Antibody compositions containing Fc variants and site-specific conjugation properties

Abstract

Disclosed herein are antibodies that bind to Her2, EGFR, Trop2, CDH3, or other TAAs that contain a triple mutation at L234A, L235A, and L328C, and methods for making such triple mutant antibodies. As a result, the triple mutant antibodies contain modified effector functions due to Fc silencing and can be administered to patients to form antibody-drug conjugates (ADCs) by site-specific conjugation at L328C, providing a method for treating cancer, immunological, and neurological disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 973,475, filed October 4, 2019, the contents of which are incorporated by reference in their entirety herein.

[0002] Sequence listing submission The contents of the following paper copy submissions are fully incorporated herein by reference in their entirety: Paper Copy of Sequence Listing Recorded on October 2, 2020.

[0003] STATEMENT OF RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

[0004] FIELD OF THEINVENTION The invention described herein relates to antibodies, antigen-binding fragments thereof, antibody drug conjugates (ADCs), and antibody boron conjugates (ABCs) 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 useful in the treatment of cancer and immunological and neurological disorders. [Background technology]

[0005] 2. Background of the Invention Cancer is the second leading cause of death worldwide after coronary artery disease. Millions of people die from cancer every year, with well over half a million people dying from cancer each year 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 attributable to cancer in general are increasing. By 2040, it is estimated that there will be more than 16 million cancer deaths annually worldwide (source; International Agency for Research on Cancer, 2018), and thus surpass heart disease as the leading cause of death, unless medical developments change the current trend.

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

[0007] Although cancer treatments have improved over the past decades and survival rates have increased, cancer heterogeneity still requires new therapeutic strategies that utilize multiple treatment modalities. This is especially true for the treatment of solid tumors (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma) in anatomically important sites that are sometimes limited to standard radiation therapy and / or chemotherapy. Nevertheless, adverse effects of these therapies are chemoresistance and radioresistance that promote locoregional recurrence, distant metastases, and second primary tumors, in addition to severe side effects that reduce the patient's quality of life.

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

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

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

[0011] Furthermore, antibody-drug conjugates (ADCs) are a new class of targeted therapeutics with improved therapeutic index over traditional chemotherapy. Drugs and linkers have become the focus of ADC development in addition to (monoclonal) antibodies (mAbs) and target selection. However, recently, the importance of conjugate homogeneity has been investigated. It has been reported that the pharmacological profile of ADCs can be improved by applying site-specific conjugation techniques that utilize surface-exposed cysteine ​​residues engineered into antibodies, which are then conjugated to linker-drugs, resulting in site-specifically conjugated ADCs with defined drug-to-antibody ratios (DARs). Compared to heterogeneous mixtures made using traditional lysine and cysteine ​​conjugation methodologies, site-specifically conjugated ADCs generally showed at least comparable in vivo potency, improved PK, and expanded therapeutic window.

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

[0013] All prior art methods disclosed so far have focused on sites of conjugation of linker drugs at surface / solvent exposed positions, at positions exhibiting high thiol reactivity, and at specific constant region positions of monoclonal antibodies with the aim of improving uniformity and pharmacokinetic properties.

[0014] Although the traditional lysine and cysteine ​​conjugation methods described above have led to FDA-approved antibody-drug conjugates, which are used to construct the majority of the numerous 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.

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

[0016] In view of the 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 of treating cancer(s), immune disorders, and other diseases utilizing engineered antibodies with triple mutations that reduce antibody effector function and contain site-specific conjugation points. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Publication No. 2006 / 034488 [Patent Document 2] International Publication No. 2014 / 124316 [Patent Document 3] US Patent Application Publication No. 2017 / 0080103 [Non-patent literature]

[0018] [Non-Patent Document 1] G. KOHLER and C. MILSTEIN, Nature 256:495-497 (1975) [Non-Patent Document 2] PMALZARI et al., Annual Rev. Immunol., 6:555-580 (1988) [Non-Patent Document 3] HEZAREH et al., J.Virol.75(24):12161-8(Dec.2001) [Non-Patent Document 4] OGANESYAN et al., Acta Crystallographica.D.Biol Crystallogr,64:(Pt.6):700-4(Jun 2008) [Non-Patent Document 5] SUTHERLAND et al., Blood 122(8):1455-1463 (2013) Summary of the Invention [Means for solving the problem]

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

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

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

[0022] In another embodiment, the present disclosure teaches methods to synthesize triple mutant antibodies and conjugate drug moieties at site-specific locations thereto.

