRn binding is not substantially affected by Fc variants and / or triple mutant Mabs. FcRn binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with recombinant human FcRn (Sino Biological, Inc., Beijing, China) in 1×PBS (pH 6.0) coating buffer. The plates were then washed and blocked with ELISA blocking buffer. The plates were incubated for 2 hours at room temperature with test samples (i.e., trastuzumab, panitumumab, cetuximab or nimotuzumab, sacituzumab, or wild-type or Fc variants of anti-CDH3(5836)) serially diluted 3-fold in 1×PBS (pH 6.0) containing 1% BSA and 0.05% Tween®-20 in a concentration range of 0.04 - 90 μg / ml (or 0.04 - 30 μg / ml). After washing to remove unbound antibody, the bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-human Fc specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells. In those wells, color development was proportional to the amount of test sample. The optical density (OD) of the color was measured at 650 nm and used to determine the amount of test sample bound to FcRn. Detectable binding of antibodies over the range of test concentrations was analyzed using one-site binding non-linear regression analysis with GraphPad Prism.

[0406] The results show that inhibition of FcRn binding was not substantially affected by Fc variants including, but not limited to, triple mutant antibodies, compared to wild-type antibodies (Figures 23, 24, 25, 26, 27, and 28).

[0407] Example 7: Kinetic and affinity analysis for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and FcRn using the Octet HTX system The binding affinities of different Fc receptors for anti-Her2 antibodies and Fc variants were measured using the Octet HTX System (Molecular Devices) at 25 °C. Briefly, a panel of human FcR His-tagged recombinant proteins (FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA-F, FcγRIIIA-V, FcγRIIIB, and FcRn) was loaded onto an anti-Penta His (H1S1K) biosensor. The loaded sensors were immersed in serial dilutions (starting at 300 nM, 1:2 dilution, 7 points) of Mab test samples in PBS at pH 7.4 containing 0.1% BSA, 0.02% Tween®-20 or in a buffer composed at pH 6.0 for FcRn analysis. Kinetic constants were calculated using a monovalent (1:1) binding model. Antibodies known to bind to the FcR panel were used as positive controls. k dis / k on The equilibrium dissociation constant (K D ), defined as the ratio of, was determined by analyzing sensogram curves obtained at several different concentrations.

[0408] The results, as summarized in Figure 29, show the effect of introducing single or triple mutations into the Fc domain on binding to FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA (F158 and V158 variants), and FcyRIIIB, as well as the effect on FcRn binding. For FcγRI binding, the substantial decrease in K D values observed for the triple mutants was mainly due to an increase in the antibody dissociation rate (k d ) (Figure 29). The rapid dissociation of the triple mutants from their antigen is also shown in the chromatogram (Figure 30). Only a slight decrease in the association rate (k on ) was observed for the triple mutants compared to wild-type or single mutant antibodies. Due to undetectable binding of the triple mutants, K d values could not be determined for the other Fcγ receptors (Figure 29). In contrast, the K DThe value is not impaired for the triple mutant, suggesting that the decrease in binding affinity for the triple mutant is specific to the Fc gamma receptor isoform.

[0409] Example 8: Kinetic analysis of binding to FcγRI (dissociation rate) using Octet. Additional binding experiments were performed at 25 °C on an Octet HTX. Briefly, a panel of human FcR His-tagged recombinant proteins (FcRI) was loaded onto anti-Penta His (H1S1K) biosensors. The loaded sensors were immersed in serial dilutions (starting at 300 nM, 1:2 dilution, 7 points) of Mab test samples in PBS at pH 7.4 containing 0.1% BSA, 0.02% Tween®-20 or in a buffer composed at pH 6.0 for FcRn analysis. Rate constants were calculated using a monovalent (1:1) binding model. An antibody known to bind to the FcR panel was used as a positive control.

[0410] The results show that the triple mutant dissociation rate is faster for FcγRI when compared to the wild type and single mutants (Figure 30).

