Antibody compositions comprising FC mutations and site-specific conjugation properties

Abstract

Disclosed herein are antibodies that bind Her2, EGFR, Trop2, CDH3 or other TAAs containing triple mutations at L234A, L235A and L328C and methods for making such triple mutation antibodies. Thus, the triple mutation antibody contains effector functions modified by Fc silencing and can be site-specifically conjugated at L328C to form an antibody-drug conjugate (ADC), which can be administered to a patient and provide a method for treating cancer, immune and neurological disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 973,475, filed on October 4, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] Submission of sequence listing

[0004] The contents of the following paper copy submissions are incorporated herein by reference in their entirety: Paper copy of the Sequence Listing recorded on October 2, 2020.

[0005] STATEMENT OF RIGHTS TO THIS INVENTION MADE UNDER FEDERALLY SPONSORED RESEARCH

[0006] not applicable. Technical Field

[0007] The invention described herein relates to antibodies, antigen-binding fragments thereof, antibody drug conjugates (ADCs) and antibody boron conjugates (ABCs) that have been engineered to contain multiple functional properties, including Fc silencing and site-specific conjugation. The invention further relates to prognostic, preventive and therapeutic methods and compositions for treating cancer and immunological and neurological disorders. Background Art

[0008] Cancer is the second leading cause of death worldwide after coronary heart disease. Millions of people die from cancer each year, with more than half a million deaths in the United States alone, with 1,688,780 new cancer cases diagnosed in 2017 (American Cancer Society). While deaths from heart disease have declined significantly, deaths from cancer are generally on the rise. It is estimated that by 2040, more than 16 million people will die from cancer each year worldwide (source; International Agency for Research on Cancer, 2018), so unless medical developments change current trends, cancer will surpass heart disease as the largest cause of death.

[0009] Several cancers have 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 sexes of all ages worldwide (GLOBOCAN 2018). These and almost all other cancers share a common lethal feature, namely that they metastasize to sites distant from the primary tumor, with few exceptions. Moreover, even for those cancer patients who initially survive the primary cancer, the general experience is that their lives have been dramatically altered. Many cancer patients experience intense anxiety due to the awareness of the possibility of recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Moreover, many cancer patients experience a recurrence of their disease.

[0010] Although cancer therapies have improved over the past few decades and survival rates have increased, the heterogeneity of cancer still requires new treatment strategies that utilize multiple treatment modalities. This is especially true when treating solid tumors at critical anatomical sites (e.g., glioblastomas, head and neck squamous cell carcinomas, and lung adenocarcinomas), which are sometimes limited to standard radiation therapy and / or chemotherapy. Nevertheless, the adverse effects of these therapies are chemo- and radioresistance, which promote local recurrence, distant metastasis, and second primary tumors, in addition to severe side effects that reduce the patient's quality of life.

[0011] 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 now approved for use as therapies for transplantation, cancer, infectious diseases, cardiovascular disease, and inflammation. Different isotypes have different effector functions. This functional difference is reflected in the different 3-dimensional structures of various immunoglobulin isotypes (PM ALZARI et al., Annual Rev. Immunol., 6: 555-580 (1988)).

[0012] In general, antibodies work through a variety of mechanisms, most of which are related to other weapons of the immune system. Antibodies can simply block the interaction of molecules, or they can activate the classical complement pathway (known as complement-dependent cytotoxicity or CDC) through the interaction of C1q on the C1 complex with clustered antibodies. Crucially, antibodies also act as a link between antibody-mediated and cell-mediated immune responses through the engagement of Fc receptors.

[0013] Fc engineering approaches have been used to identify key interaction sites of the Fc domain with Fcγ receptors and C1q, and then mutating these positions to reduce or eliminate binding to improve therapeutic properties, such as modulating effector function and reducing toxicity, etc. See HEZAREH et al., J. Virol., 75(24):12161-8 (December 2001) and OGANESYAN et al., Acta Crystallographica. D. Biol Crystallogr, 64:(No. 6):700-4 (June 2008).

[0014] In addition, antibody-drug conjugates (ADCs) are a class of emerging targeted therapeutic agents with improved therapeutic indexes compared to traditional chemotherapy. In addition to (monoclonal) antibodies (mAbs) and target selection, drugs and linkers have always been the focus of ADC development. However, recently, the importance of conjugate homogeneity has been explored. It is reported that the pharmacological characteristics of ADCs can be improved by applying site-specific conjugation technology, which utilizes surface-exposed cysteine ​​residues that are engineered into antibodies that are then conjugated to linker drugs, thereby producing site-specific conjugated ADCs with a defined drug / antibody ratio (DAR). Relative to the heterogeneous mixture produced using conventional lysine and cysteine ​​conjugation methods, site-specific conjugated ADCs generally exhibit at least equivalent in vivo efficacy, improved PK, and an expanded therapeutic window.

[0015] The prior art discloses several methods for obtaining site-specific conjugation and the resulting ADCs, see for example WO2006 / 034488 (Genentech), Sutherland et al., Blood, 122(8): 1455-1463 (2013), WO2014 / 124316 (Novartis), and US2017 / 0080103 (Synthon Biopharmaceuticals).

[0016] In all prior art approaches published to date, emphasis has been placed on site-conjugation of linker drugs at surface / solvent exposed positions, at positions exhibiting high thiol reactivity, and at positions particularly in the constant regions of monoclonal antibodies, with the goal of improving homogeneity and pharmacokinetic properties.

[0017] Although the conventional lysine and cysteine ​​conjugation methods described above have resulted in FDA-approved antibody-drug conjugates and are being used to construct most of the large number of ADCs currently in preclinical and clinical trials, 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.

[0018] In summary, it is obvious to those skilled in the art that a new therapeutic paradigm is needed for the treatment of cancer and immune diseases. By using modern antibody engineering techniques and new conjugation methods, a new class of antibodies can be achieved with the overall goal of achieving more effective treatments, reducing side effects and lowering production costs.

[0019] In view of current deficiencies known in the art, it is an object of the present invention to provide new and improved antibodies and binding ligands and methods for treating cancer, immune disorders and other diseases utilizing antibodies engineered with triple mutations to reduce antibody effector function and comprising site-specific conjugation points. Summary of the invention

[0020] The present invention provides antibodies, antigen binding fragments, antibody drug conjugates (ADC), antibody immune modification conjugates and antibody boron conjugates (ABC) 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 and CDH3. In some embodiments, the present invention includes fully human antibodies conjugated to therapeutic agents. In some embodiments, the present invention includes fully human antibodies conjugated to boronated compounds. In some embodiments, the antibodies have been engineered to reduce effector function via Fc silencing. In some embodiments, the antibodies contain site-specific mutations that can be conjugated to a drug moiety. In some embodiments, the antibodies include triple mutations, thereby silencing Fc, and inserting site-specific mutations to conjugate to a drug moiety. In other embodiments, the triple mutations include L234A, L235A, and L328C.

[0021] The present invention further provides various immunogenic or therapeutic compositions, such as antibodies, antibody drug conjugates and strategies for treating cancers expressing Her2, EGFR, Trop2, CDH3 and other tumor associated antigens (TAAs) such as those listed in Table IV.

[0022] In another embodiment, the present disclosure teaches a method for synthesizing a triple mutant antibody.

[0023] In another embodiment, the present disclosure teaches a method of synthesizing a triple mutant antibody and conjugating a drug moiety at a site-specific position thereof.

[0024] In another embodiment, the present disclosure teaches methods of synthesizing single mutant antibodies.

[0025] In another embodiment, the present disclosure teaches methods of synthesizing single mutant antibodies and conjugating drug moieties at site-specific positions thereof.

[0026] In another embodiment, the present disclosure teaches methods of treating cancer, immune disorders, and other diseases in humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .Anti-Her2 Mab and Fc variants: Fc variants did not affect target binding.

[0028] Figure 2 .Anti-EGFR Mab No. 1 and Fc variants: The Fc variant did not affect target binding.

[0029] Figure 3 .Anti-EGFR Mab No. 2 and Fc variants: The Fc variant did not affect target binding.

[0030] Figure 4 .Anti-EGFR Mab No. 3 and Fc variants: Fc variants did not affect target binding.

[0031] Figure 5 .Anti-Trop2 Mab and Fc variants: Fc variants did not affect target binding.

[0032] Figure 6 .Anti-CDH3 Mab and Fc variants: Fc variants did not affect target binding.

[0033] Figure 7 . Anti-Her2 Mabs and Fc variants: Complete inhibition of FcγRI binding was observed for the triple mutant.

[0034] Figure 8 . Anti-EGFR Mab No. 1 and Fc variants: Complete inhibition of FcγRI binding was observed for the triple mutant.

[0035] Fig. 9 . Anti-EGFR Mab No. 2 and Fc variants: Complete inhibition of FcγRI binding was observed for the triple mutant.

[0036] Fig.10 . Anti-EGFR Mab No. 3 and Fc variants: Almost complete inhibition of FcγRI binding was observed for the triple mutant.

[0037] Fig.11 . Anti-Trop2 Mabs and Fc variants: Complete inhibition of FcγRI binding was observed for the triple mutant.

[0038] Fig.12 . Anti-CDH3 Mabs and Fc variants: Almost complete inhibition of FcγRI binding was observed for the triple mutant.

[0039] Fig.13 . Anti-Her2 Mabs and Fc variants: Inhibition of FcγRIIA binding was observed for single and triple mutants.

[0040] Fig.14 . Anti-EGFR Mab No. 1 and Fc variants: Inhibition of FcγRIIA binding was observed for single and triple mutants.

[0041] Fig.15 . Anti-Trop2 mAbs and Fc variants: Inhibition of FcγRIIA binding was observed for single and triple mutants.

[0042] Fig.16 . Anti-CDH3 Mabs and Fc variants: Inhibition of FcγRIIA binding was observed for single and triple mutants.

[0043] Fig.17 . Anti-Her2 Mabs and Fc variants: Inhibition of FcγRIIIa binding was observed for single and triple mutants.

[0044] Fig.18 . Anti-EGFR Mab No. 1 and Fc variants: Inhibition of FcγRIIIa binding was observed for single and triple mutants.

[0045] Fig.19 . Anti-EGFR Mab No. 2 and Fc variants: Inhibition of FcγRIIIa binding was observed for single and triple mutants.

[0046] Fig. 20 . Anti-EGFR Mab No. 3 and Fc variants: Inhibition of FcγRIIIa binding was observed for single and triple mutants.

[0047] Fig.21 . Anti-Trop2 Mabs and Fc variants: Inhibition of FcγRIIIa binding was observed for single and triple mutants.

[0048] Fig. 22 . Anti-CDH3 Mabs and Fc variants: Inhibition of FcγRIIIa binding was observed for single and triple mutants.

[0049] Fig.23 . Anti-Her2 Mabs and Fc variants: No significant reduction in FcRn binding was observed for the Fc variants.

[0050] Fig.24 . Anti-EGFR Mab No. 1 and Fc variants: No significant reduction in FcRn binding was observed for the Fc variants.

[0051] Fig.25 . Anti-EGFR Mab No. 2 and Fc variants: No significant reduction in FcRn binding was observed for the Fc variants.

[0052] Fig.26 . Anti-EGFR Mab No. 3 and Fc variants: No significant reduction in FcRn binding was observed for the Fc variants.

[0053] Fig. 27 . Anti-Trop2 Mabs and Fc variants: No significant reduction in FcRn binding was observed for the Fc variants.

[0054] Fig.28 . Anti-CDH3 Mabs and Fc variants: No significant reduction in FcRn binding was observed for the Fc variants.

[0055] Fig.29 .Binding kinetic analysis of anti-Her2 antibodies and Fc variants to FcγRI, IIA, IIB, IIIA, IIIB and FcRn. The triple mutant showed significant binding inhibition to all FcγR isoforms but not to FcRn

[0056] Fig.30 . Analysis of the binding kinetics of anti-Her2 antibodies and Fc variants to FcγRI. The triple mutant has a faster dissociation rate compared to the wild type and single mutants.

[0057] Fig.31 . Anti-HER2 mAbs and Fc variants: A reduction in ADCC was observed for the Fc variants.

[0058] Fig.32 . Anti-EGFR mAb No. 1 and Fc variants: A reduction in ADCC was observed for the Fc variants.

[0059] Fig.33 . Anti-Trop2 mAb and Fc variants: A reduction in ADCC was observed for the Fc variants.

[0060] Fig.34 . Anti-CDH3 mAb and Fc variants: A reduction in ADCC was observed for the Fc variants.

[0061] Fig.35. Anti-HER2 mAbs and Fc variants: A reduction in CDC was observed for the Fc variants.

[0062] Fig.36 . Anti-EGFR mAb No. 1 and Fc variants: A decrease in CDC was observed for the Fc variants.

[0063] Fig.37 . Anti-Trop2 mAb and Fc variants: A reduction in CDC was observed for the Fc variants.

[0064] Fig.38 . Anti-CDH3 mAb and Fc variants: A reduction in CDC was observed for the Fc variant.

[0065] Fig.39 . Anti-HER2 mAbs and Fc variants: A reduction in C1q binding was observed for the Fc variants.

[0066] Fig.40 . Anti-EGFR mAb No. 1 and Fc variants: A reduction in C1q binding was observed for the Fc variants.

[0067] Fig.41 . Anti-Trop2 mAb and Fc variants: A reduction in C1q binding was observed for the Fc variants.

[0068] Fig.42 . Anti-CDH3 mAb and Fc variants: A reduction in C1q binding was observed for the Fc variants.

[0069] Fig.43 . Reverse phase column chromatogram of anti-Her2 Mab triple mutant conjugated with a proprietary payload (LOL1). Overlay UV traces of unconjugated triple mutant anti-HER2 mAb (peak 1), conjugated triple mutant anti-HER2 mAb (peak 2).

[0070] Fig.44 Intact mass analysis of LOL1-conjugated triple mutant anti-HER2 mAb confirmed that the conjugate was 100% site-specific. (A). Full spectrum, (B). Enlarged LC region, (C). Enlarged HC region.

[0071] Fig.45 Reverse phase column chromatograms of L328C variants of anti-HER2 mAb (A) and anti-EGFR mAb (B) conjugated to vcMMAE. The heavy chain (H1) with a single payload is the major species, indicating that the conjugation is site-specific and that the payload resides on Cys328 and not within the hinge region.

[0072] Fig.46. Reverse phase column chromatograms of the L328C variant of anti-TROP2 mAb conjugated with vcMMAE. (A). Light chain (peak 1), unconjugated heavy chain (peak 2), conjugated heavy chain with 1 payload (peak 3); conjugated heavy chain with 2 and 3 payloads (peaks 4-5). (B). Overlay UV214nm traces of mAb and ADC. Unconjugated L328C variant of anti-TROP2 antibody (black); vcMMAE conjugated L328C variant of anti-TROP2 antibody (red).

[0073] Fig.47 . Reversed phase HPLC analysis of triple mutant anti-TROP2 mAbs conjugated with vcMMAE. (A). Light chain (peak 1), unconjugated heavy chain (peak 2), conjugated heavy chain with 1 payload (peak 3); conjugated heavy chain with 2 and 3 payloads, respectively (peaks 4-5). (B). Overlay UV214nm traces of mAb and ADC. Unconjugated L328C variant of anti-TROP2 antibody (black); vcMMAE conjugated L328C variant of anti-TROP2 antibody (red).

[0074] Fig.48 . Reverse phase column chromatograms of L234A, L235A, L328C variants of anti-EGFR mAb conjugated with vcMMAE. (A). Spectrum of unconjugated anti-EGFR mAb No. 3 with triple Fc mutations. (B). Spectrum of unconjugated anti-EGFR mAb No. 1 with triple Fc mutations. (C). Spectrum of vcMMAE-conjugated anti-EGFR mAb No. 3 with triple Fc mutations. (D). Spectrum of vcMMAE-conjugated anti-EGFR mAb No. 1 with triple Fc mutations.

[0075] Fig.49 .Based on reverse phase column chromatography data ( Fig.48 An example of peak assignment and DAR calculation for mAb (provided in ). Unconjugated mAb (control) is used to identify the retention time and UV250 / 280 ratio of the heavy and light chains. These parameters are necessary for correct peak assignment and DAR determination.

[0076] Fig.50 Summary of analytical properties of the resulting ADCs: Average DAR, monomer percentage by size exclusion chromatography (SEC), and DAR1 heavy chain percentage are shown for vcMMAE or LOL1 conjugated mAbs and single or triple Fc variants.

[0077] Fig.51 Characteristic product ions after .vcMMAE-peptide fragmentation.

[0078] Fig.52. Sequence coverage of anti-EGFR antibody No. 1 and triple mutant conjugated with vcMMAE. (A). Heavy chain, (B). Light chain.

[0079] Fig.53 Representative TIC chromatograms of peptides after digestion with trypsin. (A) Non-conjugated anti-EGFR antibody No. 1 with triple mutations, (B) vcMMAE-conjugated anti-EGFR antibody No. 1 with triple mutations.

[0080] Fig.54 . The conjugation site of the anti-EGFR antibody No. 1 with triple mutations was confirmed by MS / MS of the ACPAPIEK heavy chain peptide. The complete MS (A, C) and MS / MS fragmentation (B, D) of the ACPAPIEK-carbamidomethyl (e.g., alkylated mAb, A, B) and ACPAPIEK-vcMMAE (C, D) peptides are shown. The fragments representing the peptide or vcMMAE are indicated by solid or hollow arrows, respectively.

[0081] Fig.55 . Confirmation of the conjugation site in anti-EGFR antibody No. 1 ADC with single mutations via MS / MS of ACPAPIEK heavy chain peptides. The complete MS (A, C) and MS / MS fragmentation (B, D) of ACPAPIEK-carbamidomethyl (e.g., alkylated mAb, A, B) and ACPAPIEK-vcMMAE (C, D) peptides are shown. Fragments representing peptides or vcMMAE are indicated by solid or hollow arrows, respectively.