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

[0024] In another embodiment, the present disclosure teaches methods to synthesize single mutant antibodies and conjugate drug moieties at site-specific locations thereto.

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

[0026] [Figure 1] Anti-Her2 Mab and Fc variants: The Fc variants do not affect target binding.

[0027] [Diagram 2] Anti-EGFR Mab No. 1 and Fc variants: The Fc variants have no effect on target binding.

[0028] [Diagram 3] Anti-EGFR Mab No. 2 and Fc variants: The Fc variants have no effect on target binding.

[0029] [Figure 4] Anti-EGFR Mab No. 3 and Fc variants: The Fc variants have no effect on target binding.

[0030] [Diagram 5] Anti-Trop2 Mab and Fc variants: The Fc variants do not affect target binding.

[0031] [Figure 6] Anti-CDH3 Mab and Fc variants: The Fc variants do not affect target binding.

[0032] [Figure 7] Anti-Her2 Mab and Fc variant: Complete inhibition of FcγRI binding is observed for the triple mutant.

[0033] [Figure 8] Anti-EGFR Mab No. 1 and Fc variant: Complete inhibition of FcγRI binding is observed for the triple mutant.

[0034] [Figure 9] Anti-EGFR Mab No. 2 and Fc variant: Complete inhibition of FcγRI binding is observed for the triple mutant.

[0035] [Figure 10] Anti-EGFR Mab No. 3 and Fc variants: Near complete inhibition of FcγRI binding is observed for the triple mutant.

[0036] [Figure 11] Anti-Trop2 Mab and Fc variants: Complete inhibition of FcγRI binding is observed for the triple mutant.

[0037] [Figure 12] Anti-CDH3 Mab and Fc variant: Near complete inhibition of FcγRI binding is observed for the triple mutant.

[0038] [Figure 13]Anti-Her2 Mab and Fc variants: Inhibition of FcγRIIA binding is observed for the single and triple mutants.

[0039] [Figure 14] Anti-EGFR Mab No. 1 and Fc variants: Inhibition of FcγRIIA binding is observed for the single and triple mutants.

[0040] [Figure 15] Anti-Trop2 Mab and Fc variants: Inhibition of FcγRIIA binding is observed for single and triple mutants.

[0041] [Figure 16] Anti-CDH3 Mab and Fc variants: Inhibition of FcγRIIA binding is observed for the single and triple mutants.

[0042] [Figure 17] Anti-Her2 Mab and Fc variants: Inhibition of FcγRIIIa binding is observed for the single and triple mutants.

[0043] [Figure 18] Anti-EGFR Mab No. 1 and Fc variants: Inhibition of FcγRIIIa binding is observed for the single and triple mutants.

[0044] [Figure 19] Anti-EGFR Mab No. 2 and Fc variants: Inhibition of FcγRIIIa binding is observed for the single and triple mutants.

[0045] [Figure 20] Anti-EGFR Mab No. 3 and Fc variants: Inhibition of FcγRIIIa binding is observed for the single and triple mutants.

[0046] [Figure 21]Anti-Trop2 Mab and Fc variants: Inhibition of FcγRIIIa binding is observed for single and triple mutants.

[0047] [Figure 22] Anti-CDH3 Mab and Fc variants: Inhibition of FcγRIIIa binding is observed for the single and triple mutants.

[0048] [Diagram 23] Anti-Her2 Mab and Fc variants: No substantial reduction in FcRn binding is observed with the Fc variants.

[0049] [Figure 24] Anti-EGFR Mab No. 1 and Fc variants: No substantial reduction in FcRn binding is observed with the Fc variants.

[0050] [Diagram 25] Anti-EGFR Mab No. 2 and Fc variants: No substantial reduction in FcRn binding is observed with the Fc variants.

[0051] [Figure 26] Anti-EGFR Mab No. 3 and Fc variants: No substantial reduction in FcRn binding is observed with the Fc variants.

[0052] [Figure 27] Anti-Trop2 Mab and Fc variants: No substantial reduction in FcRn binding is observed with the Fc variants.

[0053] [Figure 28] Anti-CDH3 Mab and Fc variants: No substantial reduction in FcRn binding is observed with the Fc variants.