[0411] Example 9: ADCC analysis of Fc variants using flow cytometry. To evaluate Fc variant-mediated antibody-dependent cell cytotoxicity (ADCC), flow cytometry ADCC assay was used. Briefly, human cancer cells were used as target cells and cryopreserved PBMCs were used as effector cells. Human cancer cells were labeled with 8 uM carboxyfluorescein succinimidyl ester (CFSE) dye (Selleckchem, cat# S8269 lot# S826901) for 30 minutes, washed twice with cell culture medium, added to U-bottom 96-well plates, and appropriate concentrations of Fc variants were added. ADCC was initiated by adding human PBMCs as effector cells (effector:target (E:T) ratio of 4 - 8:1). The plates were further incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. Cells were washed twice and stained with a 1:500 dilution of Fixable Viability Dye (FVD, eBioscience, CA) to stain dead cells. After incubation for 30 minutes, cells were washed with PBS containing 2% FBS and then subjected to flow cytometry analysis using Attune Nxt (Thermo Fisher Scientific). For each experiment, measurements were performed in triplicate. % cytotoxicity was determined using the percentage of dead target cells (CFSE positive and FVD positive) among total target cells (CFSE positive). Data were shown as % cytotoxicity bar graphs using GraphPad Prism software.

[0412] The results show a decrease in ADCC with Fc variants that had a significantly reduced triple mutant compared to wild type for each mAb (Figures 31, 32, 33, and 34).

[0413] Example 10: CDC analysis of Fc variants using flow cytometry. Complement-dependent cytotoxicity (CDC) was determined by a lactate dehydrogenase (LDH) release assay using fetal rabbit serum as a complement source and human cancer cells as target cells. Briefly, target cells (20 × 10e per well 3(number) was dispensed into a 96-well U-bottom plate and pre-incubated on ice for 30 minutes with the Fc variant. Then, diluted complement was added and incubation was continued at 37 °C (5% CO2, humidified atmosphere) for 4 hours. The assay was performed in triplicate regardless of the presence or absence of antibody. Maximum release was prepared using target cells lysed with lysis solution. The LDH activity in the supernatant was measured using a non-radioactive cytotoxicity assay kit (Promega cat# G1781). The released LDH activity indicating cell death was determined by optical density reading at 490 nm using a spectrophotometer (Cytation1 Biotek). Percentage cytotoxicity was calculated according to the formula: Cytotoxicity (%) = 100 × (experimental release - spontaneous release) / (maximum release - spontaneous release). The data are shown as a % cytotoxicity bar graph created using GraphPad Prism software.

[0414] The results show that a decrease in CDC was observed for each MAb with Fc variants in which the triple mutants were significantly reduced (Figures 35, 36, 37, and 38).

[0415] Example 11: C1q binding of Fc variants using ELISA. C1q binding was evaluated using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with 1 μg / ml test samples (i.e., trastuzumab, panitumumab, sacituzumab, and wild-type or Fc variants of anti-CDH3-5836) in 1×PBS (pH 7.4) coating buffer, 60 μl / well. The plates were then washed and blocked with ELISA blocking buffer (PBS containing 1% BSA). The plates were incubated for 2 (two) hours at room temperature with diluted human C1q (sigma Cat#C1740 lot#SCC6462) in the concentration range of 0.625 - 40 μg / ml, serially diluted 2-fold in 1×PBS containing 1% BSA and 0.05% Tween®-20. After washing the plates, 5 μg / ml rabbit anti-h C1q (Dako A0136 lot#20047640) in 1% BSA and 0.05% Tween® (PBS-T) in PBS, 60 μl / well was added and incubated for 1.0 hour at room temperature. After washing to remove unbound antibody, the bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit HRP-specific polyclonal antibody (Jackson ImmunoResearch, cat#111-036-046 West Grove, PA). A slow kinetic substrate solution of tetramethylbenzidine (TMB) was added and the optical density (OD) of the color was measured at 650 nm. Data representing detectable binding of the antibody over the range of test concentrations were analyzed by one-site binding non-linear regression analysis using GraphPad Prism software.

[0416] The results show a decrease in C1q binding was observed with the Fc variants and a significant decrease in the triple mutants for each MAb (Figures 39, 40, 41, and 42).