[0082] Fig.56 Cytotoxicity mediated by the L328C variant of anti-HER2 antibody conjugated to vcMMAE. HCC1954 (Her2 / neu positive) cells were incubated for 72 to 96 hours, and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0083] Fig.57 Cytotoxicity mediated by the L328C variant of anti-EGFR antibody No. 1 conjugated to vcMMAE. MDA-MB-468 (EGFR positive) cells were incubated for 72 to 96 hours, and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0084] Fig.58 Cytotoxicity mediated by L234A, L235A, L328C variants of anti-EGFR antibody No. 1 conjugated to vcMMAE. MDA-MB-468 (EGFR positive) cells were incubated for 72 to 96 hours, and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0085] Fig.59 Cytotoxicity mediated by L234A, L235A, L328C variants of anti-EGFR antibody No. 3 conjugated to vcMMAE. MDA-MB-468 (EGFR positive) cells were incubated for 72 to 96 hours, and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0086] Fig.60 Cytotoxicity mediated by the L328C variant of anti-Trop2 antibody conjugated to vcMMAE SK BR3 (TROP2 positive) cells were incubated for 72 to 96 hours, and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0087] Fig.61 Cytotoxicity mediated by L234A, L235A, L328C variants of anti-Trop2 antibodies conjugated to vcMMAE SK BR3 (TROP2 positive) cells were incubated for 72 to 96 hours and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay. DETAILED DESCRIPTION

[0088] Chapter Outline

[0089] I.) Definition

[0090] II.) Antibodies

[0091] III.) Fc mutations that modify effector function

[0092] IV.) Antibody-drug conjugates

[0093] V.) Site-specific conjugation formats of ADCs

[0094] VI.) Connecting subunits

[0095] VII.) Elongation subunit

[0096] VIII.) Amino Acid Unit

[0097] IX.) Spacer unit

[0098] X.) Drug Unit

[0099] XI.) Methods for determining the cytotoxic effect of ADCs

[0100] XII.) Treatment of cancers expressing Her2, EGFR, Trop2, CDH3 and TAA

[0101] XIII.) Combination Therapy

[0102] XIV.) Kits / Products

[0103] I.) Definition:

[0104] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which the invention belongs, unless the context clearly indicates otherwise. In some cases, for the sake of clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the meanings commonly understood in the art.

[0105] When a trade name is used herein, the trade name also refers to the product formulation, the generic drug, as well as the active pharmaceutical ingredient of the trade name product, unless the context indicates otherwise.

[0106] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" mean cancer that has extended into the capsule of associated tissues, and are intended to encompass 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. Surgery is generally not recommended for patients with locally advanced disease, and these patients have a much worse prognosis than patients with clinically localized (organ-confined) cancer.

[0107] The term "substituted" means that the specified group or moiety bears one or more substituents. The term "unsubstituted" means that the specified group bears no substituents. The term "optionally substituted" means that the specified group is unsubstituted or substituted with one or more substituents. Where the term "substituted" is used to describe a structural system, substitution is intended to occur at any valence-permitted position on the system.

[0108] Unless otherwise expressly indicated, the term "antibody" is used in the broadest sense. Therefore, an "antibody" can be naturally occurring or artificial, such as a monoclonal antibody produced by conventional hybridoma or transgenic mouse technology. Her2, EGFR, Trop2, CDH3 and / or TAA antibodies include monoclonal and polyclonal antibodies and fragments of the antigen binding domains and / or one or more complementary determining regions containing these antibodies. As used herein, the term "antibody" refers to an antibody or fragment thereof that specifically binds to Her2, EGFR, Trop2, CDH3 and / or any TAA and / or exhibits a desired biological activity in any form, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they specifically bind to Her2, EGFR, Trop2, CDH3 and / or exhibit a desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Therefore, 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, the variable regions combining to 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 that can be used for the present methods and compositions can be produced in cell culture, in bacteriophages, in yeast, or in various animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees and apes. Therefore, in one embodiment, the antibody of the present invention is a mammalian antibody. Phage technology can be used to separate initial antibodies or generate variants with altered specificity or affinity properties. Such techniques are conventional and well known in the art. In one embodiment, antibodies are produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector comprising a DNA sequence encoding an antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one VL and one VH region in a host cell.Exemplary descriptions of recombinant means for antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shepard et al., Monoclonal Antibodies (MONOCLONAL ANTIBODIES) (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles and Practice (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 improve the efficacy of the antibodies in mediating the desired function. Therefore, within the scope of the present invention, recombinant means can be used to modify antibodies by substitution. Typically, substitution will be a conservative substitution. For example, at least one amino acid in the constant region of an antibody can be replaced by a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and ANGAL et al., Mol. Immunol., 30: 105-08 (1993). Amino acid modification includes deletion, addition and substitution of amino acids. In some cases, such changes are made to reduce undesirable activity, such as complement-dependent cytotoxicity. Antibodies are often labeled by covalent or non-covalent bonding to provide a detectable signal. Various labeling and conjugation techniques are known and widely reported in science and patent literature. These antibodies can be screened for binding to normal or defective FLT3. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996). Suitable antibodies with desired biological activity can be identified using the following in vitro assays and the following in vivo assays, including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-to-cell communication, apoptosis, transport, signal transduction, and the in vivo assays such as inhibition of tumor growth. The antibodies provided herein can also be used for diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to specific antigens without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, antibodies can be used in competitive binding assays. They can also be used to quantify Her2, EGFR, Trop2 and / or CDH3 or their receptors.

[0109] 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 retains 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 a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, which is a fragment of a V L 、V H , C L and C H1 (ii) a F(ab′)2 fragment, which is a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a fragment consisting of V H and C H1 (iv) a single-arm V L and V H The Fv fragment composed of the structural domain, (v) dAb fragment (WARD et al. (1989), Nature, 341: 544-546), which consists of V H domains; and (vi) separated complementarity determining regions (CDRs). In addition, although the two domains V L and V H are encoded by different genes, but they can be joined using recombinant methods via a synthetic linker, allowing them to be made into a single protein chain, with V L and V H The domains are paired to form monovalent molecules (called single-chain Fv (scFv); 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 encompassed 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.

[0110] As used herein, the term "Fc" refers to the region including the hinge region, CH2 and / or CH3 domains.

[0111] As used herein, any form of "antigen" can be used to generate antibodies specific to Her2, EGFR, Trop2, CDH3 and / or any TAA of the present invention. Therefore, the triggering antigen can be a single epitope, multiple epitopes or a single whole protein or in combination with one or more immunogenicity enhancers known in the art. The triggering antigen can be a separated full-length protein, a cell surface protein (e.g., immunized with cells transfected with at least a portion of the antigen) or a soluble protein (e.g., immunized only with the extracellular domain portion of the protein). Antigens 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, in the context of an antigen, the term "part" refers to the minimum number of amino acids or nucleic acids that constitute the immunogenic epitope of the antigen of interest, as appropriate. Any genetic vector suitable for transforming cells of interest can be used, including but not limited to adenovirus vectors, plasmids and non-viral vectors, such as cationic lipids. In one embodiment, the antibody of the method and composition herein specifically binds to at least a portion of the extracellular domain of the target of interest.

[0112] The antibodies or antigen binding fragments thereof provided herein may constitute or be part of a "biologically active agent". As used herein, the term "biologically active 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 is capable of binding to a desired antigenic epitope and exerting a biological effect directly or indirectly. Direct effects include, but are not limited to, regulation, stimulation and / or inhibition of growth signals, regulation, stimulation and / or inhibition of anti-apoptotic signals, regulation, stimulation and / or inhibition of apoptosis or necrosis signals, regulation, stimulation and / or inhibition of ADCC cascades, and regulation, stimulation and / or inhibition of CDC cascades and / or Fc silencing.

[0113] As used herein with respect to antigen binding proteins, the term "specific binding" means that the antigen binding protein binds to a target and a discrete domain or discrete amino acid sequence within the target, without binding to other (e.g., unrelated) proteins or non-significant binding to other (e.g., unrelated) proteins. However, this term does not exclude the fact that antibodies or their binding fragments may also cross-react with closely related molecules. The antibodies and fragments thereof described herein, as well as antibody drug conjugates including these, can specifically bind to Her2, EGFR, Trop2, CDH3 and / or TAA disclosed herein with an affinity that is at least 2, 5, 10, 50, 100 or 1000 times greater than their affinity for binding to closely related molecules.

[0114] "Bispecific" antibodies can also be used in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, that has binding specificity for at least two different antigenic epitopes. In one embodiment, the epitope is from the same antigen. In another embodiment, the epitope is from two different antigens. Methods for making bispecific antibodies are known in the art. For example, coexpression of two immunoglobulin heavy chain / light chain pairs can be used to recombinantly produce bispecific antibodies. See, for example, MILSTEIN et al., Nature, 305:537-39 (1983). Alternatively, chemical bonds can be used to prepare bispecific antibodies. See, for example, Brennan et al., Science, 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., HOLLINGER et al., Proc. Natl. Acad. Sci. USA, 90:6444-48 (1993); GRUBER et al., J. Immunol., 152:5368 (1994).

[0115] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind to the target antigen and / or exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and MORRISON et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0116] As used herein, the terms "cancer" and "neoplasm / tumor" are used interchangeably, and whether in the singular or plural form, refer to cells that have undergone malignant transformation, which makes them pathological to the host organism. Primary cancer cells (i.e., cells obtained from the vicinity of the site of malignant transformation) can be easily distinguished from non-cancerous cells by mature techniques, particularly histological examinations. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells and in vitro cultures and cell lines derived from cancer cells. When referring to a cancer that is usually manifested as a solid tumor, a "clinically detectable" tumor is a tumor that can be detected based on a tumor mass; for example, by procedures such as CAT scans, MR imaging, X-rays, ultrasound or palpation, and / or due to the expression of one or more cancer-specific antigens in a sample available from a patient. Tumors can be hematopoietic tumors, such as blood cell tumors, i.e., liquid tumors. Specific examples of such tumor-based clinical conditions include leukemias, such as chronic myeloid leukemia or acute myeloid leukemia; myelomas, such as multiple myeloma; lymphomas, and the like.

[0117] The term "therapeutic agent" refers to all agents that provide a therapeutic benefit and / or have therapeutic effectiveness as defined herein. Therapeutic agents can, for example, reverse, ameliorate, alleviate, inhibit or limit the progression of a disease, disorder or condition or reduce its severity, or affect or improve (improve / ameliorate) 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-tumor agents, and "drug unit" agents as defined herein.

[0118] The term "anti-neoplastic agent" refers to all agents as defined herein that provide a therapeutic benefit and / or are therapeutically effective in the treatment of tumors or cancer.

[0119] 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; for example, nitrogen mustards, ethyleneimine compounds, and alkyl sulfonates; antimetabolites, such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors, such as anti-tubulin agents, such as vinca alkaloids, auristatins, and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biomolecules, and small molecules.

[0120] The terms "complementarity determining region" and "CDR" are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and binding affinity. Typically, there are three (3) CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three (3) CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3).

[0121] The precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of 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, Maryland ("Kabat" numbering scheme); AL-LAZIKANI et al. (1997), JMB, 273, 927-948 ("Chothia" numbering scheme); MACCALLUM et al., J. Mol. Biol., 262:732-745 (1996); "Antibody-antigen interactions: Contact analysis and binding site topology" (Chothia, 1996). topography), J. Mol. Biol., 262, 732-745 (“contact” numbering scheme); LEFRANC MP et al., “IMGT unique numbering for immunoglobulin and Tcell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol., 2003 Jan; 27(1): 55-77 (“IMGT” numbering scheme); and HONEGGER A. and PLICKTHUN A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J. Mol. Biol., 262, 732-745 (“contact” numbering scheme); LEFRANC MP et al., “IMGT unique numbering for immunoglobulin and Tcell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol., 2003 Jan; 27(1): 55-77 (“IMGT” numbering scheme); and HONEGGER A. and PLICKTHUN 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 (AHo numbering scheme).

[0122] The boundaries of a given CDR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Josiah scheme is based on structural information. The numbering of the Kabat and Josiah schemes is based on the most common antibody region sequence lengths, where insertions are represented by inserted letters, such as "30a", and there are deletions in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The contact scheme is based on the analysis of composite crystal structures and is similar to the Josiah numbering scheme in many aspects.

[0123] Therefore, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof, such as a variable region and individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or region thereof should be understood to encompass complementarity determining regions defined by any of the known schemes described above. In some instances, schemes for identifying one or more specific CDRs are specified, such as CDRs defined by Kabat, Josiah, or contact methods.

[0124] As used herein, the term "conservative substitution" refers to the substitution of amino acids and / or amino acid sequences known to those skilled in the art, and can generally be carried out without changing the biological activity of the resulting molecule. It is recognized by those skilled in the art that, generally, single amino acid substitutions in the non-essential regions of a polypeptide will not significantly change biological activity (see, for example, Watson et al., Molecular Biology of Genes, Benjamin / Cummings Publishing Company, page 224 (4th edition, 1987)). This exemplary substitution is preferably carried out according to those listed in Table II and Table III. For example, this change includes replacing any one of these hydrophobic amino acids with isoleucine (I), valine (V) and leucine (L); replacing glutamic acid (E) with aspartic acid (D), and vice versa; replacing asparagine (N) with glutamine (Q), and vice versa; and replacing threonine (T) with serine (S), and vice versa. Other substitutions may also be considered conservative, depending on the context of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). The relatively hydrophobic methionine (M) is often interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the significant feature of the amino acid residue is its charge and the different pK values ​​of the two amino acid residues are not significant. Other changes may be considered "conservative" in certain circumstances (see, e.g., Table III herein; pages 13-15 in "Biochemistry", 2nd ed. Lubert Stryer, ed. (Stanford University); HENIKOFF et al., PNAS, 1992, Vol. 89, 10915-10919; LEI et al., J Biol Chem, May 19, 1995; 270(20): 11882-6). Other substitutions are also permissible and may be determined empirically or based on known conservative substitutions.

[0125] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of a cell, the function of a cell and / or causes destruction of a cell. The term is intended to include radioactive isotopes, 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, but are not limited to, auristatins, chlortetracycline, maytansinoids, ricin, ricin A chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunomycin, tacrolimus, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraquinone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, saponin, officinalis) inhibitors and glucocorticoids and other chemotherapeutic agents, as well as radioisotopes such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 or 213 , P 32 , and radioactive isotopes of Lu, including Lu 177 .

[0126] Antibodies, including antibodies of the present invention, may also be conjugated to any of the above-mentioned cytotoxic agents as well as an anti-cancer prodrug activating enzyme capable of converting the prodrug to its active form.

[0127] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, said fragments comprising two identical polypeptide chains (V H -V L ) is connected to the light chain variable region (V L )'s heavy chain variable region (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 of another chain and create two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO93 / 11161; and HOLLINGER et al., Proc. Natl. Acad. Sci. USA, 90:6444-48 (1993).

[0128] The term "homolog" refers to a molecule that exhibits homology to another molecule by, for example, having the same or similar sequence of chemical residues at corresponding positions.

[0129] The term "identity" or "sequence identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

[0130] " Percentage identity " between two sequences varies with the number of identical positions shared by the sequences (i.e. the total number of the number of identical positions / positions of % identity==multiplied by 100), which takes into account the number of spaces and the length of each space, which all need to be introduced to carry out the best comparison of two sequences. As described below, a mathematical algorithm can be used to complete the comparison of sequences and the determination of the percentage identity between two sequences. The GAP program in the GCG software package can be used to determine the percentage identity between two nucleotide sequences 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. It is also possible to use Meyers et al., Computer Applications in Biological Sciences (Comput.Appi.Biosci.), 4:11-17 (1988) algorithm, using PAM120 weight residue table, gap length penalty 12, gap penalty 4 to determine the percentage identity between two nucleotides or amino acid sequences, the algorithm has been incorporated into the ALIGN program (version 2.0). In addition, the percent identity between two amino acid sequences can be determined using the NEEDLEMAN et al., J. Mol. Biol., 48:444-453 (1970) algorithm, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6, which algorithm has been incorporated into the GAP program in the GCG software package.

[0131] For example, a polynucleotide sequence may be identical to a reference polynucleotide sequence that is 100% identical to a reference sequence, or it may comprise up to a certain integer number of nucleotide changes compared to the reference sequence, for example, having 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 insertions, and wherein the changes may occur at the 5' or 3' terminal position of the reference nucleotide sequence or at any position between these terminal positions, interspersed individually between nucleotides in the reference sequence or interspersed in one or more consecutive groups within the reference sequence. The number of nucleotide changes is determined by multiplying the total number of nucleotides in the reference polynucleotide sequence described herein by the numerical percentage of the corresponding identity percentage (divided by 100) and subtracting the product from the total number of nucleotides in the reference polynucleotide sequence, or: n n ≤x n -(x ny ), where n n is the number of nucleotide changes, x n is the total number of nucleotides in a reference polynucleotide sequence described herein (for exemplary reference polynucleotide sequences, see the nucleic acid sequences in the "Sequence Listing"), and 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%, which is the symbol of the multiplication operator, and wherein x n Any non-integer product of y is rounded down to the nearest integer and then replaced by x n minus the integer. Similarly, a polypeptide sequence may be identical to a polypeptide reference sequence described herein, i.e., have 100% identity, or it may comprise up to a certain integer number of nucleotide changes compared to the reference sequence such that the % identity is less than 100%, for example, having at least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identity. Such changes are selected from the group consisting of at least one nucleotide deletion, substitution, including conservative and non-conservative substitutions, or insertion, and wherein the changes may occur at the amino or carboxyl terminal position of the reference polypeptide sequence or anywhere between these terminal positions, interspersed individually between the amino acids in the reference sequence or interspersed in one 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 the corresponding percent identity (divided by 100), and then subtracting the product from the total number of amino acids in the polypeptide reference sequence described herein, or: na ≤x a -(x ay ), where n a is the number of amino acid changes, x a is the total number of amino acids in the reference polypeptide sequence, and 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%, which is the sign of the multiplication operator, and wherein x a Any non-integer product of y is rounded down to the nearest integer and then replaced by x a The integer is subtracted. The identity percentage can be determined over the entire sequence length. As defined herein, the term "greater than 75% identity" encompasses greater than 75%, 80%, 85%, 95% and 99% identity and all discrete values ​​and discrete subranges within this range.

[0132] In one embodiment, the antibodies provided herein are “human antibodies.” As used herein, the term “human antibody” refers to antibodies in which substantially the entire sequence 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 trioma technology, human B cell technology (see, e.g., KOZBOR et al., Immunol. Today, 4:72 (1983), EBV transformation technology (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 partially to fully human antibodies are known in the art, and any such techniques may be used. According to a particularly preferred embodiment, fully human antibody sequences are made in transgenic mice engineered to express human heavy and light chain antibody genes. An exemplary description of making transgenic mice that produce human antibodies is found in Application No. WO 02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix) and its successors (progeny). Then, B cells from transgenic mice producing the desired antibodies can be fused to make hybridoma cell lines for continuous antibody production. See, for example, U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; 5,545,806; and JAKOBOVITS, Advanced Drug Delivery Review (Adv. Drug Del. Rev.), 31: 33-42 (1998); GREEN et al., Journal of Experimental Medicine (J. Exp. Med.), 188: 483-95 (1998).

[0133] As used herein, the term "humanized antibody" refers to an antibody form containing 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. Typically, a humanized antibody will include substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. A humanized antibody will optionally also include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. See, e.g., CABILLY, U.S. Pat. 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).

[0134] As used herein, the terms "inhibit" or "inhibition of" mean to reduce by a measurable amount, or to prevent entirely.

[0135] 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 present invention, the mammal is a mouse. In another embodiment of the present invention, the mammal is a human.