[0054] [Figure 29]Binding kinetics analysis of anti-Her2 antibodies and Fc variants for FcγRI, IIA, IIB, IIIA, IIIB and FcRn. The triple mutant shows substantial binding inhibition to all FcγR isoforms but not to FcRn.

[0055] [Diagram 30] Binding kinetics analysis of anti-Her2 antibody and Fc variants for FcγRI. The dissociation rate is faster for the triple mutant compared to the wild type and single mutations.

[0056] [Diagram 31] Anti-HER2 mAb and Fc variants: Decreased ADCC is observed with the Fc variants.

[0057] [Diagram 32] Anti-EGFR mAb No. 1 and Fc variants: Decreased ADCC is observed with the Fc variants.

[0058] [Diagram 33] Anti-Trop2 mAb and Fc variants: Decreased ADCC is observed with the Fc variants.

[0059] [Diagram 34] Anti-CDH3 mAb and Fc variants: Decreased ADCC is observed with the Fc variants.

[0060] [Diagram 35] Anti-HER2 mAb and Fc variants: Decreased CDC is observed with Fc variants.

[0061] [Diagram 36] Anti-EGFR mAb No. 1 and Fc variants: Decreased CDC is observed with the Fc variants.

[0062] [Figure 37]Anti-Trop2 mAb and Fc variants: Decreased CDC is observed with the Fc variants.

[0063] [Figure 38] Anti-CDH3 mAb and Fc variants: Decreased CDC is observed with the Fc variants.

[0064] [Figure 39] Anti-HER2 mAb and Fc variants: Decreased C1q binding is observed with the Fc variants.

[0065] [Diagram 40] Anti-EGFR mAb No. 1 and Fc variants: Decreased C1q binding is observed with the Fc variants.

[0066] [Diagram 41] Anti-Trop2 mAb and Fc variants: Decreased C1q binding is observed with the Fc variants.

[0067] [Diagram 42] Anti-CDH3 mAb and Fc variants: Decreased C1q binding is observed with the Fc variants.

[0068] [Diagram 43] Reversed-phase column chromatography profile of anti-Her2 Mab triple mutant conjugated with a unique payload (LOL1). Overlaid UV traces of unconjugated triple mutant anti-HER2 mAb (peak 1) and conjugated triple mutant anti-HER2 mAb (peak 2).

[0069] [Diagram 44] Intact mass analysis of LOL1-conjugated triple mutant anti-HER2 mAb confirmed that the conjugate was 100% site-specific. (A) Full spectrum, (B) Expanded LC region, (C) Expanded HC region.

[0070] [Diagram 45] Reverse-phase column chromatography profiles for the L328C variants of anti-HER2 mAb (A) and anti-EGFR mAb (B) conjugated with vcMMAE. The heavy chain with a single payload (H1) is the predominant species suggesting that the conjugation is site-specific and that the payload is on Cys328 and not within the hinge region.

[0071] [Figure 46] Reverse-phase column chromatography profile for the L328C variant of anti-TROP2 mAb conjugated with vcMMAE. (A) Light chain (peak 1), unconjugated heavy chain (peak 2), conjugated heavy chain with one payload (peak 3); conjugated heavy chain with two and three payloads (peaks 4-5). (B) Overlaid UV 214 nm traces of mAb and ADC. Unconjugated L328C variant of anti-TROP2 antibody (black); vcMMAE-conjugated L328C variant of anti-TROP2 antibody (red).

[0072] [Figure 47] Reverse-phase HPLC analysis of triple mutant anti-TROP2 mAb conjugated with vcMMAE. (A) Light chain (peak 1), unconjugated heavy chain (peak 2), conjugated heavy chain with one payload (peak 3); conjugated heavy chain with two and three payloads, respectively (peaks 4-5). (B) Overlaid UV 214 nm traces of mAb and ADC. Unconjugated L328C variant of anti-TROP2 antibody (black); vcMMAE-conjugated L328C variant of anti-TROP2 antibody (red)

[0073] [Figure 48]Reverse phase column chromatography profiles for L234A, L235A, L328C variants of anti-EGFR mAbs conjugated with vcMMAE. (A) Profile for unconjugated anti-EGFR mAb No. 3 with triple Fc mutations. (B) Profile for unconjugated anti-EGFR mAb No. 1 with triple Fc mutations. (C) Profile for vcMMAE-conjugated anti-EGFR mAb No. 3 with triple Fc mutations. (D) Profile for vcMMAE-conjugated anti-EGFR mAb No. 1 with triple Fc mutations.