[0417] Example 12: Generation and Characterization of Site-Specific ADCs Generation of Site-Specific Thiol The hinge disulfide of the Fc variant was reduced with DTT in PBS at room temperature for 15 (fifteen) minutes and the antibody was purified from the excess reducing agent by desalting into a pH 6.5 buffer using a PD-10 column. The predicted free thiols were confirmed by Ellman's test. Subsequently, the hinge disulfide was reformed using a 35-fold excess of dehydroascorbic acid (DHA) relative to the antibody concentration. The progress of reoxidation was monitored by Ellman's test. When the thiol-to-antibody ratio reaches approximately 2.0, maleimide-payload is added.

[0418] Preparation of Conjugate To generate the site-specific ADCs of the present invention, antibody conjugates using unique linker(s) designated LOL1 were prepared by adding maleimide-linker to the activated site-specific antibody. The activation process (i.e., liberation of free thiols) is as described above (generation of site-specific thiols). The linker solution in DMSO is added to the thiol with 1.2 molar equivalents of linker and the reaction is stirred at room temperature for 45 - 60 minutes. The resulting conjugate is purified into a histidine formulation buffer (pH 6) using a PD-10 or similar desalting column and analyzed.

[0419] Conjugate Evaluation To verify the ability to produce site - specific ADCs using either a single - variant (i.e., L328C) antibody or a triple - variant (i.e., L234A, L235A, L328C) antibody, three techniques were used: (i) intact mass spectrometry, (ii) peptide mapping, and (iii) reverse - phase HPLC. Reverse - phase HPLC is the main method for cleaving cysteine - based conjugates, and was used to confirm that the payload was conjugated mainly or solely to the heavy chain and only at a single position within the heavy chain, thus ensuring that the product was site - specific. Intact analysis by mass spectrometry was used to confirm that the conjugated heavy chain had the exact predicted mass (i.e., HC + payload). On the other hand, peptide mapping was used to confirm that the payload was tethered to a specific position within the heavy chain. Here, intact mass spectrometry was utilized to demonstrate that the LOL - 1 conjugate anti - HER2 mAb with triple mutations was composed of a single - conjugated heavy chain and an unconjugated light chain (Figure 44). Peptide mapping of both single - variant and triple - variant vcMMAE - conjugated anti - EGFR mAb No.1, each having either a single or triple mutation, revealed that the ACPAPIEK peptide within the heavy chain was both the site of the L328C mutation and the site of conjugation (Figures 54, 55).

[0420] Mass spectrometry is a low - throughput technique, while reverse - phase HPLC is a high - throughput technique that shows the relative composition of ADCs. In monoclonal antibodies, there are only eight (8) cysteines available for conjugation that exist in wild - type human IgG1, making them particularly useful for ADCs conjugated via cysteine side chains and for the conjugates themselves.

[0421] Generally, each Cys-based ADC carries a finite number of payloads that need to be known. ADCs utilizing vcMMAE as the payload are generally accepted to carry 2(2)-4(4) payloads anywhere for being efficient in tumor killing. Further, their compositions are a distribution of payloads in the range of 0 (non-conjugated) - 8 (fully conjugated). Antibodies engineered to carry a defined number (typically two per mAb) of payloads (i.e., site-specific ADCs lacking a defined distribution and high payload-to-mAb species) have been shown to be advantageous in clinical efficacy, safety, or stability.

[0422] Therefore, the inventors use an analytical method in which reverse-phase chromatography assay is the main technique to decouple the composition of the ADC resulting from the conjugation of vcMMAE. The various species (i.e., heavy chain, light chain, and conjugate species) are identified based on both retention time and UV250 / 280 ratio, and the DAR (drug-to-mAb ratio) is calculated. Further, the inventors have shown that the payload is mainly or completely tethered to the heavy chain, and the heavy chain with a single payload is the main species and a major attribute of site-specific conjugation.

[0423] The materials and methods for analysis were performed as follows.