[0136] The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread to regional lymph nodes or distant sites, and are intended to encompass stage D disease under the AUA system and stage T×N×M+ under the TNM system.

[0137] As used herein, the term "modified" refers to the presence of changes to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such changes or modifications can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or by co-translation, or by post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide.

[0138] "Molecular recognition" refers to a chemical event in which a host molecule is able to form a complex with a second molecule (i.e., a guest). This process occurs through non-covalent chemical bonds, including but not limited to hydrogen bonding, hydrophobic interactions, and ionic interactions.

[0139] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies for (or specific to) different epitopes. In one embodiment, polyclonal antibodies contain a variety of monoclonal antibodies with different epitope specificities, affinities or avidities in a single antigen containing multiple antigenic epitopes. The modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibodies to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be manufactured by the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or can be manufactured by a recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described by CLACKSON et al., Nature, 352:624-628 (1991) and MARKS et al., J. Mol. Biol., 222:581-597 (1991). These monoclonal antibodies will typically bind with a Kd of at least about 1 μM, more typically 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, typically determined by ELISA.

[0140] "Non-natural amino acid" or otherwise written as "nnAA" refers to an amino acid that is not one of the twenty (20) common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term nnAA are "non-naturally encoded amino acid," "non-natural amino acid," "non-naturally occurring amino acid." Furthermore, the term nnAA includes, but is not limited to, amino acids that are not naturally occurring and that can be obtained by synthesis or that can be obtained by modifying non-natural amino acids.

[0141] "Pharmaceutically acceptable excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives and the like.

[0142] "Pharmaceutically acceptable" refers to non-toxic, inert compositions that are physiologically compatible with humans and other mammals.

[0143] The term "polypeptide" means a polymer of at least about 4, 5, 6, 7 or 8 amino acids. Throughout the specification, the standard three-letter (see Table II) or single-letter names of amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".

[0144] As used herein, the term "single-chain Fv" or "scFv" or "single-chain" antibody refers to a V fragment comprising an antibody. H and V L The Fv polypeptide further comprises a V domain, wherein these domains are present in a single polypeptide chain. H and V L The polypeptide linker between the domains enables the sFv to form the desired structure for antigen binding. For a review of sFv, see PLUCKTHUN, THE PHARMACOLOGY OF MONOCLONALANTIBODIES, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0145] As used herein, the terms "specificity", and "specifically binds / binds specifically" refer to the selective binding of an antibody to a target antigen epitope. The binding specificity of an antibody can be tested by comparing the binding to an appropriate antigen with the binding to an unrelated antigen or antigen mixture under a given set of conditions. If the antibody binds to an appropriate antigen at least 2, 5, 7 times and preferably 10 times more than the binding to an unrelated antigen or antigen mixture, it is considered specific. In one embodiment, a specific antibody is an antibody that binds to Her2, EGFR, Trop2, CDH3 antigens but does not bind to unrelated antigens. In another embodiment, the 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 greater amino acid homology with the Her2, EGFR, Trop2, CDH3 antigens. In another embodiment, the 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 greater identity percentages with the amino acid sequence of the Her2, EGFR, Trop2, CDH3 antigens. In another embodiment, the specific antibody is an antibody that binds to human Her2, EGFR, Trop2, CDH3 antigens and to murine Her2, EGFR, Trop2, CDH3 antigens, but the degree of binding to human antigens is higher. In another embodiment, the specific antibody is an antibody that binds to human Her2, EGFR, Trop2, CDH3 antigens and to primate Her2, EGFR, Trop2, CDH3 antigens, but the degree of binding to human antigens is higher. In another embodiment, the specific antibody binds to human Her2, EGFR, Trop2, CDH3 antigens and to any non-human Her2, EGFR, Trop2, CDH3 antigens, but to a greater extent to the human antigens, or any combination thereof.

[0146] As used herein, "treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as prolonging survival, reducing morbidity and / or alleviating side effects that are by-products of alternative treatment modalities; as is readily understood in the art, complete eradication of the disease is preferred, but not a requirement for therapeutic action.

[0147] The term "variant" refers to a molecule that exhibits a different type or specification than that described, such as a protein having one or more different amino acid residues at corresponding positions of a specifically described protein (e.g., Her2, EGFR, Trop2, CDH3 proteins). Analogs are an example of variant proteins. Splicing isoforms and single nucleotide polymorphisms (SNPs) are additional examples of variants.

[0148] II.) Antibodies

[0149] Another aspect of the present invention provides antibodies that bind to Her2, EGFR, Trop2, CDH3 and other TAAs disclosed herein. In one embodiment, the antibody binds to Her2, EGFR, Trop2, CDH3 and other TAA-related proteins.

[0150] As known in the art, the Her2, EGFR, Trop2, CDH3 and other TAA antibodies of the present invention are particularly useful in cancer (see, e.g., Table I) for prognostic determination, imaging, diagnosis and treatment methods. In one embodiment, the Her2, EGFR, Trop2, CDH3 and other TAA binding assays disclosed herein are used to detect cancer, such as in immunoassays. Similarly, such antibodies can be used for the treatment and / or prognosis of cancer (e.g., cancers listed in Table I) (e.g., when combined with therapeutic agents in ADC), to the extent that Her2, EGFR, Trop2, CDH3 and / or other TAAs are also expressed or overexpressed in these other cancers. In addition, antibodies (e.g., single-chain antibodies) expressed intracellularly show therapeutic effects in treating cancers involving the expression of Her2, EGFR, Trop2, CDH3 and other targets.

[0151] Various methods for preparing antibodies, particularly monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by using Her2, EGFR, Trop2, CDH3 and other TAA-related proteins, peptides or fragments to immunize suitable mammalian hosts in a separated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Harlow and Lane, eds. (1988); Harlow, Antibodies, Cold Spring Harbor Press, New York (1989)). In addition, fusion proteins of Her2, EGFR, Trop2, CDH3 and other TAAs, such as Her2, EGFR, Trop2, CDH3 GST fusion proteins, can also be used. In a specific embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Her2, EGFR, Trop2, CDH3 is produced and then used as an immunogen to generate suitable antibodies. In another embodiment, Her2, EGFR, Trop2, CDH3 and other TAA-related proteins are synthesized and used as immunogens.

[0152] In addition, naked DNA immunization techniques known in the art (with or without purified Her2, EGFR and other TAA-related proteins or Her2, EGFR, Trop2, CDH3 and other TAA-expressing cells) are used to generate immune responses to encoded immunogens (for review, see DONNELLY et al., 1997, Ann. Rev. Immunol., 15:617-648).

[0153] Preferred methods for generating 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 cases, direct conjugation using, for example, carbodiimide reagents is used; in other examples, linking agents, such as those provided by Pierce Chemical Co., Rockford, Illinois, are effective. As understood in the art, administration of Her2, EGFR, Trop2, CDH3 and other TAA immunogens is typically performed by injection over an appropriate period of time and using a suitable adjuvant. During the immunization program, antibody titers can be used to determine the adequacy of antibody formation.

[0154] 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 secreting the desired monoclonal antibodies are prepared using standard hybridoma techniques of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as generally known. Immortalized cell lines secreting the desired antibodies are screened by immunoassays, wherein the antigens are Her2, EGFR, Trop2, CDH3 and other TAA-related proteins. When suitable immortalized cell cultures are identified, cells can be amplified and antibodies produced from in vitro cultures or ascites.

[0155] The antibodies or fragments of the present invention can also be produced by recombinant means. Regions specifically bound to the desired regions of Her2, EGFR, Trop2, CDH3 and other TAA proteins can also be produced in the context of chimeric or complementary determining region (CDR) transplanted antibodies of multiple species sources. Humanized or human Her2, EGFR, Trop2, CDH3 and other TAA antibodies can also be produced, and are preferably used in therapeutic contexts. Methods for humanizing mice and other non-human antibodies by replacing the corresponding human antibody sequences with one or more non-human antibody CDRs 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.

[0156] In one embodiment, human monoclonal antibodies of the invention can be prepared using VelocImmune mice in which genomic sequences bearing endogenous mouse variable segments at immunoglobulin heavy chain (VH, DH, and JH segments) and / or kappa light chain (VK and JK) loci have been replaced in whole or in part with human genomic sequences (Regeneron, Tarrytown, New York) bearing unrearranged germline variable segments of human immunoglobulin heavy chain (VH, DH, and JH) and / or kappa light chain (VK and JK) loci. See, e.g., U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

[0157] In addition, human antibodies of the invention can be generated using the HuMAb mouse (Medarex, Inc.), which contains a human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences and targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., LONBERG et al. (1994), Nature, 368(6474):856-859).

[0158] In another embodiment, the fully human antibodies of the present invention can be produced using mice carrying human immunoglobulin sequences on transgenic and transchromosomes, such as mice carrying human heavy chain transgenic and human light chain transchromosomes. Such mice are referred to herein as "KM mice", such mice are described in TOMIZUKA et al. (2000), Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 97: 722-727 and PCT publications WO 02 / 43478 granted to TOMIZUKA et al.

[0159] The human monoclonal antibodies of the present invention can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are well established in the art. See, for example: U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to LADNER et al.; 5,427,908 and 5,580,717 to DOWER et al.; 5,969,108 and 6,172,197 to McCafferty et al.; and 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to GRIFFITHS et al.

[0160] Human monoclonal antibodies of the invention can also be prepared using SCID mice, in which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767 to WILSON et al.

[0161] In addition, human antibodies of the present invention can be made by using a technique that uses transgenic mice that are inactivated for antibody production and engineered with human heavy and light chain loci, known as Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.). An exemplary description of making transgenic mice that produce human antibodies can be found in U.S. Pat. No. 6,657,103. See also U.S. Pat. Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; 5,545,806; and MENDEZ et al., Nature Genetics, 15: 146-156 (1998); KELLERMAN, SA and GREEN, LL, Curr. Opin. Biotechnol, 13, 593-597 (2002).

[0162] Any of the above production methods produces antibodies with a certain ability to bind to Her2, EGFR, Trop2, CDH3 and other TAAs, or homologs or fragments or polypeptide sequences with 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with Her2, EGFR, Trop2, CDH3 and other TAAs.

[0163] Binding affinity (K) of antibodies, binding fragments thereof and antibody drug conjugates comprising the same to Her2, EGFR, Trop2, CDH3 and other TAAs D ) 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 It can be between 5 and 10 nM; or between 1 and 2 nM. K D It may be between 1 micromolar and 500 micromolar or between 500 micromolar and 1 nM.

[0164] The binding affinity of the 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 through the 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 through the surface. Those skilled in the art can identify other suitable assays known in the art to measure binding affinity.

[0165] The engineered antibodies of the present invention comprise a V H and / or V L The framework residues within the antibody have been modified (for example, to improve the properties of the antibody). Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "reverse mutate" one or more framework residues into the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to restore the framework region sequence to its germline configuration, somatic mutations can be "reverse mutated" into germline sequences (for example, "reverse mutated" from leucine to methionine) by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "reverse mutation" antibodies are also intended to be encompassed by the present invention.

[0166] Another type of framework modification involves mutating one or more residues in the framework region or even in one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization," and is described in more detail in U.S. Patent Publication No. 2003 / 0153043 by CARR et al.

[0167] In addition to or as an alternative to modifications made within the framework or CDR regions, the antibodies of the invention may be engineered to include modifications within the Fc region, typically to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular toxicity. In addition, the Her2, EGFR, Trop2, CDH3 and other TAA MAbs of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the MAb. Each of these embodiments is described in more detail below.

[0168] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 to BODMER et al. The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to promote the assembly of light and heavy chains or to increase or decrease the stability of Her2, EGFR, Trop2, CDH3 and other TAA MAbs.

[0169] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of 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 so that the antibody's SpA binding is impaired relative to the native Fc hinge domain Staphylococcus A protein (SpA) binding. This method is described in more detail in U.S. Pat. No. 6,165,745 to Ward et al.

[0170] In another embodiment, Her2, EGFR, Trop2, CDH3 and other TAA MAbs are modified to extend their biological half-life. Various methods are possible. For example, mutations can be introduced, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, in order to extend the biological half-life, antibodies can be changed in CH1 or CL regions to contain salvage receptor binding epitopes of two loops of the CH2 domain taken from the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to PRESTA et al.

[0171] In other embodiments, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function 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 so that the affinity of the antibody for the effector ligand is changed but the antigen binding ability of the parent antibody is retained. The effector ligand with changed affinity can be, for example, an Fc receptor or C1 component of complement. This method is more specifically described in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

[0172] In a preferred embodiment, Her2, EGFR, Trop2, CDH3 and other TAA Mabs comprise triple substitutions at the following positions: L234A, L235A, L328C.

[0173] In a preferred embodiment, the Her2 Mab comprises triple substitutions at the following positions: L234A, L235A, L328C.

[0174] In a preferred embodiment, the EGFR Mab comprises triple substitutions at the following positions: L234A, L235A, L328C.

[0175] In a preferred embodiment, Trop2 Mab comprises triple substitutions at the following positions: L234A, L235A, L328C.

[0176] In a preferred embodiment, the CDH3 Mab comprises triple substitutions at the following positions: L234A, L235A, L328C.

[0177] In another preferred embodiment, the TAA Mab comprises triple substitutions at the following positions: L234A, L235A, L328C.

[0178] In another embodiment, Her2, EGFR, Trop2, CDH3 and other TAA Mabs comprise a single substitution at the following position: L328C.

[0179] In another embodiment, the Her2 Mab comprises a single substitution at the following position: L328C.

[0180] In another embodiment, the EGFR Mab comprises a single substitution at the following position: L328C.

[0181] In another embodiment, the Trop2 Mab comprises a single substitution at the following position: L328C.

[0182] In another embodiment, the CDH3 Mab comprises a single substitution at the following position: L328C.

[0183] In another embodiment, the TAA Mab of the present disclosure comprises a single substitution at the following position: L328C.

[0184] In another embodiment, Her2, EGFR, Trop2, CDH3 and other TAA Mabs comprise double substitutions at the following positions: L234A, L235A.

[0185] In another embodiment, the Her2 Mab comprises double substitutions at the following positions: L234A, L235A.

[0186] In another embodiment, the EGFR Mab comprises double substitutions at the following positions: L234A, L235A.

[0187] In another embodiment, Trop2 Mab comprises double substitutions at the following positions: L234A, L235A.

[0188] In another embodiment, the CDH3 Mab comprises double substitutions at the following positions: L234A, L235A.

[0189] In another embodiment, the TAA Mab of the present disclosure comprises double substitutions at the following positions: L234A, L235A.

[0190] The reactivity of Her2, EGFR, Trop2, CDH3 and other TAA antibodies can be determined 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 extracts thereof as appropriate. Her2, EGFR, Trop2, CDH3 and other TAA antibodies or fragments thereof 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 chelators or enzymes.

[0191] III.) Fc mutations that modify effector function

[0192] The Fc region of an antibody (i.e., the end of the heavy chain of an antibody across domains CH2, CH3, and part of the hinge region) has limited variability and is involved in influencing the physiological effects exerted by the antibody. The effector functions attributed to the Fc region of an antibody vary with the class and subclass of the antibody, and include antibodies binding to specific Fc receptors ("FcR") on cells via the Fc region, thereby triggering various biological responses.

[0193] These receptors typically have an extracellular domain, a transmembrane region, and an intracellular domain that mediates certain signal transduction events that are bound to Fc. These receptors are expressed in a variety of 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 Fc / FcγR complexes recruits these effector cells to the site of antigen binding, thereby typically causing intracellular signal transduction events and important subsequent immune responses, such as release, B cell activation, endocytosis, engulfment, and cytotoxic attacks of inflammatory mediators. The ability to mediate cytotoxicity and engulf effector functions is the potential mechanism of antibody destruction of targeted cells. The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells and subsequently cause lysis of the target cells 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 FcγRs recognize bound antibodies on target cells and subsequently cause engulfment of the target cells is called antibody-dependent cell-mediated phagocytosis (ADCP). In addition, overlapping sites on the Fc region of the molecule also control the activation of the non-cell-dependent cytotoxic function mediated by complement, which is also called complement-dependent cytotoxicity (CDC).

[0194] Additionally, the complement inflammatory cascade is part of the innate immune response and is essential to an individual's ability to defend against infection. Another important Fc ligand is the complement protein C1q. Fc binding to C1q mediates a process known as complement-dependent cytotoxicity (CDC). C1q is able to bind 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.

[0195] In many cases, the combination and stimulation of the effector function mediated by the Fc region of immunoglobulin are very beneficial. However, in some instances, it may be more advantageous to reduce or even eliminate the effector function. This is especially true for those antibodies designed to be used for delivering drugs (e.g., toxins and isotopes) to target cells, where the effector function mediated by Fc / FcγR brings healthy immune cells to the vicinity of the deadly payload, thereby causing normal lymphoid tissue to be exhausted together with target cells (see Hutchins et al., Proceedings of the National Academy of Sciences (PNAS USA), 92 (1995) 11980-11984. In these cases, using antibodies that recruit complement or effector cells poorly will have great benefits (see also U.S. Patent No. 6,194,551; U.S. Patent No. 5,885,573 and PCT Publication WO 04 / 029207).

[0196] In other examples, for example, in the case of blocking the interaction of widely expressed receptors with their cognate ligands as the goal, it would be advantageous to reduce or eliminate all antibody effector functions to reduce unwanted toxicity. In addition, in examples where therapeutic antibodies show promiscuous binding in many human tissues, it is prudent to limit the targeting of effector functions to multiple tissues to limit toxicity. Finally, the reduction in the affinity of antibodies to FcγRII receptors will be particularly beneficial for antibodies to induce platelet activation and aggregation via FcγRII receptor binding, which will be a serious side effect of such antibodies. See TAM et al., Antibodies, 6:12 (2017). See also WEBER et al., Pharm Res, 35:169 (2018).

[0197] Although certain subclasses of human immunoglobulins lack specific effector functions, there are no known naturally occurring immunoglobulins lacking all effector functions. Another approach is to engineer or mutate the key residues responsible for effector functions in the Fc region. See SCHLOTHAUER et al., Protein Engineering, Design and Selection (Protein Eng. Design, and Selection), Vol. 29, No. 10, pp. 457-466 (2016) and WANG et al., Protein Cell, 9 (1), pp. 63-73 (2018). In fact, some research entities have tried this effort. See, e.g., PCT Publications WO 2009 / 100309 (Medimmune), WO 2006 / 076594 (Xencor), WO 1999 / 58572 (Cambridge University), US 2006 / 0134709 (Macrogenics), WO 2006 / 047350 (Xencor), WO 2006 / 053301 (Xencor), U.S. Pat. No. 6,737,056 (Genentech), U.S. Pat. No. 5,624,821 (Scotgen Pharmaceuticals), US 2010 / 0166740 (Roche), and US 8,969,526 (Roche Glycart AG).