[0074] [Figure 49] An example of peak assignment and DAR calculation based on reversed-phase column chromatography data (provided in Figure 48). Unconjugated mAb (control) was used to confirm both retention times and UV250 / 280 ratios for both heavy and light chains. These parameters were necessary for correct peak assignment and DAR determination.

[0075] [Figure 50] A summary of the analytical attributes of the resulting ADCs: average DAR, percent monomer by size exclusion chromatography (SEC), and percent DAR1 heavy chain are shown for vcMMAE- or LOL1-conjugated mAbs with single or triple Fc variants.

[0076] [Figure 51] Characteristic daughter ions after fragmentation of vcMMAE-peptides.

[0077] [Figure 52] Sequence coverage of anti-EGFR antibody No. 1 with the triple mutant conjugated with vcMMAE. (A) Heavy chain, (B) Light chain.

[0078] [Figure 53]Representative TIC chromatograms of peptides after trypsin digestion for (A) unconjugated anti-EGFR antibody No. 1 with a triple mutation, (B) vcMMAE-conjugated anti-EGFR antibody No. 1 with a triple mutation.

[0079] [Figure 54] MS / MS of the ACPAPIEK heavy chain peptide confirmed the conjugation site for anti-EGFR antibody No. 1 with the triple mutation. Full 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. Fragments representing the peptide or vcMMAE are indicated by solid or open arrows, respectively.

[0080] [Figure 55] Confirmation of the site of conjugation in anti-EGFR antibody No. 1 with a single mutant ADC by MS / MS of the ACPAPIEK heavy chain peptide. Full 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. Fragments representing the peptide or vcMMAE are indicated by solid or open arrows, respectively.

[0081] [Figure 56] Cytotoxicity mediated by the L328C variant of anti-HER2 antibody conjugated with vcMMAE. HCC1954 (Her2 / neu positive) cells were incubated for 72–96 h and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0082] [Figure 57]Cytotoxicity mediated by the L328C variant of anti-EGFR antibody No. 1 conjugated with vcMMAE. MDA-MB-468 (EGFR positive) cells were incubated for 72–96 h and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0083] [Figure 58] Cytotoxicity mediated by the L234A, L235A, and L328C variants of anti-EGFR antibody No. 1 conjugated with vcMMAE. MDA-MB-468 (EGFR positive) cells were incubated for 72–96 h and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0084] [Figure 59] Cytotoxicity mediated by the L234A, L235A, and L328C variants of anti-EGFR antibody No. 3 conjugated with vcMMAE. MDA-MB-468 (EGFR positive) cells were incubated for 72–96 h and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0085] [Figure 60] Cytotoxicity mediated by the L328C variant of anti-Trop2 antibody conjugated with vcMMAE. SK BR3 (TROP2 positive) cells were incubated for 72–96 h and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay.

[0086] [Figure 61] Cytotoxicity mediated by the L234A, L235A, and L328C variants of anti-Trop2 antibodies conjugated with vcMMAE. SK BR3 (TROP2 positive) cells were incubated for 72–96 h and the percentage of viable cells was measured using the CellTiter-Glo cell viability assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] Detailed Description of the Invention Section Overview I.) Definition II.) Antibodies III.) Fc Mutations That Alter Effector Function IV.) Antibody-Drug-Conjugates V.) Site-Specific Conjugation Formats for ADCs VI.) Linker Units VII.) Stretcher Unit VIII.) Amino Acid Units IX.) Spacer Units X.) Drug Units XI.) Methods for determining the cytotoxic effect of ADCs XII.) Treatment of cancer(s) expressing Her2, EGFR, Trop2, CDH3 and TAA XIII.) Combination Therapy XIV.) Kits / Manufactured Products

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

[0089] When trade names are used herein, reference to the trade name also refers to the product formulation, generic drug, and active pharmaceutical ingredient(s) of the trade name product, unless the context dictates otherwise.