[0424] Intact mass analysis Briefly, the sample was prepared by adding 1 / 20 volume of 200 mM DTT and incubating at 37 °C for 1 hour. The sample was analyzed using an Agilent 1260 LC and an Agilent 6520 Q-TOF mass spectrometer. The liquid chromatography column was an ID 2 mm × length 10 cm column packed with 10 μm PLRP-S polystyrene reverse phase packing. The solvents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was a 1-minute hold at 1% B, a 14-minute ramp to 70% B, a 1-minute ramp to 90% B, a 1-minute hold at 90% B, and a 1-minute ramp back to 1% B. Data were acquired using Agilent Mass Hunter Data Acquisition software and analyzed using Agilent Mass Hunter Qualitative Analysis by Bioconfirm. The reverse stacking was performed using the maximum entropy model.

[0425] The results showed that the LOL1-conjugated anti-HER2 antibody with triple mutations resulted in a distinct retention shift, suggesting that only one positional isomer was formed, indicating very site-specific conjugation (Figure 43). Further confirmation by intact mass spectrometry showed that the above LOL1-conjugated triple mutant anti-HER2 antibody was site-specific (Figure 44). Only the unconjugated light chain (LC) with an average mass of 23,440 Daltons (Da) was detected (Figure 44). The peak at 23,462 Da represents the sodium adduct normally formed in the LC-MS system. For the heavy chain (HC), conjugated species with an average mass of 49,487 Da (reflecting the C-terminal lysine deletion (-K) typically observed in the Chinese hamster ovary (CHO) cell culture system used for antibody production) and a product of 49,613 Da (reflecting the HC with intact untreated C-terminal lysine (+K)) were found. Neither unconjugated HC species nor HC with a second conjugation site was found (Figure 44). In summary, intact mass spectrometry confirmed that (i) only a single LOL1 payload was conjugated to the heavy chain, (ii) no conjugation to the light chain was detected, and (iii) such conjugates could only form with the engineered Cys-328 and not with any of the hinge region cysteines.

[0426] The results in Figures 43 and 44 confirm that the ADC conjugated with the unique payload LOL-1 is 100% site-specific with L328C for the triple Fc variant.

[0427] Reverse-phase chromatography analysis Furthermore, antibodies and / or antibody-drug conjugates were analyzed by reverse-phase chromatography using the following protocol. Briefly, the ADC or mAb was reduced with DTT and analyzed using an Acquity C4 Wide Pore column (100×2.1 mm, 300 Å, Waters) on a Waters H-class UPLC system. The column was maintained at 80 °C. The mAb light and heavy chains were separated with an acetonitrile (0.1% TFA) gradient.

[0428] The results in Figure 45 show that the heavy chain (H1) with a single payload is the major species suggesting that conjugation is site-specific and the payload is present on Cys328 and not within the hinge region. Furthermore, Figure 46 shows the reverse-phase column chromatography profile for the L328C variant of anti-Trop2 Mab conjugated to vcMMAE. Greater than 80% site-specific conjugation is shown to occur on the Cys328 heavy chain.

[0429] Figure 47 shows the reverse-phase column chromatography profile for the L234A, L235A, L328C triple variant of anti-Trop2 Mab conjugated to vcMMAE. Greater than 70% site-specific conjugation is shown to occur on the Cys328 heavy chain. Figure 48 shows the reverse-phase column chromatography profile for the L234A, L235A, L328C variant of anti-EGFR Mab conjugated to vcMMAE. DAR1 is shown to be the predominant species exceeding 80% - 90% of the site-specific conjugation occurring on the Cys328 heavy chain.

[0430] Figure 49 shows peak assignment and DAR calculation based on the RP-HPLC data provided in Figure 49. Using the non-conjugated Mab (control), retention times and UV250 / 280 ratios were identified for both the heavy and light chains. Note that these parameters are required for both peak assignment and DAR determination. The DAR was calculated as follows.

Number

[0431] Figure 50 shows a summary of the analytical attributes including the average DAR, percent monomer peak determined by SEC, and percent of DAR1 species for vcMMAE or LOL1 conjugate mAbs with single or triple Fc variants.

[0432] Peptide mapping To confirm that the payload is tethered at specific positions within the heavy chain, peptide mapping analysis was performed using the following protocol.