[0198] The binding of IgG to activating and inhibitory Fcγ receptors or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcγR and complement C1q binding and have unique sequences. Substitution of human IgG1 and IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces 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).

[0199] In addition, 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 to C1q and activate complement. In addition, it has been reported that replacing Pro329 with Ala results in reduced binding to 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 exhibiting wild-type-like binding to FcγRI and FcγRII, and only a very small reduction in binding to the FcγRIIIA receptor (see EP 1,068,241, Genentech).

[0200] Furthermore, OGANESYAN et al., Acta Cristallographica, D64 (2008) 700-704, introduced the triple mutation L234F / L235E / P331S into the lower hinge and C2H domain and showed a reduction in binding activity to human IgG1 molecules and to human C1q receptor, FcγRI, FcγRII and FcγRIIIA.

[0201] Against the foregoing background, the present invention relates to a method for making a polypeptide comprising an Fc variant, wherein the Fc is silenced and / or inhibited. "Parent", "starting", "non-variant" or "wild-type" polypeptides are prepared using techniques that can be used in the art to generate polypeptides or antibodies comprising an Fc region. In a preferred embodiment of the present 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 Her2, EGFR, Trop2, CDH3 and / or any TAA of the present disclosure (see Table IV).

[0202] One embodiment of the present invention encompasses a polypeptide comprising an Fc region of an antibody, comprising an addition, substitution or deletion of at least one amino acid residue of the Fc region, resulting in a reduction or elimination of affinity for at least one Fc receptor. The Fc region interacts with many 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 a variety of effector functions and downstream signaling events, including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0203] Thus, in certain embodiments, the variants of the invention have reduced or eliminated affinity for Fc receptors responsible for effector function compared to a polypeptide having the same amino acid sequence as a polypeptide comprising an Fc variant of the invention but not comprising an addition, substitution or deletion of at least one amino acid residue in the Fc region (also referred to herein as a "wild-type polypeptide"). In certain embodiments, polypeptides comprising Fc variants of the invention include at least one or more of the following properties: reduced or eliminated effector (ADCC and / or CDC and / or ADCP) function, reduced or eliminated binding to Fc receptors, reduced or eliminated binding to C1q, or reduced or eliminated toxicity. More specifically, embodiments of the invention provide anti-Her2, anti-EGFR, anti-Trop2, anti-CDH3, and anti-TAA (tumor-associated antigens (Table IV)) antibodies with reduced affinity for Fcγ receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB) and / or complement protein C1q.

[0204] In one embodiment, the invention includes Her2, EGFR, Trop2, CDH3 and TAA MAbs comprising triple mutations.

[0205] In one embodiment, an antibody of the invention comprises an Fc region comprising at least one addition, substitution or deletion of an amino acid residue at position 328, wherein the numbering system for the constant region is that of the EU index as set forth in KABAT et al., NIH Publication, 91 (1991) 3242, National Technical Information Service, Springfield, VA.

[0206] In one embodiment, an antibody of the invention comprises an Fc region comprising at least one addition, substitution or deletion of an amino acid residue at position 234, wherein the numbering system for constant regions is that of the EU index as set forth by KABAT et al., NIH Publication, 91 (1991) 3242, National Technical Information Service, Springfield, VA.

[0207] In one embodiment, an antibody of the invention comprises an Fc region comprising at least one addition, substitution or deletion of an amino acid residue at position 235, wherein the numbering system for the constant region is that of the EU index as set forth by KABAT et al., NIH Publication, 91 (1991) 3242, National Technical Information Service, Springfield, VA.

[0208] In a certain aspect of the invention, the polypeptide comprising an Fc variant comprises an antibody. In another aspect of the invention, the polypeptide comprising an Fc variant comprises a human IgG1, IgG2, IgG3 or IgG4 Fc region. In yet another aspect of the invention, the variant is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0209] In another specific embodiment, the polypeptide comprises a human IgG1 region.

[0210] In a specific embodiment, the polypeptide of the present invention includes an Fc variant of a wild-type human Fc polypeptide, wherein the variant includes an amino acid substitution at position L328C, wherein the numbering of the residues in the IgG Fc region is the numbering of the EU index as in Kabat. In another embodiment, the variant includes at least one other amino acid substitution. In another embodiment, the variant includes at least one other amino acid substitution. In another embodiment, the variant includes three (3) amino acid substitutions at L234A, L235A and L328C.

[0211] In a specific embodiment, the anti-Her2 MAbs of the invention include an Fc variant of a wild-type human Fc Her2 Mab comprising three (3) amino acid substitutions at L234A, L235A, and L328C.

[0212] In a specific embodiment, the anti-EGFR MAb of the invention comprises an Fc variant of a wild-type human Fc EGFR Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0213] In a specific embodiment, the anti-Trop2 MAb of the invention comprises an Fc variant of wild-type human Fc Trop2 Mab comprising three (3) amino acid substitutions at L234A, L235A and L328C.

[0214] In a specific embodiment, the anti-CDH3 MAbs of the invention include an Fc variant of a wild-type human Fc CDH3 Mab comprising three (3) amino acid substitutions at L234A, L235A, and L328C.

[0215] In a specific embodiment, the TAA MAbs of the invention include an Fc variant of a wild-type human Fc TAA Mab comprising three (3) amino acid substitutions at L234A, L235A, and L328C.

[0216] In one aspect, a polypeptide comprising an Fc variant does not affect target binding compared to the unmodified antibody.

[0217] In one aspect, polypeptides comprising an Fc variant of the invention exhibit inhibition of FcγRI binding compared to an unmodified antibody.

[0218] In one aspect, polypeptides comprising an Fc variant of the invention exhibit inhibition of FcγRII binding compared to an unmodified antibody.

[0219] In one aspect, polypeptides comprising an Fc variant of the invention exhibit inhibition of FcγRIIIa binding compared to an unmodified antibody.

[0220] In one aspect, polypeptides comprising an Fc variant of the invention do not substantially affect FcRn binding compared to an unmodified antibody.

[0221] As will be appreciated by those skilled in the art, and the surprising realization of the present invention, the need for triple mutation, specifically the addition of amino acid substitutions at L234A and L235A, is due to the following observations. First, L328C by itself exhibits only a partial reduction in Fc effector function. Greater Fc silencing can be achieved by combining additional mutations. 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 exhibits almost complete inhibition of FcγR interaction for all isotypes tested.

[0222] As observed, even though FcγR binding was completely abolished, the triple mutation (L328C in combination with L234A / L235A) nor meeting The triple mutations did not affect target antigen binding, as comparable binding activity was observed compared to the wild-type counterpart. Antibody stability and expression also remained comparable to the wild-type antibody. Thus, the triple mutations allowed for complete inhibition of Fc effector function without compromising target specificity, antibody quality, and yield.

[0223] Furthermore, as discussed below, specific triple mutations can promote efficient site-specific conjugation.

[0224] IV.) Antibody Drug Conjugates

[0225] In another aspect, the invention provides antibody-drug conjugates (ADC), which include antibodies conjugated to therapeutic agents. Therapeutic agents can be cytotoxic agents, cytostatic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., enzyme activity toxins from bacteria, fungi, plants or animals, or fragments thereof) or radioactive isotopes (i.e., radioactive conjugates). In another aspect, the invention further provides methods using ADC. In one aspect, ADC includes any of the above-mentioned Her2, EGFR, Trop2, CDH3 and other TAA MAbs covalently attached to cytotoxic agents or detectable agents or attached via oxime bonds.

[0226] In another embodiment, the ADC comprises Her2, EGFR, Trop2, CDH3 and other TAA Mabs further comprising triple substitutions at the following positions: L234A, L235A, L328C.

[0227] In a preferred embodiment, the ADC includes Her2, EGFR, Trop2, CDH3 and other TAA Mabs, which further include triple substitutions at the following positions: L234A, L235A, L328C, and further include site-specific conjugation at L328C.

[0228] As background, antibody-drug conjugates are used for local delivery of cytotoxic or cytostatic agents, i.e., delivery of drugs to kill or inhibit tumor cells in cancer therapy (Syrigos and Epenetos, (1999) Anticancer Res. Research, 19:605-614; NICULESCU-DUVAZ and SPRINGER, (1997) Adv. Drg. Del. Rev., 26:151-172; U.S. Pat. No. 4,975,278), which allows targeted delivery of drug moieties to tumors and their intracellular accumulation, where systemic administration of these unconjugated drug agents may cause unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (BALDWIN et al., (1986) Lancet, (March 15, 1986) pp. 603-05; Thorpe, (1985) Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review). Review), Monoclonal Antibodies '84: Biological and Clinical Applications, A. PINCHERA et al. (eds.), pp. 475-506). Thus, maximum efficacy with minimal toxicity is 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), maytansine alkaloids (EP 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 may affect their cytotoxic and cytostatic effects through mechanisms involving 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.

[0229] Examples of antibody drug conjugates are ADCETRIS (brentuximab, Seattle Genetics), (ibritumomab tiuxetan, Biogen / Idec), MYLOTARG TM (Gemmuzumab uzumab, Wyeth Pharmaceuticals), (ado trastuzumab, Genentech), (inotuzumab, Pfizer / Wyeth), POLIVY (polotuzumab, Genentech / Roche), mocantuzumab (Immunogen, Inc.), and MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.).

[0230] In addition, therapeutic agents are described herein, including but not limited to chemotherapeutic agents that can be used to generate ADCs. 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, α-sarcin, tung oil protein, dianthin protein, American pokeweed protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, saponin inhibitor, gelonin, milinomycin, restrictocin, phenomycin, enomycin and trichothecenes. See, for example, WO 93 / 21232 published on October 28, 1993. A variety of radionuclides can be used to produce radioactive conjugated antibodies. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re. Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., 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, a ricin immunotoxin can be prepared as described in Vitetta et al., (1987) Science, 238:1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies (WO 94 / 11026).

[0231] Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozotocin, vincristine and 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Pat. No. 5,877,296).

[0232] 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, α-sarcin, Aleurites fordii protein, dianthus protein, pokeweed protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, Curcas toxin, croton toxin, saponin inhibitor, gelonin, milinomycin, restrictocin, phenomycin, enomycin and trichothecenes. See, for example, WO 93 / 21232 (published October 28, 1993).

[0233] The present invention further contemplates ADCs formed between an antibody and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

[0234] For selective destruction of tumors, antibodies can include highly radioactive atoms. A variety of radioactive isotopes can be used to produce radioconjugated antibodies. Examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 88 、Sm 53 、Bi 212 , P 32 , Pb 212 When the conjugate is used for detection, it may include a radioactive atom for scintigraphic studies, such as tc 99m or I 123 , or spin labels 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, again mentioned.

[0235] Radioactive or other labels may be incorporated into the conjugate in a known manner. For example, the peptide may be biosynthesized, or may be synthesized by chemical amino acid synthesis using appropriate amino acid precursors, involving, for example, replacement of hydrogen with fluorine-19. 99m or I 123 、Re 186 、Re 188 andIn 111Labels such as iodine and iodine 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. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (CHATAL, CRC Press, 1989).

[0236] The present invention provides, inter alia, antibody-drug conjugate compounds for targeted delivery of therapeutic agents. The inventors have found that the antibody-drug conjugate compounds have potent cytotoxic and / or cytostatic activity against cells expressing Her2, EGFR, Trop2, CDH3 and other TAAs.

[0237] The antibody-drug conjugate compound includes an antibody unit covalently linked to at least one drug unit. The drug unit can be directly covalently linked or linked to the antibody unit via a linker unit (-LU-). In addition, the drug unit is conjugated at a site-specific position at L328C.

[0238] In some embodiments, the antibody drug conjugate compound has the formula:

[0239] L-(LU-D) p (I)

[0240] or a pharmaceutically acceptable salt or solvate thereof; wherein:

[0241] oL is an antibody unit, such as Her2, EGFR, Trop2, CDH3 or another TAA MAb of the invention; and wherein the MAb comprises triple mutations at the following positions: L234A, L235A and L328C;

[0242] o(LU-D) is a Linker unit-Drug unit moiety, wherein:

[0243] οLU - is a connector unit, and

[0244] o-D is a drug unit having cytostatic or cytotoxic activity on target cells; and

[0245] The range of οp is 1 to 20 or 1-50.

[0246] Furthermore, the Drug unit moiety was conjugated at a site-specific position on the MAb at L328C.

[0247] In some embodiments, the antibody drug conjugate compound has the formula:

[0248] L-(A a -W w —Y y -D) p (II)

[0249] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0250] o L is an antibody unit, such as Her2, EGFR, Trop2, CDH3 or another TAA MAb, and wherein the MAb comprises triple mutations at the following positions: L234A, L235A, and L328C; and

[0251] ο-A a -W w -Y y - is a connection subunit (LU), wherein:

[0252] o-A- is an elongation subunit,

[0253] οa is 0 or 1 or 2 or 3,

[0254] o each -W- is independently an amino acid unit,

[0255] οw is an integer from 0 to 12,

[0256] o-Y- is a self-immolative spacer unit,

[0257] οy is 0, 1 or 2;

[0258] o-D is a drug unit having cytostatic or cytotoxic activity on target cells; and

[0259] οp is an integer from 1 to 20 or 1-50.

[0260] Furthermore, the Drug unit moiety was conjugated at a site-specific position on the MAb at L328C.

[0261] For compositions including multiple antibodies, drug loading is represented by p, which is the average number of drug molecules per antibody. The drug loading range for each antibody is 1 to 20 drugs (D). The average number of drugs per antibody in the preparation of the conjugated reactants can be characterized by conventional means, such as mass spectrometry, ELISA assays and HPLC. The quantitative distribution of antibody-drug conjugates represented by p can also be determined. In some instances, the separation, purification and characterization of homogeneous antibody-drug conjugates with p being a certain value and antibody-drug conjugates with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis. In an exemplary embodiment, p is 2 to 8.

[0262] The generation of antibody-drug conjugate compounds can be accomplished by any technique known to those skilled in the art. In short, the antibody-drug conjugate compound includes Her2, EGFR, Trop2, CDH3 or another TAA MAb, and wherein the MAb includes a triple mutation as an antibody unit, a drug and an optional linker, wherein the triple mutation is located at the following positions: L234A, L235A and L328C; the linker joins the drug and the binding agent together. In a preferred embodiment, the drug unit is conjugated at a site-specific position at L328C.

[0263] Many different reactions can be used for the covalent attachment of drugs and / or linkers to binding agents. This is usually accomplished by the reaction of the amino acid residues of the binding agent (e.g., antibody molecule), which contain the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine ​​and the various parts of aromatic amino acids. One of the most commonly used non-specific covalent attachment methods is the carbodiimide reaction that connects the carboxyl (or amino) group of the compound to the amino (or carboxyl) group of the antibody. In addition, bifunctional reagents such as dialdehydes or iminoesters have been used to connect the amino group of the compound to the amino group of the antibody molecule. Schiff base reaction can also be used for the attachment of drugs to binding agents. The present method involves the periodate oxidation of drugs containing diols or hydroxyl groups to form aldehydes, which then react with the binding agent. Attachment occurs via the formation of Schiff bases with the amino groups of the binding agent. Isothiocyanates can also be used as coupling agents to covalently attach drugs to binding agents. Other techniques are known to those skilled in the art and within the scope of the present invention.

[0264] In certain embodiments, the intermediate as a linker precursor reacts with the drug under appropriate conditions. In certain embodiments, a reactive group is used on the drug and / or the intermediate. The product of the reaction between the drug and the intermediate or the derivative drug is then reacted with Her2, EGFR, Trop2, CDH3 or other TAA MAbs under appropriate conditions.

[0265] V.) Site-specific conjugation formats of ADC

[0266] As one of ordinary skill in the art will appreciate, the ability to optimize payload placement and conjugate composition in the context of ADCs is an advantageous endeavor. See ABHIJIIT et al., Bioprocess Int., Mab Upstream Processing (October 2014).

[0267] In general, bioconjugation strategies involve covalently linking a protein or peptide (biologic) to a small molecule, carbohydrate, oligonucleotide, synthetic polymer, or another protein / peptide. This approach is critical to creating differentiation and driving product development in the competitive biologic market. These strategies are essential for the development of highly successful conjugate vaccines, such as Prevnar 13, Menactra, Menomune, and HibTITER. These four (4) are produced by conjugating bacterial polysaccharides to immunogenic carrier proteins. Similarly, the use of bioconjugation with half-life-extending polymeric carriers, such as polyethylene glycol (PEG), has produced current commercial drugs, such as Cimzia, Neulasta, and Somavert, which have a longer duration of action than their non-conjugated counterparts and a dosing regimen that promotes patient compliance.

[0268] As is known in the art, the choice of linker and conjugation chemistry used to make first generation ADCs was dictated by the constraints of the protein being used. Therefore, the linker was functionalized with reactive groups designed to react specifically with surface accessible nucleophilic amino acid side chains belonging to natural amino acids such as cysteine ​​(thiol) or lysine (amine).

[0269] However, treatment of antibodies with reducing agents such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) may disrupt these disulfide bonds and expose free thiols, which can then be easily conjugated to maleimide-containing linkers. Up to four interchain disulfide bonds can be reduced, exposing up to eight reactive thiol groups for conjugation. Conditions developed for thiol chemical conjugation result in complete or partial reduction of disulfide bonds, and conjugates made using this method can contain zero, two, four, six, or eight drugs per antibody molecule.

[0270] It is worth noting that, in addition to the number of drugs per antibody molecule, another level of heterogeneity also comes from the conjugation site. Therefore, the ADC with a specific drug / antibody ratio (DAR) generated by cysteine ​​conjugation is still a heterogeneous mixture of conjugates with different conjugation sites. However, it is fair to say that because only eight sites can be used for cysteine ​​conjugation (compared to up to 80 can be used for lysine-directed chemistry), the cysteine ​​conjugation method provides better control of the conjugation site, thereby contributing to better characterization. The controlled reduction-alkylation strategy has been successfully used to manufacture the approved ADCETRIS (Seattle Genetics, Bothell, Washington) products and many other ADCs currently undergoing clinical trials.

[0271] The researchers studied the in vivo effects of ADCs targeting CD30+ tumor cells with two, four, and eight monomethyl auristatin E (MMAE) toxins per antibody molecule; and demonstrated that the stoichiometry of drug loading significantly affects the pharmacokinetics (PK), efficacy, and toxicity of the drug. HAMBLETT et al. found that in their system, ADCs with four drugs per antibody were more potent than those with two drugs per antibody, but had comparable efficacy and better tolerability than those with eight drugs per antibody. The results indicate that, in general, ADCs with higher drug loadings have greater clearance, higher efficacy, and higher toxicity. This means that each ADC will have an optimal drug loading at the appropriate balance of efficacy and toxicity.