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

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

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

[0093] As used herein, the term "antigen-binding fragment" or "antibody fragment" of an antibody (or simply "antibody portion") refers to one or more fragments of a Her2, EGFR, Trop2, CDH3, and / or TAA antibody that retain the ability to specifically bind to an antigen (e.g., Her2, EGFR, Trop2, CDH3, and / or variants). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) V L , V H , C L and C H1 (ii) a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, called F(ab') 2 Fragment; (iii) V H and C H1 (iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H(v) Fv fragment consisting of V domains; H and (vi) isolated complementarity determining regions (CDRs). In addition, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but they can be synthesized using recombinant methods to L and V H The domains can be linked by a synthetic linker that allows them to be made into a single protein chain that pairs to form a monovalent molecule known as a 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.

[0094] As used herein, the term "Fc" refers to the hinge region, CH 2 and / or C.H. 3 Refers to the region that contains the domain.

[0095] As used herein, any form of "antigen" can be used to generate antibodies specific for Her2, EGFR, Trop2, CDH3, and / or any TAA of the present invention. Thus, the eliciting antigen may be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art. The eliciting antigen may be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein). The antigen may be produced in a genetically modified cell. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" in the context of an antigen refers to the minimum number of amino acids or nucleic acids to constitute an immunogenic epitope of the antigen of interest, as appropriate. Any genetic vector suitable for transformation of the cells of interest can be used, including, but not limited to, adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids. In one embodiment, an antibody of methods and compositions herein specifically binds to at least a portion of the extracellular domain of a target of interest.

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

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

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

[0099] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies 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 a 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)).

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

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

[0102] The term "anti-neoplastic agent" refers to any agent that provides a therapeutic benefit and / or is therapeutically effective, as defined herein, in the treatment of neoplasia or cancer.

[0103] The term "chemotherapeutic agent" refers to any chemical compound that is effective in inhibiting tumor growth.Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustard, ethyleneimine compounds and alkylsulfonates; antimetabolites, such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors, such as vinca alkaloids, auristatins and podophyllotoxin derivatives, antitubulin agents; 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, biological molecules and small molecules.

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

[0105] The exact amino acid sequence boundaries of a given CDR can be determined by Kabat et al., (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("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 topography," J. Mol. Biol. 262, 732-745." (Contact numbering scheme); LEFRANC MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January;27(1):55-77 (the "IMGT" numbering scheme), and HONEGGER A. and PLICKTHUN A., "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol,2001 June.8;309(3):657-70 (the AHo numbering scheme).

[0106] The boundaries of a given CDR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat scheme and the Chothia scheme is based on the most common antibody region sequence length, with insertions provided by insertion letters, e.g. "30a", and deletions appearing 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 complex crystal structures, and is similar in many ways to the Chothia numbering scheme.

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

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

[0109] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity, function, and / or causes destruction of a cell. The term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include auristatins, auromycins, maytansinoids, ricin, ricin A chain, combrestatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogenin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents and At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 or 213 , P 32 and Lu 177 Examples of radioisotopes of Lu include, but are not limited to,

[0110] Antibodies, including those of the present invention, may also be conjugated to any of the cytotoxic agents described above, as well as to anti-cancer prodrug activating enzymes capable of converting a prodrug into its active form.

[0111] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) bound to a heavy chain variable domain (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 complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097, WO 93 / 11161; and HOLLINGER et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

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

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

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

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

[0116] In one embodiment, the antibodies provided herein are "human antibodies." As used herein, the term "human antibody" refers to an antibody in which essentially the entire 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 produced using techniques such as trioma technology, human B cell technology (see, e.g., KOZBOR et al., Immunol. Today 4:72 (1983), EBV transformation technique (see, e.g., COLE et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, 2001), and the like. 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 generating such partially to fully human antibodies are known in the art, and any such technique can be used. According to a particularly preferred embodiment, fully human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. The human antibody is produced by the transgenic mouse. An exemplary description of preparing a transgenic mouse that produces human antibodies can be found in Application No. WO 02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix) and its progeny. The B cells from the transgenic mouse that produces the desired antibody can then be fused to produce a hybridoma cell line for continuous production of the antibody. See, for example, U.S. Patent Nos. 5,569,825, 5,625,126, 5,633,425, 5,661,016, and 5,545,806, as well as JAKOBOVITS, Adv. Drug Del. Rev. 31:33-42 (1998), GREEN et al., J. Exp. Med. 188:483-95 (1998).

[0117] As used herein, the term "humanized antibody" refers to a form of an antibody that contains sequences from non-human (e.g., murine) and human antibodies. Such antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being those of a human immunoglobulin sequence. The humanized antibody also optionally contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for example, 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).