[0433] (i) Sample preparation: According to the published procedures known in the art, Mab(s) and ADC(s) were reduced with DTT and the free thiols were alkylated using iodoacetamide. To obtain a tryptic digest peptide map, the above antibodies were digested as follows: The antibodies were reduced with DTT under partially denaturing conditions using 5M guanidine. Iodoacetamide was added at twice the concentration of DTT. The alkylated mAb was purified by desalinating into 100 mM phosphate buffer using a Zeba-Spin (Thermo) column. Trypsin was added to each sample, and the samples were incubated overnight at 37 °C, evaporated to dryness, and the pellet was resuspended in 100 μL of 5% ACN, 95% water, 0.1% formic acid.

[0434] (ii) LC-MS: After preparing the sample, 5.0 μL of the sample was injected into a final 3000 nanoLC equipped with a 75 μm × 2 cm trap column packed with 3 μm bulk resin of C18 (Acclaim PepMap 100, Thermo Scientific) and a 75 μm × 15 cm analytical column containing 2 μm resin of C18 (Acclaim PepMap RSLC, Thermo Scientific). The nanoLC gradient was 3 - 35% of solvent B (A = H2O containing 0.1% formic acid, B = acetonitrile containing 0.1% formic acid) over 40 minutes and 35% - 85% of solvent B over 5 minutes, with a flow rate of 300 nL / min. The nanoLC was combined with a Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The ESI voltage was set to 1.9 kV and the capillary temperature was set to 275 °C. Using an automated gain control (AGC) target of 3×10 6 at m / z 200, full spectra (m / z 350 - 2000) were acquired in profile mode with a resolution of 70,000. The 15 most abundant ions were subjected to fragmentation by high-energy collision dissociation (HCD) with 25 normalized collision energies. The MS / MS spectra were acquired in mass center mode with a resolution of 17,500 at m / z 200. The AGC target for fragment ions was set to 2×10 4 with a maximum injection time of 50 ms. Charge states 1, 7, 8, and those not assigned were excluded from the tandem MS experiments. Dynamic exclusion was set to 45.0 seconds.

[0435] The results in Figure 51 show characteristic daughter ions after fragmentation of the vcMMAE-peptide. Further, Figure 52 shows the sequence coverage of anti-EGFR antibody No 1 with triple mutations conjugated to vcMMAE. Figure 53 shows a representative TIC chromatogram. The differences are indicated by circles. Figure 54 shows the charge states of peptides containing the conjugation site for anti-EGFR antibody No 1 with triple mutations. The unconjugated control mAb has a [M+1] ion with an M / z of 886 that corresponds to the ACPAPIEK peptide from the heavy chain (A). Its +2 ion is shown in the inset. Two prominent daughter ions with M / z of 486 and 654 are shown in (B). In the vcMMAE conjugate, this peptide elutes from the reverse-phase column at a different location due to conjugation to cysteine (see previous figure). The resulting product is identified in C (showing +2 and +3 charge states). The fragment contains the same ions as above (D, black-filled arrows), confirming that this is the same ACPAPIEK peptide. Additional fragments with M / z of 686, 506, and 321 (D, open arrows) belong to VCMMAE and correspond to those in Figure 52. Finally, Figure 55 shows that the conjugation site of anti-EGFR antibody No.1 with a single mutation (L328C) was also confirmed on the ACPAPIEK peptide of the heavy chain.

[0436] Table VII shows a list of the confidence scores of the antibody fragments and peptide maps for the single mutants of the vcMMAE-conjugated anti-EGFR antibody No.1. Table VIII shows a list of the confidence scores of the antibody fragments and peptide maps for the triple mutants of the vcMMAE-conjugated anti-EGFR antibody No.1.