[0272] This seminal work established the concept that DAR is a critical design parameter for ADCs. It is clear that chemical conjugation to native cysteine ​​or lysine residues is suboptimal as they produce heterogeneous mixtures of ADCs. Heterogeneity arises from differences in DAR and conjugation sites, resulting in subpopulations of ADCs that may be less potent, more toxic, and have different disposition and PK properties. In addition, analytical characterization and control of batch-to-batch variations during manufacturing remain significant challenges for such non-selective conjugation approaches. To overcome these limitations, the concept of site-specific conjugation has been developed, with the initial goal of producing homogeneous ADCs and controlling the DAR and conjugation site.

[0273] Several site-specific conjugation technologies have been demonstrated using a variety of techniques to optimize payload placement (see, eg, ThioMab (Genentech), Seattle Genetics, and Ambrx, Inc.).

[0274] The work reported on ThioMab reveals another fundamental concept: not only is ADC homogeneity key to improving biophysical and therapeutic properties, but the actual binding site on the antibody backbone also has a significant impact on the in vivo behavior of the ADC molecule. Shen et al. made multiple homogeneous TDC conjugates with HER2-targeting antibodies using MMAE payloads, in which the engineered cysteines for conjugation were located in 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.

[0275] When studied in a mouse xenograft model, the LC conjugate exhibited the greatest efficacy, with the HC conjugate having moderate activity and the Fc conjugate having little activity. A mouse PK study revealed a similar trend, with the LC conjugate exhibiting the greatest stability and lowest clearance, followed by the HC conjugate, while the Fc-conjugated ADC was cleared fastest and provided the lowest ADC exposure. These results have been attributed to differences in the local microenvironment and solvent accessibility, which result in different stabilities of the linker systems at different sites.

[0276] Several additional site-specific bioconjugation approaches have been reported with the goal of delivering homogenous ADCs relative to first-generation lysine and cysteine ​​conjugations, but only some of these technologies offer greater versatility in finding the optimal conjugation site for a given antibody-payload combination. These include conjugation of 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); and transglutaminase (TG)-mediated conjugation with engineered glutamine tags (Pfizer, New York, NY); and conjugation of aldehyde-labeled antibodies generated by co-expressed formylglycine generating enzymes (FGE) (Redwood Bioscience, acquired by Catalent PharmaSolutions, Emeryville, CA).

[0277] Based on the above, research into technologies that provide flexible site-specific conjugation options has determined that the conjugation site significantly affects the pharmacological properties of ADCs. Therefore, it should be considered a key parameter in product design, and the ability to find the optimal site for a specific payload-antibody combination may be critical to the success of the development of one or more products. See SCHUMACHER et al., J. 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 (January / February 2014); PCT Patent Publication WO2018 / 20081 and U.S. Patent Publication No.: 2017 / 0080103.

[0278] In one embodiment, a site-specific conjugation method via conversion of Leu-328 to Cys in the Fc domain of a monoclonal antibody allows controlled conjugation without affecting target binding. In addition, it was shown that the introduction of L328C does not impair the expression level and stability of the antibody.

[0279] In another embodiment, a site-specific conjugation method via conversion of Leu-328 to Cys in the Fc domain of Her2 antibodies allows controlled conjugation without affecting target binding. In addition, it was shown that the introduction of L328C did not impair the expression level and stability of the antibody.

[0280] In another embodiment, a site-specific conjugation method via conversion of Leu-328 to Cys in the Fc domain of an EGFR antibody allows controlled conjugation without affecting target binding. In addition, it was shown that the introduction of L328C did not impair the expression level and stability of the antibody.

[0281] In another example, a site-specific conjugation approach via conversion of Leu-328 to Cys in the Fc domain of the Trop2 antibody allowed controlled conjugation without affecting target binding. In addition, it could be shown that the introduction of L328C did not impair the expression level and stability of the antibody.

[0282] In another embodiment, a site-specific conjugation method via conversion of Leu-328 to Cys in the Fc domain of the CDH3 antibody allows controlled conjugation without affecting target binding. In addition, it was shown that the introduction of L328C did not impair the expression level and stability of the antibody.

[0283] In another embodiment, a site-specific conjugation method that converts Leu-328 to Cys in the Fc domain of a TAA antibody (e.g., a TAA listed in Table IV) allows controlled conjugation without affecting target binding. In addition, it was shown that the introduction of L328C does not impair the expression level and stability of the antibody.

[0284] The site-specific conjugation method mediated by L328C was selected to make the drug product more homogeneous and improve conjugation efficiency. Various therapeutic modalities including antibody drug conjugates (ADCs) can benefit from site-specific conjugation because it can prevent the formation of heterogeneous mixtures, which can negatively affect in vivo efficacy and therapeutic index. Similarly, attaching boron-containing entities to specific sites of antibody molecules can improve the efficacy of boron neutron capture therapy (BNCT), a non-invasive treatment modality for treating cancer.

[0285] The conversion of Leu-328 to Cys in the Fc domain of the monoclonal antibody allows controlled conjugation without affecting target binding. In addition, the introduction of L328C does not impair the expression level and stability of the antibody. Quality assessment via size exclusion chromatography shows a main peak of >99%, comparable to the wild-type counterpart. Therefore, no aggregation tendency usually associated with the introduction of unpaired cysteines was observed for L328C. Compared with the complex and inefficient production process required for non-specific conjugated counterparts, the uniform product formation with 100% conjugation efficiency mediated by L328C means a simpler manufacturing process. The clear and homogeneous composition mediated by L328C conjugation greatly reduces the therapeutic burden and enables a simpler manufacturing process, which can accelerate the discovery and development of ADCs and antibody boron conjugates for BNCT applications.

[0286] Furthermore, L328C also exhibits the additional benefit of reducing Fc effector function. Safety burdens associated with infusion reactions triggered 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 to improve clinical safety can be further developed for L328C-mediated conjugation or as a naked antibody, which may be combined with other variants previously shown to reduce Fc effector function.

[0287] VI.) Connecting subunits

[0288] Typically, the 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 other embodiments, the linker unit is non-cleavable, and the drug is released, for example, by antibody degradation.

[0289] In a preferred embodiment, the linker is conjugated at the site-specific position L328C.

[0290] In some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease, including but not limited to a lysosomal or endosomal protease. The linker can also be cleaved by a cleavage agent present in the extracellular environment (e.g., near a cell membrane or tissue space). The linker can be, for example, a peptidyl linker that is cleaved by an extracellular peptidase or protease, including but not limited to a cathepsin family enzyme or a matrix metalloproteinase.

[0291] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH sensitive linker can be hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., oximes, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that can be hydrolyzed in lysosomes can be used. (See, e.g., U.S. Patent 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).

[0292] In other embodiments, the linker can be cleaved under reducing conditions known in the art. (See, e.g., Thorpe et al., 1987, Cancer Res., 47:5924-5931; WAWRZYNCZAK et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW VOGEL, ed., Oxford University Press, 1987. See also U.S. Pat. No. 4,880,935). The linker can also be cleaved under reducing conditions found in cells (or extracellularly). For example, in a preferred embodiment, a specific linker N-O bond can be formally reduced and broken to result in the cleavage of the linker.

[0293] In other embodiments, the linker unit is non-cleavable and the drug is released by antibody degradation. (See PCT Publication No. WO2012 / 166560 (Ambrx, Inc.), which is incorporated herein by reference in its entirety and for all purposes).

[0294] In other non-mutually exclusive embodiments, the linker facilitates cellular internalization as known in the art.

[0295] Various exemplary linkers that can be used with the compositions and methods of the present invention are described in WO 2004 / 010957, US Publication No. 2006 / 0074008, US Publication No. 20050238649, and US Publication No. 2006 / 0024317 (each of which is incorporated herein by reference in its entirety and for all purposes).

[0296] VII.) Elongation subunit

[0297] When present, the stretcher unit (A) is capable of connecting the antibody unit to the amino acid unit (-W-) (if present), to the spacer unit (-Y-) (if present); or to the drug unit (-D). Useful functional groups that may exist naturally or via chemical manipulation on Her2, EGFR, Trop2, CDH3 or other TAA MAbs include, but are not limited to, keto, aldehyde, sulfhydryl, amino, hydroxyl, anomeric hydroxyl of a carbohydrate, and carboxyl. Suitable functional groups are keto, aldehyde, sulfhydryl and amino. In one example, the keto group is located on a non-natural amino acid (nnAA) incorporated into the MAb of the present invention. In another example, the aldehyde group is located on the nnAA incorporated into the MAb of the present invention. In another example, the sulfhydryl group can be generated by reduction of intramolecular disulfide bonds of Her2, EGFR, Trop2, CDH3 or other TAA MAbs. In another embodiment, the sulfhydryl group can be generated by the reaction of the amino group of the lysine moiety of Her2, EGFR, Trop2, CDH3 or other TAA MAbs with 2-iminothiolane (Trouble reagent) or other sulfhydryl generating reagents. In certain embodiments, Her2, EGFR, Trop2, CDH3 or other TAA MAbs are recombinant antibodies and are engineered to carry one or more lysines. In certain other embodiments, recombinant Her2, EGFR, Trop2, CDH3 or other TAA MAbs are engineered to carry additional sulfhydryl groups, such as additional cysteine.

[0298] In a preferred embodiment, the Her2, EGFR, Trop2, CDH3 or other TAA MAb comprises an Fc modification at L234A, L235A, and further comprises another modification at L328C, and further comprises a site-specific conjugation at L328C.

[0299] In a preferred embodiment, the stretcher unit is located at L328C.

[0300] VIII.) Amino Acid Unit

[0301] When present, the Amino Acid unit (-W-) links the Stretcher unit to the Spacer unit (if the Spacer unit is present), links the Stretcher unit to the Drug Moiety (if the Spacer unit is not present), and links the Antibody unit to the Drug unit (if the Stretcher unit and Spacer units are not present).

[0302] In certain embodiments, the Amino Acid unit may include natural amino acids. In other embodiments, the Amino Acid unit may include non-natural amino acids.

[0303] In some embodiments, the Amino Acid unit can be enzymatically cleaved by one or more enzymes including a cancer or tumor associated protease to release the Drug unit (-D), which in one embodiment is protonated in vivo after release to provide Drug (D).

[0304] In a preferred embodiment, the amino acid unit is located at L328C.

[0305] IX.) Spacer unit

[0306] When present, the spacer unit (-Y-) connects the amino acid unit to the drug unit (when the amino acid unit is present). Alternatively, when the amino acid unit is not present, the spacer unit connects the stretcher unit to the drug unit. When both the amino acid unit and the stretcher unit are not present, the spacer unit also connects the drug unit to the antibody unit. There are two general types of spacer units: 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).

[0307] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (HAY et al., 1999, Bioorg. Med. Chem. Lett., 9:2237) and o- or p-aminobenzyl acetals. Spacers that undergo cyclization upon amide bond hydrolysis may be used, such as 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 amide (AMSBERRY et al., 1990, J. Org. Chem., 55:5867). 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-immolative spacer.

[0308] In a preferred embodiment, the amino acid unit is located at L328C.

[0309] X.) Drug Unit

[0310] The drug unit (D) can be any therapeutic agent. For example, the drug unit can be a moiety or chemotherapeutic agent with cytotoxicity, cytostasis or immunomodulatory (e.g., immunosuppressive). D is a drug unit (part) with atoms that can form a bond with a spacer unit (if present), an amino acid unit (if present), an extender unit (if present), or an antibody unit. In certain embodiments, the drug unit D has a nitrogen atom that can form a bond with a spacer unit (if used). As used herein, the terms "drug unit" and "drug moiety" are synonymous and can be used interchangeably.

[0311] XI.) Methods for determining the cytotoxic effect of ADCs

[0312] Methods for determining whether a drug or antibody-drug conjugate exerts a cytostatic and / or cytotoxic effect on a cell are known. Typically, the cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing the target protein of the antibody-drug conjugate to a 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 the viability (proliferation), cytotoxicity, and apoptosis induction (caspase activation) of antibody-drug conjugates.

[0313] 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 / well in a 96-well plate for 72 hours and exposed to 0.5 μCi of thymidine for the last 8 hours of the 72-hour period. 3 H-thymidine. Measured in the presence and absence of ADC 3 H-thymidine incorporation in cultured cells.

[0314] To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased plasma membrane permeability; cell swelling and plasma membrane rupture. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous nucleases. Determination of any of these effects on cancer cells indicates that the ADC can be used to treat cancer.

[0315] Cell viability can be measured by measuring the uptake of dyes such as neutral red, trypan blue or ALAMAR TMBlue (see, e.g., Page et al., 1993, Intl. J. Oncology, 3:473-476). In this assay, cells are incubated in medium containing the dye, the cells are washed, and the remaining dye is measured spectrophotometrically, which reflects the uptake of the dye by the cells. Cytotoxicity can also be measured using the protein-bound dye sulforhodamine B (SRB) (SKEHAN et al., 1990, J. Natl. Cancer Inst., 82:1107-12).

[0316] Alternatively, tetrazolium salts such as MTT can be used in quantitative colorimetric assays of mammalian cell survival and proliferation by detecting living cells but not dead cells (see, e.g., MOSMANN, 1983, J. Immunol. Methods, 65:55-63).

[0317] Apoptosis can be quantified by measuring, for example, DNA fragmentation.

[0318] Commercial photometric assays for quantitative in vitro determination of DNA fragmentation can be used. Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, No. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0319] Apoptosis can also be determined by measuring morphological changes in cells. For example, as with necrosis, the loss of plasma membrane integrity can be determined by measuring the absorption of certain dyes (e.g., fluorescent dyes, such as acridine orange or ethidium bromide). The method for measuring the number of apoptotic cells has been described by Duke and Cohen, Current Protocols in Immunology (ed., COLIGAN et al., 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 chromatin condensation and marginalization of the cells along the inner nuclear membrane can be observed. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage.

[0320] The presence of apoptotic cells can be measured in both the attached and "floating" compartments of the culture. For example, the two compartments can be collected by removing the supernatant, trypsinizing the attached cells, combining the preparations after a centrifugation wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation). (See, e.g., PIAZZA et al., 1995, Cancer Research, 55:3110-16).

[0321] In vivo, the effect of Her2, EGFR, Trop2, CDH3 or other TAA MAb therapeutic compositions can be evaluated in a suitable animal model. For example, a xenogeneic cancer model 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 WO98 / 16628 and U.S. Patent No. 6,107,540 describe various xenograft models of human prostate cancer, which can summarize the development of primary tumors, micrometastases and the formation of osteoblast metastases representing late-stage disease characteristics. Efficacy can be predicted using assays that measure the inhibition of tumor formation, tumor regression or metastasis, etc.

[0322] In vivo assays to evaluate apoptosis promotion can be used to evaluate therapeutic compositions. In one embodiment, xenografts from tumor-bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in tumors of treated mice provides an indication of the therapeutic efficacy of the composition.

[0323] The therapeutic composition used in the practice of the aforementioned method can be formulated into a pharmaceutical composition including a carrier suitable for the desired delivery method. Suitable carriers include any material that retains the anti-tumor function of the therapeutic composition when combined with the therapeutic composition and does not usually react with the patient's immune system. Examples include but are not limited to any of a variety of standard pharmaceutical carriers, such as sterile phosphate buffered saline, bacteriostatic water, etc. (generally referring to Remington's Pharmaceutical Sciences 16th edition, A. Osal (A. Osal) edited, 1980).

[0324] The therapeutic formulation can be dissolved 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, in situ, and the like. Preferred formulations for intravenous injection include solutions of the therapeutic composition in preservative bacteriostatic water, sterile non-preservative water, and / or diluents containing 0.9% sterile sodium chloride for injection, USP in polyvinyl chloride or polyethylene bags. The therapeutic protein formulation can be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water (e.g., containing a benzyl alcohol preservative) or sterile water prior to injection.

[0325] Dosages and administration regimens for treating cancer using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors understood in the art.

[0326] In one embodiment, due to the modification of Her2, EGFR, Trop2, CDH3 or other TAA MAbs, the pharmaceutical composition of the present invention may include more than one ADC species of the present invention. For example, the present invention includes a pharmaceutical composition comprising an ADC of the present invention, wherein Her2, EGFR, Trop2, CDH3 or other TAA MAbs are antibodies with partial or complete removal of C-terminal lysine, antibodies with N-terminal post-translational modifications, antibodies lacking heavy chain C-terminal lysine and having N-terminal post-translational modifications, and / or antibodies with heavy chain C-terminal lysine and no N-terminal post-translational modifications.

[0327] In a preferred embodiment, the Her2, EGFR, Trop2, CDH3 or other TAA MAb is a triple mutation at L234A, L235A and L328C, wherein L328C is the position for site-specific conjugation.

[0328] XII.) Treatment of cancers expressing Her2, EGFR, Trop2, CDH3 and TAA

[0329] Identification of Her2, EGFR, Trop2, CDH3 or other TAAs as proteins that are normally expressed in a restricted set of tissues or cells, but are also expressed in cancers such as those listed in Table I, opens up a variety of therapeutic approaches to treat such cancers.

[0330] It is noteworthy that targeted anti-tumor therapy is useful even if the targeted protein is expressed on normal tissues or cells, even on vital normal organ tissues. Vital organs are organs that are essential for maintaining life, such as the heart or colon. Non-vital organs are organs that can be removed so that the individual can still survive. Examples of non-vital organs are ovaries, breasts, and prostates.

[0331] Expression of a target protein in normal tissues, even critical normal tissues, does not undermine the utility of a targeting agent for that protein as a therapeutic agent for certain tumors where that protein is also overexpressed. For example, expression in critical organs is not harmful in itself. Furthermore, organs that are considered dispensable, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some critical organs are not affected by expression in normal organs due to immunoprivilege. Immunoprivileged organs are organs that are protected from the blood by the blood-organ barrier and therefore cannot be treated with immunotherapy. Examples of immunoprivileged organs are the brain and testes.

[0332] Therefore, therapeutic methods that inhibit the activity of Her2, EGFR, Trop2, CDH3 or other TAA proteins can be used for patients with cancers expressing Her2, EGFR, Trop2, CDH3 or other TAAs (such as, for example, those listed in Table I). These therapeutic methods are generally divided into three categories. The first category regulates the function of Her2, EGFR, Trop2, CDH3 or other TAAs because it is related to tumor cell growth, thereby resulting in inhibition or delay of tumor cell growth or induction of its killing. The second category includes various methods for inhibiting the binding or association of Her2, EGFR, Trop2, CDH3 or other TAA proteins with their binding partners or other proteins. The third category 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.

[0333] Therefore, the presence and level of Her2, EGFR, Trop2, CDH3 or other TAA expression in cancer patients can be evaluated, preferably using immunohistochemical evaluation of tumor tissue, quantitative Her2, EGFR, Trop2, CDH3 or other TAA imaging or other techniques that reliably indicate the presence and extent of Her2, EGFR, Trop2, CDH3 or other TAA expression. If applicable, immunohistochemical analysis of tumor biopsy or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

[0334] XIII.) Combination Therapy

[0335] In one embodiment, when Her2, EGFR, Trop2, CDH3 or other TAA ADC is used to treat a tumor including a human tumor with a chemotherapeutic agent or radiation or a combination thereof, there is a synergistic effect. In other words, when combined with a chemotherapeutic agent or radiation or a combination thereof, the inhibition of tumor growth by Her2, EGFR, Trop2, CDH3 or other TAA ADC is enhanced beyond expectations. For example, the synergistic effect may be manifested as a greater inhibition of tumor growth by combined treatment than expected by Her2, EGFR, Trop2, CDH3 or other TAA ADC alone; or an additive effect is obtained by treatment with Her2, EGFR, Trop2, CDH3 or other TAA ADC and chemotherapeutic agents or radiation. Preferably, the synergistic effect is manifested as a relief of cancer in the case where relief is not expected by treatment with Her2, EGFR, Trop2, CDH3 or other TAA ADC or by treatment with a cumulative combination of Her2, EGFR, Trop2, CDH3 or other TAA ADC and chemotherapeutic agents or radiation.

[0336] Methods for inhibiting tumor cell growth using Her2, EGFR, Trop2, CDH3 or other TAA ADC and chemotherapy or radiation or a combination of the two include administering Her2, EGFR, Trop2, CDH3 or other TAA ADC before, during or after starting chemotherapy or radiotherapy, and any combination thereof (i.e. before and during the start of chemotherapy or radiotherapy, before and after the start of chemotherapy or radiotherapy, during and after the start of chemotherapy or radiotherapy, or before, during and after the start of chemotherapy or radiotherapy). For example, Her2, EGFR, Trop2, CDH3 or other TAA ADCs are typically administered 1 to 60 days, preferably 3 to 40 days, and more preferably 5 to 12 days before starting radiotherapy and / or chemotherapy. However, depending on the treatment regimen and specific patient needs, the method is performed in a manner that will provide the most effective treatment and ultimately prolong the patient's life.

[0337] Administration of chemotherapeutic agents can be accomplished in a variety of ways, including systemic administration by parenteral and enteral routes. In one embodiment, Her2, EGFR, Trop2, CDH3 or other TAA ADC and chemotherapeutic agents are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), actinomycin, chloramphenicol (nitrogen mustard), streptozotocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunomycin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxizo), docetaxel (taxotere), a interleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol acetate, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uramustine, vinorelbine, gemcitabine, chlorambucil, tacrolimus, and combinations thereof.

[0338] The radiation source used in combination with Her2, EGFR, Trop2, CDH3 or other TAA ADCs can be outside or inside the patient being treated. When the source is outside the patient, the therapy is called external beam radiation therapy (EBRT). When the radiation source is inside the patient, the therapy 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.

[0339] The above treatment regimens can be further combined with additional cancer therapeutic agents and / or regimens, such as additional chemotherapies, cancer vaccines, signal transduction inhibitors, agents useful for treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies (Pfizer) as described in WO / 2005 / 092380), or other ligands and cytokines that inhibit tumor growth by binding to IGF-1R.

[0340] When mammals receive other chemotherapy, the above-mentioned chemotherapeutic agents can be used. In addition, growth factor inhibitors, biological response modifiers, anti-hormone therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors and anti-androgens can be used. For example, anti-hormones, such as anti-estrogens, such as Nolvadex (tamoxifen) can be used; or anti-androgens, such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-'-(trifluoromethyl) propionanilide).

[0341] The above treatment methods can be combined with any of a variety of surgical, chemotherapy or radiotherapy regimens. The treatment methods of the present invention can use reduced doses of chemotherapy (or other therapies) and / or reduce the frequency of administration, which is beneficial to all patients, especially those patients who cannot tolerate the toxicity of chemotherapeutic agents well.

[0342] XIV.) Kit / Products

[0343] For the use in laboratory, prognosis, prevention, diagnosis and treatment applications described herein, the kit is within the scope of the present invention. Such a kit may include a carrier, package or container, which is separated to receive one or more containers, such as vials, tubes, etc., each container including one of the individual elements to be used in the method and a label or insert including instructions for use (e.g., uses described herein). For example, a container may include a Her2, EGFR, Trop2, CDH3 or other TAA Mab or several Her2, EGFR, Trop2, CDH3 or other TAA MAbs disclosed herein. The kit may include a container including a drug unit. The kit may contain all or part of Her2, EGFR, Trop2, CDH3 or other TAA ADCs and / or diagnostic assays for detecting cancer and / or other immune disorders.

[0344] The kits of the invention will typically include the above-mentioned container and one or more other containers associated therewith, said other containers comprising materials required from a commercial and user perspective, including buffers, diluents, filters, needles, syringes; carriers, packages, containers, vials and / or test tube labels listing the contents and / or instructions for use, and package inserts with instructions for use.

[0345] The label may be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as prognosis, prevention, diagnosis or laboratory application, and may also indicate instructions for use in vivo or in vitro, such as those described herein. Instructions for use and / or other information may also be included on an insert or label included with or on the kit. The label may be on or associated with the container. When the letters, numbers or other characters forming the label are molded or etched into the container itself, the label may be on the container; when present in a container or carrier that also holds the container, the label may be associated with the container, such as a package insert. The label may indicate that the composition is used for diagnosis, treatment, prevention or prognosis of a condition, such as cancer or other immune disorders.

[0346] The terms "kit" and "article of manufacture" may be used synonymously.

[0347] In another embodiment of the present invention, the article contains a composition, such as Her2, EGFR, Trop2, CDH3 or other TAA ADC disclosed herein. The 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 of a variety of materials, such as glass, metal or plastic. The container can hold one or more Her2, EGFR, Trop2, CDH3 or other TAA ADCs and / or one or more therapeutic doses of Her2, EGFR, Trop2, CDH3 or other TAA ADCs.

[0348] The container may alternatively contain a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be Her2, EGFR, Trop2, CDH3 or other TAA Mab or ADC of the present disclosure.

[0349] The article of manufacture may further include a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and / or dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes and / or a package insert with instructions and / or directions for use.

[0350] Exemplary Embodiments

[0351] In the examples provided:

[0352] 1) An antibody composition comprising a triple mutation, wherein the triple mutation comprises an L234A modification, an L235A modification and an L328C modification, and wherein the triple mutation modifies the Fcγ receptor binding and antibody effector function.

[0353] 2) The antibody according to technical solution 1, wherein the antibody comprises an EGFR antibody.

[0354] 3) The antibody according to technical solution 1, wherein the antibody comprises a Her2 antibody.

[0355] 4) The antibody according to technical solution 1, wherein the antibody comprises a Trop2 antibody.

[0356] 5) The antibody according to technical solution 1, wherein the antibody comprises a CDH3 antibody.

[0357] 6) The antibody according to technical solution 1, wherein the antibody comprises a GPNMB antibody.

[0358] 7) The antibody according to technical solution 1, wherein the antibody comprises a DLL3 antibody.

[0359] 8) The antibody according to technical solution 1, wherein the antibody comprises an ENPP3 antibody.

[0360] 9) The antibody according to technical solution 1, wherein the antibody comprises a SLITRK6 antibody.

[0361] 10) The antibody according to technical solution 1, wherein the antibody comprises CA9 antibody.

[0362] 11) The antibody according to technical solution 1, wherein the antibody comprises a PSMA antibody.

[0363] 12) The antibody according to technical solution 1, wherein the antibody comprises a CDH6 antibody.

[0364] 13) The antibody according to technical solution 1, wherein the antibody comprises a glypican 3 antibody.

[0365] 14) The antibody according to technical solution 1, wherein the antibody comprises an EDNRB antibody.

[0366] 15) The antibody according to technical solution 1, wherein the antibody comprises a NECTIN-4 antibody.

[0367] 16) The antibody according to technical solution 1, wherein the antibody comprises SLC34A2 antibody.

[0368] 17) The antibody according to technical solution 1, wherein the antibody comprises a Her3 antibody.

[0369] 18) The antibody according to technical solution 1, wherein the antibody comprises an NRP1 antibody.

[0370] 19) The antibody according to technical solution 1, wherein the antibody comprises a tumor-associated antigen (TAA) antibody.

[0371] 20) The Her2 antibody according to technical solution 3, further comprising the antibody heavy chain comprising SEQ ID NO:2.

[0372] 21) The Her2 antibody according to technical solution 3, further comprising the antibody heavy chain comprising SEQ ID NO: 3.

[0373] 22) The Her2 antibody according to technical solution 3, further comprising the antibody heavy chain comprising SEQ ID NO:4.

[0374] 23) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:7.

[0375] 24) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:8.

[0376] 25) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:9.

[0377] 26) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:12.

[0378] 27) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:13.

[0379] 28) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:14.

[0380] 29) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:17.

[0381] 30) The EGFR antibody according to technical solution 2 further comprises the antibody heavy chain comprising SEQ ID NO:18.

[0382] 31) The EGFR antibody according to technical solution 2, further comprising the antibody heavy chain comprising SEQ ID NO:19.

[0383] 32) The Trop2 antibody according to technical solution 4, further comprising the antibody heavy chain comprising SEQ ID NO:22.

[0384] 33) The Trop2 antibody according to technical solution 4, further comprising the antibody heavy chain comprising SEQ ID NO:23.

[0385] 34) The Trop2 antibody according to technical solution 4, further comprising the antibody heavy chain comprising SEQ ID NO:24.

[0386] 35) The CDH3 antibody according to technical solution 5, further comprising the antibody heavy chain comprising SEQ ID NO:27.

[0387] 36) The CDH3 antibody according to technical solution 5, further comprising the antibody heavy chain comprising SEQ ID NO:28.

[0388] 37) The CDH3 antibody according to technical solution 5 further comprises the antibody heavy chain comprising SEQ ID NO:29.

[0389] 38) An antibody-drug conjugate (ADC), comprising:

[0390] (i) an antibody composition comprising a triple mutation, wherein the triple mutation comprises an L234A modification, an L235A modification and an L328C modification, and wherein the triple mutation modifies the antibody effector function;

[0391] (ii) a linker; and

[0392] (iii) a Drug unit, wherein the Drug unit is specifically conjugated at the L328C site.

[0393] 39) The ADC according to technical solution 38, wherein the antibody composition includes EGFR antibody.

[0394] 40) The ADC according to technical solution 38, wherein the antibody composition includes Her2 antibody.

[0395] 41) The ADC according to technical solution 38, wherein the antibody composition includes Trop2 antibody.

[0396] 42) The antibody according to technical solution 38, wherein the antibody comprises a CDH3 antibody.

[0397] 43) The ADC according to technical solution 38, wherein the antibody composition includes a tumor-associated antigen (TAA) antibody.

[0398] 44) The ADC according to technical solution 38, wherein the TAA is listed in Table IV.

[0399] 45) According to the ADC described in technical solution 38, it further includes an extension subunit.

[0400] 46) According to the ADC described in technical solution 38, it further includes a spacer unit.

[0401] 47) According to the ADC described in technical solution 38, it further includes an amino acid unit.

[0402] 48) A product comprising the antibody according to technical solution 1.

[0403] 49) A product comprising the ADC according to technical solution 38.

[0404] 50) A pharmaceutical composition comprising a therapeutically effective amount of the ADC according to technical solution 38 and a pharmaceutically acceptable excipient.

[0405] 51) A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to Technical Solution 1 and a pharmaceutically acceptable excipient.

[0406] 52) A method of treating cancer in an individual, comprising,

[0407] (i) administering a therapeutically effective amount of the ADC according to technical solution 38 to the individual, wherein the cancer comprises cells expressing the cancer listed in Table I.

[0408] 53) A method of treating cancer in an individual, comprising,

[0409] (i) administering a therapeutically effective amount of the antibody according to technical solution 1 to the individual, wherein the cancer comprises cells expressing the cancer listed in Table I.

[0410] 54) A method of treating a disease in an individual, comprising,

[0411] (i) administering a therapeutically effective amount of the ADC according to technical solution 38 to the individual, wherein the disease comprises cells expressing the cancer listed in Table V.

[0412] 55) A method of treating a disease in an individual, comprising,

[0413] (i) administering a therapeutically effective amount of the antibody according to technical solution 1 to the individual, wherein the disease comprises cells expressing the cancer listed in Table V.

[0414] 56) An antibody-boron conjugate (ABC), comprising,

[0415] (i) an antibody composition comprising a triple mutation, wherein the triple mutation comprises an L234A modification, an L235A modification and an L328C modification, and wherein the triple mutation modifies the antibody effector function;

[0416] (ii) a linker; and

[0417] (iii) a Drug unit, wherein the Drug unit comprises a boronated composition, and wherein the Drug unit is specifically conjugated at position L328C.

[0418] 57) ABC according to technical solution 56, wherein the antibody composition includes EGFR antibody.

[0419] 58) ABC according to technical solution 56, wherein the antibody composition includes Her2 antibody.

[0420] 59) ABC according to technical solution 56, wherein the antibody composition includes Trop2 antibody.

[0421] 60) ABC according to technical solution 56, wherein the antibody composition includes CDH3 antibody.

[0422] 61) An antibody according to technical solution 56, wherein the antibody comprises a GPNMB antibody.

[0423] 62) An antibody according to technical solution 56, wherein the antibody comprises a DLL3 antibody.

[0424] 63) The antibody according to technical solution 56, wherein the antibody comprises an ENPP3 antibody.

[0425] 64) An antibody according to technical solution 56, wherein the antibody comprises a SLITRK6 antibody.

[0426] 65) The antibody according to technical solution 56, wherein the antibody includes CA9 antibody.

[0427] 66) An antibody according to technical solution 56, wherein the antibody comprises a PSMA antibody.

[0428] 67) An antibody according to technical solution 56, wherein the antibody comprises a CDH6 antibody.

[0429] 68) An antibody according to technical solution 56, wherein the antibody comprises a phosphatidylinositol protein glycan 3 antibody.

[0430] 69) An antibody according to technical solution 56, wherein the antibody comprises an EDNRB antibody.

[0431] 70) An antibody according to technical solution 56, wherein the antibody comprises a NECTIN-4 antibody.

[0432] 71) An antibody according to technical solution 56, wherein the antibody comprises SLC34A2 antibody.

[0433] 72) An antibody according to technical solution 56, wherein the antibody comprises a Her3 antibody.

[0434] 73) An antibody according to technical solution 56, wherein the antibody includes an NRP1 antibody.

[0435] 74) ABC according to technical solution 56, wherein the antibody composition includes tumor-associated antigen (TAA) antibodies.

[0436] 75) The ABC according to technical solution 56 further includes the antibody heavy chain comprising SEQ ID NO:2.

[0437] 76) The ABC according to technical solution 56 further includes the antibody heavy chain comprising SEQ ID NO:3.

[0438] 77) The ABC according to technical solution 56 further includes the antibody heavy chain comprising SEQ ID NO:4.

[0439] 78) The ABC according to technical solution 56 further includes the antibody heavy chain comprising SEQ ID NO:7.

[0440] 79) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:8.

[0441] 80) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:9.

[0442] 81) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:12.

[0443] 82) The ABC according to technical solution 56 further comprises the antibody heavy chain comprising SEQ ID NO:13.

[0444] 83) The ABC according to technical solution 56 further includes the antibody heavy chain comprising SEQ ID NO:14.

[0445] 84) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:17.

[0446] 85) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:18.

[0447] 86) The ABC according to technical solution 56 further comprises the antibody heavy chain comprising SEQ ID NO:19.

[0448] 87) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:22.

[0449] 88) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:23.

[0450] 89) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:24.

[0451] 90) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:27.

[0452] 91) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:28.

[0453] 92) According to the ABC described in technical solution 56, it further includes the antibody heavy chain comprising SEQ ID NO:29.

[0454] 93) According to ABC described in technical solution 56, it further includes an extension unit.

[0455] 94) According to ABC described in technical solution 56, it further includes a spacer unit.

[0456] 95) According to ABC described in technical solution 56, it further includes an amino acid unit.

[0457] 96) A pharmaceutical composition comprising a therapeutically effective amount of ABC according to technical solution 56 and a pharmaceutically acceptable excipient.

[0458] 97) A method of treating cancer in an individual, comprising,

[0459] (i) administering a therapeutically effective amount of ABC according to technical solution 56 to the individual, wherein the cancer comprises cells expressing the cancer listed in Table I.

[0460] Examples:

[0461] Various aspects of the present invention are further described and illustrated by the following several examples, which are not intended to limit the scope of the present invention.

[0462] Example 1: Methods of generating antibodies.

[0463] Anti-Her2, anti-EGFR, anti-Trop2 and anti-CDH3 MAbs are generated by obtaining the amino acid sequence and codon-optimizing the corresponding nucleotide sequence. (See Table VI—Antibody Sequences 1-30). Gene fragments are synthesized and cloned into the restriction enzyme sites of the double HC and LC expression vector boxes to achieve stable expression. For transient constructs, separate expression vectors for HC and LC are generated and co-transfected with a HC / LC ratio of 1 to 3. Fc variants are constructed via site-directed mutagenesis or partial gene fragment synthesis, and then subcloned using techniques known in the art. Stable or transient expression vectors are constructed and purified using an endotoxin-free DNA purification kit before transfection. Stably transfected Chinese hamster ovary (CHO) cells undergo a selection and recovery process to generate a stable pool expressing recombinant antibodies and Fc variants. For antibody generation, a fed-batch production process with a typical culture duration of 8 to 12 days is used for stable transfection pools, and for transient expression, transfected cells are cultured for 6-8 days before harvesting after transfection. The harvested cell culture fluid is then subjected to protein-A affinity purification, and the purified material is buffer exchanged into phosphate buffered saline (PBS).The quality of the recombinant antibodies can be assessed by size exclusion chromatography, SDS-PAGE, and other methods known in the art.

[0464] Example 2: Single and / or triple mutant MAbs do not affect target binding.

[0465] Target antigen binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with 0.05–0.075 ug recombinant human soluble extracellular domain (ECD) of Her2 / ErbB2 or EGFR or Trop2 (Sino Biologicals, Inc, Beijing, China) or 0.075 ug recombinant human CDH3 full length (Abnova, Taiwan, China). Subsequently, the plates were washed and blocked with ELISA blocking buffer. The plates were then incubated with test samples (i.e., wild-type or Fc variants of anti-HER2, anti-EGFR, anti-TROP2, and anti-CDH3 mAbs, with concentrations ranging from 0.0001-6.667 nM for anti-HER2 and anti-EGFR mAbs, and 0.0003–20 nM for anti-TROP2 and anti-CDH3 mAbs, serially diluted 3-fold in 1x PBS containing 1% BSA, 0.05% Tween-20) at room temperature for two (2) hours. After washing to remove unbound antibody, 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, where 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 antibody binding over the test concentration range was analyzed using single-site binding nonlinear regression analysis with GraphPad Prism.