[0437] Example 13: Cytotoxicity of Fc single mutants and / or triple mutant conjugates of vcMMAE The cytotoxic effects of Fc single mutants or triple mutant antibodies conjugated with vcMMAE against tumor cell lines were measured using the CellTiterGLo assay kit (Promega cat#G7571). The CellTiterGlo® Luminescent Cell Viability Assay is a homogeneous method for determining the number of live cells in culture based on the quantification of ATP present, which signals the presence of metabolically active cells. Briefly, human tumor cells (6000 human cancer cells / well in 100 μl cell culture medium) in an opaque-walled multi-well plate were incubated at 37 °C (5% CO2, humidified atmosphere) for 3 - 4 days with serial dilutions of Fc single mutant or triple mutant antibody vcMMAE conjugates. After incubation, 100 μl of CellTiter-Glo® substrate was added to the wells. Luminescence was recorded after 10 minutes using a Cytation1 (Biotek) plate reader. Percent survival was calculated according to the formula: Survival (%) = 100 × (Experimental RLU - RLU of medium only) / (RLU of cells only - RLU of medium only), and the data were analyzed by non-linear regression log(inhibitor) vs. response (3 parameters) curve fitting using GraphPad Prism software. The cell lines tested in the cytotoxicity assay had positive surface expression for each of the targets of the mAbs used in the assay. Specifically, the HCC1954 cell line was tested for anti-HER2 mAb with a single mutant (L328C). The MDA MB468 cell line was tested for anti-EGFR mAb with either a single mutant or a triple mutant. The SK BR3 cell line was evaluated for anti-TROP2 antibody with either a single mutant or a triple mutant.

[0438] The results in FIGS. 56, 57, 58, 59, 60 and 61 show that the in vitro cytotoxicity of antibodies with single or triple Fc mutants is enhanced by their conjugation with vcMMAE, an MMAE derivative having a cleavable dipeptide valine-citrulline (vc) linker that promotes efficient and selective drug cleavage in target cells by lysosomal cathepsin B after internalization. The results further showed that site-specific conjugated mAbs with vcMMAE exhibited potent and selective cytotoxic activity against antigen-positive tumor cell lines in a dose-dependent manner. In contrast, unconjugated mAbs with single mutants or triple mutants showed substantially little or no cytotoxic effect. Collectively, these results demonstrate the in vitro efficacy of ADCs composed of monoclonal antibodies covalently linked in a site-specific manner to cytotoxins that enable the release of cytotoxic drugs upon cell binding and internalization.

[0439] Example 14: Human Clinical Trials for the Treatment of Human Cancer by the Use of Her2, EGFR, TROP2, CDH3, or Other TAA Triple Mutant MAb and Site-Specific Conjugated ADCs

[0440] Her2, EGFR, Trop2, CDH3, or other TAA ADCs are synthesized according to the present invention, specifically accumulate in tumor cells, and are used for the treatment of specific tumors and other immunological disorders and / or other diseases (see Tables I and V). Two clinical approaches have been successfully pursued in connection with each of these indications.

[0441] I.) Adjuvant Therapy: In adjuvant therapy, the patient is treated with Her2, EGFR, Trop2, CDH3, or other TAA ADCs in combination with chemotherapeutic agents or pharmaceuticals or biopharmaceuticals or combinations thereof. The primary cancer target is treated under a standard protocol by the addition of Her2, EGFR, Trop2, CDH3, or other TAA ADCs. The protocol design addresses efficacy as evaluated by the following examples, including but not limited to, reduction of tumor burden of primary or metastatic lesions, prolongation of progression-free survival, overall survival, improvement of patient health, disease stabilization, and the ability to reduce the normal dosages of standard chemotherapy and other biological agents. These dosage reductions enable additional and / or long-term treatment by reducing the dose-related toxicities of chemotherapeutic or biological agents.

[0442] II.) Monotherapy: In relation to the use of Her2, EGFR, Trop2, CDH3, or other TAA ADCs in monotherapy of tumors, Her2, EGFR, Trop2, CDH3, or other TAA ADCs are administered to patients without chemotherapeutic agents or pharmaceuticals or biological agents. In one embodiment, the monotherapy is clinically implemented in end-stage cancer patients with extensive metastatic disease. The protocol design addresses efficacy as evaluated by the following examples, including but not limited to, reduction of tumor burden of primary or metastatic lesions, prolongation of progression-free survival, overall survival, improvement of patient health, disease stabilization, and the ability to reduce the normal dosages of standard chemotherapy and other biological agents.