[0466] The results showed that mAbs with single mutation L328C, double mutation L234A and L235A, and triple mutation L234A, L235A, and L328C bound to the target compared with the wild type, and the triple mutation did not affect target binding ( Figure 1 , 2 , 3, 4, 5 and 6), as in the case of single and double mutations.

[0467] Example 3: Inhibition of FcγRI binding was observed for the triple mutant MAb.

[0468] FcγRI binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well ELISA plate was coated with recombinant human soluble ECD of CD64 / FCGRIA (Sino Biological, Inc, Beijing, China) in 1x PBS (pH 7.4) coating buffer. Subsequently, the plate was washed and blocked with ELISA blocking buffer. The plate was incubated with the test sample (i.e., wild-type or Fc variant of trastuzumab, panitumumab, cetuximab, nimotuzumab, certolizumab or anti-CDH3 (5836)) (concentration range of 0.005-80ug / ml (or 0.002-30ug / ml), serially diluted 4-fold in 1x PBS containing 1% BSA, 0.05% Tween-20) at room temperature for two (2) hours. After washing to remove unbound antibody, 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, where 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 FcγRI. Detectable antibody binding over the test concentration range was analyzed using single-site binding nonlinear regression analysis with GraphPad Prism.

[0469] The results showed that significant inhibition of FcγRI binding was observed for the triple mutant compared to the wild type, the single mutant L328C, and the double mutants L234A and L235A ( Figure 7 , 8 , 9, 10, 11 and 12).

[0470] Example 4: Suppression of FcγRIIA (CD32) binding was observed for triple mutant MAbs.

[0471] The binding of antibodies to low-affinity FcγRIIA receptors was determined by a modified sandwich ELISA. In brief, microtiter plates were coated with 4ug / ml 100ul / well anti-his tag antibodies (Novus, NBP1-25939, batch number A4). The wells were then blocked with PBS containing BSA. Then, serial dilutions of different ratios of Fc variants and FcγRIIa mixtures (CD32) (SinoBiological, Inc, Beijing, China, catalog number 10374-H08H) were added, and the plates were incubated overnight at 4°C. After washing with PBS, horseradish peroxidase (HRP)-labeled goat anti-hIgG Fc (Jackson ImmunoResearch) was added at a 1:3,000 dilution in PBS-T and 1% BSA to detect FcγRIIA and Mab complexes captured by the His tag. Results were visualized by addition of tetramethylbenzidine (TMB) and optical density (OD) measured at 650 nm.Data representing antibody binding to FcγRIIA detectable and captured by anti-his tag Ab over the range of Fc variants tested concentrations were analyzed using single site binding nonlinear regression analysis using GraphPad Prism software.

[0472] The results showed that for all antibodies tested, suppression of FcγRIIA binding was observed for the triple mutant and the single mutant L328C compared to the wild type and the double mutants L234A and L235A ( Fig.13 , 14 , 15 and 16).

[0473] Example 5: Inhibition of FcγRIIIa binding was observed for the triple mutant MAb.

[0474] FcγRIIIa binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well ELISA plate was coated with a recombinant human soluble ECD of hCD16a / FCGRIIIA (Sino Biological, Inc, Beijing, China) in 1x PBS (pH 7.4) coating buffer. Subsequently, the plate was washed and blocked with ELISA blocking buffer. The plate was incubated with the test sample (i.e., wild-type or Fc variant of trastuzumab, panitumumab, cetuximab or nimotuzumab) (concentration range of 0.005-80ug / ml, serially diluted 4 times in 1x PBS containing 1% BSA, 0.05% Tween-20) at room temperature for two (2) hours. After washing to remove unbound antibodies, bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-human Fc-specific polyclonal antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells, where 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 antibody binding over the test concentration range was analyzed using GraphPad Prism's single-site binding nonlinear regression analysis.

[0475] The results showed that for all antibodies tested, significant inhibition of FcγRIIIa binding was observed for the triple mutant and the single mutant L328C compared to the wild type and the double mutants L234A and L235A ( Fig.17 , 18 , 19, 20, 21 and 22).

[0476] Example 6: Inhibition of FcRn binding is not substantially affected by Fc variants and / or triple mutant MAb effects.

[0477] 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 1x PBS (pH 6.0) coating buffer. Subsequently, the plates were washed and blocked with ELISA blocking buffer. The plates were incubated with test samples (i.e., wild-type or Fc variants of trastuzumab, panitumumab, cetuximab or nimotuzumab, certolizumab or anti-CDH3 (5836)) (concentration range of 0.04-90ug / ml (or 0.04-30ug / ml), serially diluted 3 times in 1x PBS (pH 6.0) containing 1% BSA, 0.05% Tween-20) at room temperature for two (2) hours. After washing to remove unbound antibody, 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, where 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 antibody binding over the test concentration range was analyzed using single-site binding nonlinear regression analysis with GraphPad Prism.

[0478] The results showed that the inhibition of FcRn binding was not substantially affected by Fc variants, including but not limited to triple mutant antibodies ( Fig.23 , 24 , 25, 26, 27 and 28).

[0479] Example 7: Using the Octet HTX system to characterize FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb Kinetic and affinity analysis of β-catenin and FcRn.

[0480] The binding affinity of different Fc receptors to anti-Her2 antibodies and Fc variants was measured at 25°C using the Octet HTX system (Molecular Devices). In brief, a human FcR His-tagged recombinant protein group (FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA-F, FcγRIIIA-V, FcγRIIIB and FcRn) was loaded onto the Anti-Penta His (H1S1K) biosensor. The loaded sensor was immersed in a serial dilution of the Mab test sample (300nM starting, 1:2 dilution, 7 points) in a pH 7.4 buffer consisting of PBS containing 0.1% BSA, 0.02% Tween-20 or a pH 6.0 buffer for FcRn analysis. The kinetic constants were calculated using a monovalent (1:1) binding model. Antibodies known to bind to the FcR group were used as positive controls. Defined as k dis / k on The equilibrium dissociation constant (K D ) was determined by analyzing the sensorgram curves obtained with several different concentrations.

[0481] like Fig.29 The results summarized in Figure 5 show the effect of introducing single or triple mutations in the Fc domain on binding to FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA (F158 and V158 variants) and FcγRIIIB, as well as the effect on FcRn binding. For FcγRI binding, the K observed for the triple mutant D The significant decrease in the value was mainly attributed to the antibody dissociation rate (k d ) Fig.29 The chromatogram also depicts the rapid dissociation of the triple mutant from its antigen ( Fig.30 Compared with the wild-type or single mutant antibodies, only the association rate (k on ). Since no binding of the triple mutant could be detected, the K values ​​of other Fcγ receptors could not be determined. d value( Fig.29 In contrast, for the triple mutant, the K D The values ​​were not impaired, indicating that the reduction in binding affinity of the triple mutant is specific for the Fcγ receptor isotype.

[0482] Example 8: Binding kinetics analysis of FcγRI (off-rate) using Octet.

[0483] Additional binding experiments were performed on Octet HTX at 25°C. Briefly, a human FcR His-tagged recombinant protein group (FcRI) was loaded onto the Anti-Penta His (H1S1K) biosensor. The loaded sensor was immersed in a serial dilution of the Mab test sample (300nM start, 1:2 dilution, 7 points) in a pH 7.4 buffer consisting of PBS containing 0.1% BSA, 0.02% Tween-20 or a pH 6.0 buffer for FcRn analysis. Kinetic constants were calculated using a monovalent (1:1) binding model. Antibodies known to bind to the FcR group were used as positive controls.

[0484] The results showed that the triple mutant of FcγRI had a faster dissociation rate than the wild type and single mutant ( Fig.30 ).

[0485] Example 9: ADCC analysis of Fc variants using flow cytometry.

[0486] In order to evaluate the antibody-dependent cellular cytotoxicity (ADCC) mediated by Fc variants, flow cytometry ADCC evaluation was adopted. In short, human cancer cells were used as target cells, and frozen PBMCs were used as effector cells. Human cancer cells were labeled with 8uM carboxyfluorescein succinimidyl ester (CFSE) dye (Selleckchem, catalog number S8269, batch number S826901) for 30 minutes, washed twice with cell culture medium, and then added to U-bottom 96 plates and added with Fc variants of appropriate concentrations. ADCC was initiated by adding human PBMCs as effector cells (effector cells: target (E: T) ratio of 4 to 8: 1). The plate was further incubated overnight at 37 ° C in a 5% CO2 humidified atmosphere. The cells were washed twice and stained with a 1:500 dilution of a fixable viability dye (FVD, eBioscience, California), which stained dead cells. After incubation for 30 minutes, cells were washed with PBS containing 2% FBS and then flow cytometry analysis was performed 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) to total target cells (CFSE positive). Data are shown as a bar graph of cytotoxicity% using GraphPad Prism software.

[0487] The results showed that a reduction in ADCC was observed for the Fc variants compared to wild-type, with the triple mutant showing significant reductions for each mAb ( Fig.31 , 32 , 33 and 34).

[0488] Example 10: CDC analysis of Fc variants using flow cytometry.

[0489] Complement-dependent cytotoxicity (CDC) was determined by lactate dehydrogenase (LDH) release assay using rabbit serum as complement source and human cancer cells as target cells. Briefly, target cells (20×10 e per well) were plated and plated with 5% paraformaldehyde (P < 0.05) for 24 h. 3 ) were distributed into 96-well U bottom plates and pre-incubated with Fc variants on ice for 30 minutes. Then, diluted complement was added and further incubated at 37°C (5% CO2, humidified atmosphere) for 4 hours. The assay was performed in triplicate with or without antibodies. The maximum release was prepared with target cells lysed with lysis solution. The supernatant LDH activity was measured with a non-radioactive cytotoxicity assay kit (Promega, catalog number G1781). The LDH activity of the released indicator cell death was determined by using a spectrophotometer (Cytation1 Biotek) at an optical density reading of 490nm. The cytotoxicity percentage was calculated according to the following formula: cytotoxicity (%) = 100x (experimental release-spontaneous release) / (maximum release-spontaneous release). Data are shown as a cytotoxicity % bar graph created using GraphPadPrism software.

[0490] The results showed that a reduction in CDC was observed for the Fc variants, with the triple mutant showing a significant reduction for each MAb ( Fig.35 , 36 , 37 and 38).

[0491] Example 11: CIq binding of Fc variants using ELISA.

[0492] C1q binding was evaluated using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA plates were coated with 60ul / well of 1ug / ml test sample (i.e., wild-type or Fc variant of trastuzumab, panitumumab, certolizumab, or anti-CDH3-5836) in 1x PBS (pH 7.4) coating buffer. Subsequently, the plates were washed and blocked with ELISA blocking buffer (PBS containing 1% BSA). The plates were incubated with diluted human C1q (sigma, catalog number C1740, lot number SCC6462) (concentration range 0.625-40ug / ml, serially diluted 2-fold in 1x PBS containing 1% BSA, 0.05% Tween-20) at room temperature for two (2) hours. After washing the plate, 60ul / well of rabbit anti-h C1q (Dako A0136, lot 20047640) (at a concentration of 5ug / ml in 1% BSA and 0.05% Tween in PBS (PBS-T)) was added and incubated at room temperature for 1.0 hour. After washing to remove unbound antibody, bound antibody was detected with horseradish peroxidase (HRP) conjugated goat anti-rabbit HRP specific polyclonal antibody (Jackson ImmunoResearch, catalog number 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 650nm. Data representing detectable antibody binding within the tested concentration range was analyzed using single site binding nonlinear regression analysis of GraphPad Prism.

[0493] The results showed that a reduction in C1q binding was observed for the Fc variants and the triple mutant had a significant reduction for each MAb ( Fig.39 , 40 , 41 and 42).

[0494] Example 12: Generation and Characterization of Site-Specific ADCs

[0495] Site-specific thiol generation

[0496] The hinge disulfides of the Fc variants were reduced with DTT in PBS for fifteen (15) minutes at room temperature and the antibody was purified from excess reducing agent by desalting the antibody into a pH 6.5 buffer using a PD-10 column. The expected free thiols were confirmed by the Ellman test. The hinge disulfides were then reformed using a 35-fold excess of dehydroascorbic acid (DHA) relative to the antibody concentration. The progress of reoxidation was monitored by the Ellman test. When the thiol / antibody ratio reached approximately 2.0, the maleimide payload was added.

[0497] Conjugate preparation

[0498] To generate the site-specific ADC of the present invention, an antibody conjugate using a proprietary linker called LOL1 is prepared by adding a maleimide linker to an activated site-specific antibody. The process of activation (i.e., release of free thiols) is as described above (generation of site-specific thiols). A DMSO solution of the linker is added to the thiol at 1.2 molar equivalents of the linker. The reaction is stirred at room temperature for 45-60 minutes. The resulting conjugate is purified into histidine formulation buffer (pH 6) using a PD-10 or similar desalting column and analyzed.

[0499] Conjugate evaluation

[0500] To verify the ability to generate site-specific ADCs using single (i.e., L328C) or triple mutant (i.e., L234A, L235A, L328C) antibodies, three techniques were used: (i) intact mass analysis, (ii) peptide mapping, and (iii) reversed-phase HPLC. Reverse-phase HPLC is the primary method used to dissect cysteine-based conjugates, which is used to confirm that the payload is primarily or exclusively conjugated to the heavy chain and is only conjugated to a single position within the heavy chain and that the product is therefore specific. Intact analysis by mass spectrometry is used to confirm that the conjugated heavy chain has the correct and expected mass (i.e., HC+payload), while peptide mapping confirms that the payload is tethered to a specific position within the heavy chain. Here, intact mass analysis is used to demonstrate that LOL-1 conjugated anti-HER2 mAb with triple mutations comprises a single conjugated heavy chain and an unconjugated light chain ( Fig.44 Peptide mapping analysis of single and triple mutant vcMMAE-conjugated anti-EGFR mAb No. 1 with single or triple mutations showed that the ACPAPIEK peptide within the heavy chain was both the L328C mutation site and the conjugation site ( Fig.54 , 55 ).

[0501] While mass analysis is a low throughput technique, reverse phase HPLC is a high throughput technique that shows the relative composition of the ADC. It is particularly useful for conjugates and ADCs conjugated via cysteine ​​side chains because for monoclonal antibodies, there are only eight (8) cysteines available for conjugation in wild type human IgG1.

[0502] Typically, each Cys-based ADC carries a limited number of payloads that need to be known. It is generally believed that ADCs utilizing vcMMAE as payload should carry two (2) to four (4) payloads at any position to effectively kill tumors. In addition, their composition is a distribution of payloads ranging from 0 (unconjugated) to 8 (fully conjugated). It has been shown that antibodies engineered to carry a limited number (typically, 2 per mAb) of payloads (i.e., site-specific ADCs lacking distribution and high payload / mAb ratio species) have advantages in clinical efficacy, safety, or stability.

[0503] Therefore, we used analytical methods to analyze the composition of ADCs produced by the conjugation of vcMMAE using reverse phase chromatography as the main technique. Each species (i.e., heavy chain, light chain, and conjugated species) was identified based on retention time and UV250 / 280 ratio, and DAR (drug / mAb ratio) was calculated. In addition, we showed that the payload was mainly or completely tethered to the heavy chain, and the heavy chain with a single payload was the main species, which is the main attribute of site-specific conjugation.

[0504] The materials and methods for the analysis are as follows:

[0505] Complete mass spectrometry analysis

[0506] Briefly, samples were prepared by adding 1 / 20 volume of 200 mM DTT and incubating at 37°C for 1 hour. Samples were analyzed using an Agilent 1260LC and an Agilent 6520Q-TOF mass spectrometer. The liquid chromatography column was a 2 mm ID x 10 cm long column filled with 10 μm PLRPS polystyrene reverse phase filler. The solvents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was 1% B for 1 minute, 70% B for 14 minutes, 90% B for 1 minute, 90% B for 1 minute, and 1% B for 1 minute. Data were collected using Agilent Mass Hunter data acquisition software and analyzed using Agilent Mass Hunter qualitative analysis software with Bioconfirm. Deconvolution used a maximum entropy model.

[0507] The results showed that LOL1-conjugated anti-HER2 antibody with triple mutations produced a significant retention shift, indicating that only one positional isomer was formed and its high degree was indicative of site-specific conjugation ( Fig.43 ). Further confirmation by intact mass analysis showed that the above LOL1-conjugated triple mutant anti-HER2 antibody was site-specific ( Fig.44 Only unconjugated light chains (LC) with an average mass of 23440 Daltons (Da) were detected ( Fig.44The 23462 Da peak represents the sodium adduct that is commonly formed in LC-MS systems. For the heavy chain (HC), the conjugated species found were a product with an average mass of 49487 Da, reflecting the absence of a c-terminal lysine (-K) typically observed in Chinese hamster ovary (CHO) cell culture systems for antibody production, and a product with an average mass of 49613 Da, reflecting HC with an intact, unprocessed c-terminal lysine (+K). No unconjugated HC species or HC with a second binding site ( Fig.44 ). In summary, intact mass analysis 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 conjugation could only be formed with the engineered Cys-328 and not with any hinge region cysteines.

[0508] Fig.43 and 44 The results in demonstrated that for the triple Fc mutant, the ADC conjugated with the proprietary payload LOL-1 was 100% site-specific at L328C.

[0509] Reversed phase chromatography

[0510] In addition, antibodies and / or antibody drug conjugates were analyzed by reverse phase chromatography using the following protocol. Briefly, ADC or mAb was reduced with DTT and HPLC was performed using a Waters H-class UPLC system using an Acquity C4 wide pore column (100x2.1 mm, The analysis was performed by HPLC-MS / ...

[0511] Fig.45 The results in showed that the heavy chain (H1) with a single payload was the major species, suggesting that the conjugation was site-specific and that the payload was located at Cys328 rather than within the hinge region. Fig.46 Reverse phase column chromatography profile of the L328C variant of anti-Trop2 Mab conjugated to vcMMAE is shown. It was shown that more than 80% of the site-specific conjugation occurred at Cys328 of the heavy chain.

[0512] Fig.47 Reverse phase column chromatograms of L234A, L235A, L328C triple mutant variants of anti-Trop2 Mab conjugated to vcMMAE are shown. It was shown that more than 70% of site-specific conjugation occurred at Cys328 heavy chain. Fig.48Reverse phase column chromatograms of L234A, L235A, L328C variants of anti-EGFR Mab conjugated with vcMMAE are shown. It was shown that DAR1 was the major species with more than 80%-90% of site-specific conjugation occurring on Cys328 heavy chain.

[0513] Fig.49 Shown based on Fig.49 Peak assignment and DAR calculation of RP-HPLC data provided in . Unconjugated Mab (control) was used to identify the retention time and UV250 / 280 ratio of heavy and light chains. It should be noted that these parameters are necessary for peak assignment and DAR determination. DAR calculation is as follows:

[0514]

[0515] in

[0516] LC0 and LC1 are the AUC of unconjugated and vcMMAE-conjugated LC, respectively.

[0517] HC n is the AUC of each heavy chain species, and n is a multiple of the vcMMAE payload

[0518] Fig.50 A summary of the analytical properties including the mean DAR, percentage of monomeric peak determined by SEC, and percentage of DAR1 species for vcMMAE or LOL1 conjugated mAbs and single or triple Fc variants is shown.

[0519] Peptide mapping

[0520] To confirm that the payload was tethered to a specific position within the heavy chain, peptide mapping was performed using the following protocol.

[0521] (i) Sample preparation:

[0522] Mabs or ADCs were reduced with DTT and free thiols were alkylated using iodoacetamide following published procedures known in the art. To obtain tryptic peptide maps, the above antibodies were digested as follows: Antibodies were reduced with DTT under partial denaturing conditions using 5M guanidine. Iodoacetamide was added at twice the concentration of DTT. Alkylated mAbs were purified by desalting into 100 mM phosphate buffer using Zeba-Spin (Thermo) columns. 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.

[0523] (ii) LC-MS:

[0524] After sample preparation, 5.0 μL of sample was injected into the Ultimate 3000 nano LC equipped with a 75 μm x 2 cm trapping column (Acclaim PepMap 100, Thermo Scientific) filled with C18 3 μm bulk resin and a 75 μm x 15 cm analytical column (Acclaim PepMap RSLC, Thermo Scientific) with C18 2 μm resin. The nanoLC gradient was 3-35% solvent B in 40 min (A = H2O with 0.1% formic acid; B = acetonitrile with 0.1% formic acid) and 35% to 85% solvent B in 5 min at a flow rate of 300 nL / min. The nanoLC was coupled to 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. The full spectrum (m / z 350–2000) was acquired in profile mode with a resolution of 70,000, m / z 200, and an automatic gain control (AGC) target of 3 × 10 6 The 15 most abundant ions were fragmented by higher energy collision dissociation (HCD) with a normalized collision energy of 25. MS / MS spectra were acquired in centroid mode with a resolution of 17,500 at m / z 200. The AGC target for the fragment ions was set to 2x10 4 , with a maximum injection time of 50 ms. Charge states 1, 7, 8 and unassigned were excluded from the tandem MS experiment. Dynamic exclusion was set to 45.0 s.

[0525] Fig.51 The results in show the characteristic daughter ions after fragmentation of vcMMAE peptide. In addition, Fig.52 Sequence coverage of anti-EGFR antibody No. 1 with triple mutations conjugated to vcMMAE is shown. Fig.53 Representative TIC chromatograms are shown. Differences are shown in circles. Fig.54The charge states of peptides containing the conjugation sites of anti-EGFR antibody No. 1 with triple mutations are shown. The unconjugated control mAb has an [M+1] ion with M / z of 886, which matches the ACPAPIEK peptide from the heavy chain (A). Its +2 ion is shown in the inset. Two major daughter ions with M / z of 486 and 654 are shown in (B). In the vcMMAE conjugate, this peptide is eluted from the reverse phase column at different positions due to conjugation with cysteine ​​(see previous figure). The resulting product is labeled C (+2 and +3 charge states are shown). The fragment contains the same ions as above (D, solid arrows), confirming that this is the same ACPAPIEK peptide. Other fragments with M / z of 686, 506 and 321 (D, hollow arrows) belong to VCMMAE and correspond to Fig.52 Finally, Fig.55 It is shown that the conjugation site of anti-EGFR antibody No. 1 having a single mutation (L328C) was also confirmed on the ACPAPIEK peptide of the heavy chain.

[0526] A list of antibody fragments and confidence scores of the peptide map of the single mutant of vcMMAE-conjugated anti-EGFR antibody No. 1 is shown in Table VII. A list of antibody fragments and confidence scores of the peptide map of the triple mutant of vcMMAE-conjugated anti-EGFR antibody No. 1 is shown in Table VIII.

[0527] Example 13: Cytotoxicity of Fc single mutant and / or triple mutant conjugates of vcMMAE

[0528] The cytotoxic effects of Fc single mutant or triple mutant antibodies conjugated to vcMMAE on tumor cell lines were measured using the CellTiter-GLo assay kit (Promega, catalog number G7571). The luminescent cell viability assay is a homogeneous method for determining the number of viable cells in culture based on the quantification of the presence of ATP, which indicates the presence of metabolically active cells. Briefly, human tumor cells (6000 human cancer cells / well in 100 μl of cell culture medium) in opaque-walled multiwell plates were incubated with serial dilutions of Fc single mutant or triple mutant antibody vcMMAE conjugates at 37°C (5% CO2, humidified atmosphere) for 3 to 4 days. After incubation, 100 μl of Substrate. Luminescence was recorded after 10 minutes using a Cytation1 (Biotek) plate reader. Percent survival was calculated according to the following formula: survival (%) = 100x (experimental RLU-medium RLU only) / (cell RLU-medium RLU only), and the data were analyzed by curve fitting of nonlinear regression log (inhibitor) versus reaction (three parameters) using GraphPadPrism software. The cell lines tested in the cytotoxicity assay had positive surface expression for the corresponding targets of the mAbs used in the assay. Specifically, the HCC1954 cell line was tested for anti-HER2 mAbs containing single mutants (L328C). MDA MB468 cell lines were tested for anti-EGFR mAbs and single or triple mutants. SK BR3 cell lines were evaluated for anti-TROP2 antibodies and single or triple mutants.

[0529] Fig.56 , 57 , 58, 59, 60 and 61, the results show that the in vitro cytotoxicity of antibodies and single or triple Fc mutants is enhanced by their conjugation with vcMMAE, which is an MMAE derivative with a cleavable dipeptide valine-citrulline (vc) linker, which promotes effective and selective drug cleavage by lysosomal cathepsin B in target cells after internalization. The results further show that site-specific conjugated mAbs with vcMMAE show potent and selective cytotoxic activity to antigen-positive tumor cell lines in a dose-dependent manner. In contrast, unconjugated mAbs and single or triple mutants show significantly less cytotoxic effects or do not show cytotoxic effects. In summary, these results demonstrate the in vitro efficacy of ADCs containing monoclonal antibodies covalently linked to cytotoxins in a site-specific manner, which achieves the release of cytotoxic drugs after cell binding and internalization.

[0530] Example 14: By using Her2, EGFR, TROP2, CDH3 or other TAA triple mutant MAbs and site-specific conjugation Human clinical trials of ADCs combined with ADCs for the treatment of human cancers.

[0531] According to the present invention, Her2, EGFR, Trop2, CDH3 or other TAA ADCs are synthesized, which specifically accumulate in tumor cells and are used to treat certain tumors and other immune disorders and / or other diseases (see Table I and Table V). For each of these indications, two clinical approaches have been successfully adopted.

[0532] I.) Adjunctive therapy: In adjuvant therapy, patients are treated with a combination of Her2, EGFR, Trop2, CDH3 or other TAA ADCs and chemotherapeutic agents or pharmaceutical agents or biological pharmaceutical agents or combinations thereof. By adding Her2, EGFR, Trop2, CDH3 or other TAA ADCs, the primary cancer target is treated according to standard regimens. The regimen design addresses the effectiveness evaluated by the following examples, including but not limited to the reduction of tumor volume of primary or metastatic lesions; improvement of progression-free survival, overall survival; improvement of patient health; disease stabilization and the ability to reduce conventional doses of standard chemotherapy and other biological agents. These dose reductions allow additional and / or extended therapy by reducing the dose-related toxicity of chemotherapeutic or biological agents.

[0533] II.) Monotherapy :Related to the use of Her2, EGFR, Trop2, CDH3 or other TAA ADC in monotherapy of tumors, Her2, EGFR, Trop2, CDH3 or other TAA ADC is administered to patients without chemotherapeutic agents or drug agents or biological drug agents. In one embodiment, monotherapy is performed clinically in terminal cancer patients with extensive metastatic disease. The design of the program addresses the effectiveness evaluated by the following examples, including but not limited to the reduction of tumor volume of primary or metastatic lesions; improvement of progression-free survival rate, overall survival rate; improvement of patient health; disease stabilization and the ability to reduce conventional doses of standard chemotherapy and other biological agents.

[0534] dose

[0535] The dosage regimen can be adjusted to provide the best desired response. For example, a single Her2, EGFR, Trop2, CDH3 or other TAA ADC injection can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the treatment situation. As used herein, "dosage unit form" refers to a physical discrete unit suitable as a unit dose for a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a desired drug carrier. The specifications of the dosage unit form of the present invention are determined by and directly depend on the following: (a) the unique characteristics of Her2, EGFR, Trop2, CDH3 or other TAA ADCs, the individual mechanisms of the irradiation mechanism (reactor) and the specific therapeutic or preventive effects to be achieved, and (b) the limitations inherent in the field of combining such compounds for the treatment of individual sensitivities.

[0536] Clinical Development Plan (CDP)

[0537] CDP uses the Her2, EGFR, Trop2, CDH3 or other TAA ADCs disclosed herein to track and develop treatments for cancer and / or immune disorders (see Tables I and V). The trial initially demonstrated safety, and then confirmed efficacy with repeated doses. The trial is open, comparing standard chemotherapy with standard therapy plus Her2, EGFR, Trop2, CDH3 or other TAA ADCs. As will be appreciated, a non-limiting criterion that can be used for patient registration is the concentration of Her2, EGFR, Trop2, CDH3 or other TAA ADCs in tumors as determined by standard detection methods known in the art.

[0538] The scope of the present invention is not limited by the embodiments disclosed herein, which are intended as a single illustration of various aspects of the present invention, and any content that is functionally equivalent is within the scope of the present invention. Various modifications to the models, methods, and life cycle methods of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the present invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of the present invention.

[0539] Table I. List of cancers to be treated .

[0540]

[0541]

[0542] Table II. Amino Acid Abbreviation .

[0543]

[0544]

[0545] Table III. Amino Acid Substitution Matrix .

[0546] Adapted from GCG software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely it is that the substitution will be found in the related natural protein.

[0547]

[0548] Table IV. Tumor-associated antigens (TAA) .

[0549]

[0550]

[0551] Table V List of diseases (non-cancer) to be treated .

[0552]

[0553] Table VI. Antibody sequences

[0554] Anti-HER2 antibody heavy chain wild type

[0555]

[0556] Anti-HER2 antibody heavy chain L328C

[0557]

[0558] Anti-HER2 antibody heavy chain L234A, L235A

[0559]

[0560] Anti-HER2 antibody heavy chain L234A, L235A, L328C

[0561]

[0562] Anti-HER2 antibody light chain wild type

[0563]

[0564] Anti-EGFR antibody No. 1 heavy chain wild type

[0565]

[0566] Anti-EGFR antibody No. 1 heavy chain L328C

[0567]

[0568] Anti-EGFR antibody No. 1 heavy chain L234A, L235A

[0569]

[0570] Anti-EGFR antibody No. 1 heavy chain L234A, L235A, L328C

[0571]

[0572] Anti-EGFR antibody light chain 1 wild type

[0573]

[0574] Anti-EGFR antibody heavy chain wild type 2

[0575]

[0576] Anti-EGFR antibody heavy chain 2 L328C

[0577]

[0578] Anti-EGFR antibody heavy chain 2 L234A, L235A

[0579]

[0580] Anti-EGFR antibody heavy chain 2 L234A, L235A, L328C

[0581]

[0582] Anti-EGFR antibody light chain 2 wild type

[0583]

[0584] Anti-EGFR antibody heavy chain wild type 3

[0585]

[0586] Anti-EGFR antibody No. 3 heavy chain L328C

[0587]

[0588] Anti-EGFR antibody No. 3 heavy chain L234A, L235A

[0589]

[0590] Anti-EGFR antibody heavy chain 3 L234A, L235A, L328C

[0591]

[0592] Anti-EGFR antibody light chain 3 wild type

[0593]

[0594] Anti-Trop2 antibody heavy chain wild type

[0595]

[0596] Anti-Trop2 antibody heavy chain L328C

[0597]

[0598] Anti-Trop2 antibody heavy chain L234A, L235A

[0599]

[0600] Anti-Trop2 antibody heavy chain L234A, L235A, L328C

[0601]

[0602] Anti-Trop2 antibody light chain wild type

[0603]

[0604] Anti-CDH3 antibody heavy chain wild type

[0605]

[0606] Anti-CDH3 antibody heavy chain L328C

[0607]

[0608] Anti-CDH3 antibody heavy chain L234A, L235A

[0609]

[0610] Anti-CDH3 antibody heavy chain L234A, L235A, L328C

[0611]

[0612] Anti-CDH3 antibody light chain wild type

[0613]

[0614] Table VII. Antibody fragments and confidence scores obtained from peptide mapping analysis of single mutants of anti-EGFR mAb No. 1.

[0615]

[0616]

[0617]

[0618] Table VIII. Antibody fragments and confidence scores obtained from peptide mapping analysis of triple mutants of anti-EGFR mAb No. 1. Note that "*" indicates a peptide with a conjugation site.

[0619]

[0620]

[0621]

[0622]

Claims

1. An antibody comprising a triple amino acid mutation, wherein the triple amino acid mutation comprises an L234A modification, an L235A modification and an L328C modification, wherein the numbering of the amino acid mutations is according to the EU index in Kabat, and wherein the triple amino acid mutation inhibits binding to FcγRI, FcγRIIA and FcγRIIIA and reduces antibody-dependent cell-mediated cytotoxicity ADCC, and wherein the triple amino acid mutation further reduces C1q binding and reduces complement-dependent cytotoxicity CDC.

2. The antibody of claim 1, wherein the antibody comprises an EGFR antibody.

3. The antibody of claim 1, wherein the antibody comprises a Her2 antibody.

4. The antibody of claim 1, wherein the antibody comprises a Trop2 antibody. The antibody of claim 1 , wherein the antibody comprises a CDH3 antibody.

6. The antibody of claim 1, wherein the antibody comprises a tumor associated antigen (TAA) antibody.

7. The antibody of claim 6, wherein the TAA is selected from the group consisting of GPNMB, DLL3, ENPP3, SLITRK6, CA9, PSMA, CDH6, Glypican 3, EDNRB, NECTIN-4, SLC34A2, Her3 and NRP-1.

8. An antibody-drug conjugate (ADC), comprising: (i) an antibody comprising a triple amino acid mutation, wherein the triple amino acid mutation comprises an L234A modification, an L235A modification, and an L328C modification, wherein the numbering of the amino acid mutations is according to the EU index as in Kabat, and wherein the triple amino acid mutation inhibits binding to FcγRI, FcγRIIA, and FcγRIIIA and reduces antibody-dependent cell-mediated cytotoxicity ADCC, and wherein the triple amino acid mutation further reduces C1q binding and reduces complement-dependent cytotoxicity CDC; (ii) a linker; and (iii) a Drug unit, wherein the Drug unit is specifically conjugated at the L328C site.

9. The ADC of claim 8, wherein the antibody comprises an EGFR antibody.

10. The ADC of claim 8, wherein the antibody comprises a Her2 antibody.

11. The ADC of claim 8, wherein the antibody comprises a Trop2 antibody.

12. The ADC of claim 8, wherein the antibody comprises a CDH3 antibody.

13. The ADC of claim 8, wherein the antibody comprises a tumor-associated antigen (TAA) antibody.

14. The ADC of claim 13, wherein the TAA is selected from the group consisting of GPNMB, DLL3, ENPP3, SLITRK6, CA9, PSMA, CDH6, Glypican 3, EDNRB, NECTIN-4, SLC34A2, Her3, and NRP-1. The ADC of claim 8 , further comprising an extension subunit. The ADC of claim 8 , further comprising a spacer unit.

17. The ADC of claim 8, further comprising an Amino Acid unit, wherein the Amino Acid unit connects the Spacer unit to the Stretcher unit.

18. A preparation comprising the antibody according to claim 1.

19. An article comprising the ADC according to claim 8.

20. A pharmaceutical composition comprising a therapeutically effective amount of the ADC according to claim 8 and a pharmaceutically acceptable excipient.

21. A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to claim 1 and a pharmaceutically acceptable excipient.

22. An antibody-boron conjugate (ABC) comprising, (i) an antibody comprising a triple amino acid mutation, wherein the triple amino acid mutation comprises an L234A modification, an L235A modification, and an L328C modification, wherein the numbering of the amino acid mutations is according to the EU index as in Kabat, and wherein the triple amino acid mutation inhibits binding to FcγRI, FcγRIIA, and FcγRIIIA and reduces antibody-dependent cell-mediated cytotoxicity ADCC, and wherein the triple amino acid mutation further reduces C1q binding and reduces complement-dependent cytotoxicity CDC; (ii) a linker; and (iii) a Drug unit, wherein the Drug unit comprises a boronated composition, and wherein the Drug unit is specifically conjugated at position L328C.

23. The ABC of claim 22, wherein the antibody comprises an EGFR antibody.

24. The ABC of claim 22, wherein the antibody comprises a Her2 antibody.

25. The ABC of claim 22, wherein the antibody comprises a Trop2 antibody.

26. The ABC of claim 22, wherein the antibody comprises a CDH3 antibody.

27. The ABC of claim 22, wherein the antibody comprises a tumor associated antigen (TAA) antibody.

28. The antibody of claim 27, wherein the TAA is selected from the group consisting of GPNMB, DLL3, ENPP3, SLITRK6, CA9, PSMA, CDH6, Glypican 3, EDNRB, NECTIN-4, SLC34A2, Her3, and NRP-1.

29. The ABC of claim 22, further comprising a Stretcher unit, wherein the Stretcher unit connects the Antibody unit to the Drug unit.

30. The ABC of claim 22, further comprising a Spacer unit, wherein the Spacer unit connects the Stretcher unit to the Drug unit.

31. The ABC of claim 22, further comprising an Amino Acid unit, wherein the Amino Acid unit connects the Spacer unit to the Stretcher unit.

32. A pharmaceutical composition comprising a therapeutically effective amount of ABC according to claim 22 and a pharmaceutically acceptable excipient.

33. Use of ABC according to claim 22 in the preparation of a medicament for the treatment of cancer in an individual, comprising: (i) administering to the individual a therapeutically effective amount of the ABC, wherein the cancer comprises cells expressing cancer, and the cancer is selected from the group consisting of acute myeloid leukemia, adrenocortical carcinoma, adult T-cell leukemia / lymphoma, B-cell Hodgkin lymphoma, bladder cancer, breast cancer, renal cell carcinoma, colorectal cancer, gastric cancer, head and neck cancer, multiple myeloma, non-small cell lung cancer, pancreatic cancer, genital cancer, esophageal cancer and glioma.

34. The use of claim 33, wherein the cancer comprises cells expressing a cancer selected from the group consisting of ovarian cancer, prostate cancer, and cervical cancer.

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