[0443] Dosage The dosing schedule can be adjusted to provide an optimal desired response. For example, a single injection of Her2, EGFR, Trop2, CDH3, or other TAA ADC may be administered, several divided doses may be administered over time, or the dose may be proportionally decreased or increased as indicated by the urgency of the treatment situation. As used herein, "unit dosage form" refers to physically discrete units suitable as unit doses for the mammalian subject to be treated. Each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined by and directly depend on (a) the unique characteristics of Her2, EGFR, Trop2, CDH3 or other TAA ADC, the individual mechanisms of the irradiation mechanism (reactor) and the specific therapeutic or prophylactic effects achieved, and (b) the limitations inherent in the art of formulating such compounds for the treatment of hypersensitivity in an individual.

[0444] Clinical Development Plan (CDP) The CDP is advanced following the treatment of cancer(s) and / or immune disorders (see Tables I and V) using the Her2, EGFR, Trop2, CDH3, or other TAA ADCs of the present disclosure. The trials first demonstrate safety and then confirm efficacy in repeated dosing. The trials are non-blinded and compare standard chemotherapy with Her2, EGFR, Trop2, CDH3, or other TAA ADC in addition to standard treatment. As will be appreciated, one non-limiting criterion that can be utilized in connection with patient enrollment is the concentration of Her2, EGFR, Trop2, CDH3, or other TAA ADC in the tumor determined by standard detection methods known in the art.

[0445] The present invention should not be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the present invention, and all functionally equivalent ones are within the scope of the present invention. In addition to what is described herein, various changes to the models, methods, and life cycle methodologies of the present invention will be apparent to those skilled in the art from the foregoing description and teachings, and are also intended to fall within the scope of the present invention. Such changes or other embodiments can be implemented without departing from the true scope and spirit of the present invention.

[0446]

Table 1

[0447]

Table 2

[0448]

Table 3

[0449]

Table 4

[0450]

Table 5

[0451] Table VI Antibody Sequences Anti-HER2 Ab Heavy Chain Wild-Type EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) Anti-HER2 Ab heavy chain L328C EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2) Anti-HER2 Ab heavy chain L234A, L235A EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3) Anti-HER2 Ab heavy chain L234A, L235A, L328C EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4) Anti-HER2 Ab light chain wild type DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 5) Anti-EGFR Ab No.1 heavy chain wild type QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6) Anti-EGFR Ab No.1 heavy chain L328C QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7) Anti-EGFR Ab No. 1 heavy chain L234A, L235A QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) Anti-EGFR Ab No. 1 heavy chain L234A, L235A, L328C QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) Anti-EGFR Ab No.1 light chain wild type DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) Anti-EGFR Ab No.2 heavy chain wild type QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) Anti-EGFR Ab No.2 Heavy Chain L328C QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12) Anti-EGFR Ab No. 2 Heavy Chain L234A, L235A QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13) Anti-EGFR Ab No. 2 Heavy Chain L234A, L235A, L328C QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) Anti-EGFR Ab No.2 light chain wild type DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15) Anti-EGFR Ab No.3 heavy chain wild type QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 16) Anti-EGFR Ab No. 3 heavy chain L328C QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 17) Anti-EGFR Ab No. 3 heavy chain L234A, L235A QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) Anti-EGFR Ab No. 3 heavy chain L234A, L235A, L328C QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19) Anti-EGFR Ab No. 3 light chain wild type DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20) Anti-Trop2 Ab heavy chain wild type QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21) Anti-Trop2 Ab heavy chain L328C QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) Anti-Trop2 Ab heavy chain L234A, L235A QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) Anti-Trop2 Ab heavy chain L234A, L235A, L328C QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24) Anti-Trop2 Ab light chain wild type DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25) Anti-CDH3 Ab heavy chain wild type QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 26) Anti-CDH3 Ab heavy chain L328C QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 27) Anti-CDH3 Ab heavy chain L234A, L235A QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28) Anti-CDH3 Ab heavy chain L234A, L235A, L328C QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29) Anti-CDH3 Ab light chain wild type DIQMTQSPSSLSASVGDRVTITCRASQTISNTLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLSWFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30)

[0452]

Table 7-1

Table 7-2

Table 7-3

[0453]

Table 8-1

Table 8-2

Table 8-3

Claims

【Claim 1】 The invention described in the specification.

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