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

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

[0120] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has spread to regional lymph nodes or distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM+ under the TNM system.

[0121] The term "modified" as used herein refers to the presence of an alteration to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. Such an alteration or modification can be obtained by post-synthetic modification of a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide, or by co-translational or post-translational modification of a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide.

[0122] "Molecular recognition" refers to a chemical event by which a host molecule can 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 bonds, hydrophobic interactions, and ionic interactions.

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

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

[0125] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.

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

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

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

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

[0130] As used herein, "treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as extending survival time, reducing morbidity, and / or reducing side effects that are a by-product of alternative therapies.As is readily understood in the art, complete eradication of a disease, although preferred, is not a requirement for treatment actions.

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

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

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

[0134] Various methods for preparing antibodies, particularly monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing suitable mammalian hosts with Her2, EGFR, Trop2, CDH3, and other TAA-related proteins, peptides, or fragments, either in isolated form or in immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of Her2, EGFR, Trop2, CDH3, and fusion proteins of other TAAs, such as Her2, EGFR, Trop2, CDH3 GST fusion proteins, can also be used. In certain embodiments, GST fusion proteins containing all or most of the amino acid sequence of Her2, EGFR, Trop2, CDH3 are produced and then used as immunogens to generate suitable antibodies. In another embodiment, Her2, EGFR, Trop2, CDH3, and other TAA associated proteins are synthesized and used as immunogens.

[0135] Additionally, naked DNA immunization techniques known in the art can be used (with or without purified Her2, EGFR and other TAA associated proteins or Her2, EGFR, Trop2, CDH3 and other TAA expressing cells) to generate an immune response against the encoded immunogen (for review, see DONNELLY et al., 1997, Ann. Rev. Immunol. 15:617-648).

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

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

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

[0139] In one embodiment, the human monoclonal antibodies of the invention can be prepared using a VelocImmune mouse in which a genomic sequence having endogenous mouse variable segments at the immunoglobulin heavy chain (VH, DH and JH segments) and / or kappa light chain (VK and JK) loci has been replaced in whole or in part with a human genomic sequence having unrearranged germline variable segments at the human immunoglobulin heavy chain (VH, DH and JH) and / or kappa light chain (VK and JK) loci (Regeneron, Tarrytown, NY). See, e.g., U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

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

[0141] In another embodiment, the fully human antibodies of the invention can be produced using mice carrying human immunoglobulin sequences on a transgene and transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice", are described in TOMIZUKA et al., (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO 02 / 43478 to TOMIZUKA et al.

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

[0143] Human monoclonal antibodies of the invention can also be prepared using SCID mice into 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. Patent Nos. 5,476,996 and 5,698,767 to WILSON et al.

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

[0145] Any of the above production methods will result in antibodies with some 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, 9, 98 or 99% sequence identity to Her2, EGFR, Trop2, CDH3 and other TAAs.

[0146] The binding affinity (K ) of antibodies, their binding fragments, and antibody-drug conjugates containing them against 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 can be between 5 and 10 nM, or between 1 and 2 nM. D can be from 1 micromolar to 500 micromolar or from 500 micromolar to 1 nM.

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

[0148] The engineered antibodies of the invention include H and / or V LThese include those in which modifications have been made to framework residues within the framework region (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutations may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis (e.g., "backmutation" of leucine to methionine). Such "backmutated" antibodies are also intended to be encompassed by the present invention.

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

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

[0151] In another embodiment, the Fc hinge region of the antibody is mutated to reduce 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 has impaired Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in more detail in US Patent No. 6,165,745 by WARD et al.

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

[0153] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of Her2, EGFR, Trop2, CDH3 and other TAA MAbs. For example, one or more amino acids selected from amino acid specific residues can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260, both by Winter et al.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0173] III.) Fc Mutations That Alter Effector Function The Fc region of an antibody (i.e., the end of the antibody's heavy chain spanning domains CH2, CH3 and part of the hinge region) has limited variability and is involved in the physiological role played by antibodies. The effector functions attributed to the Fc region of an antibody vary depending on the class and subclass of the antibody and include binding of the antibody via the Fc region to specific Fc receptors ("FcR") on cells, which leads to various biological responses.

[0174] These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that may mediate some signaling events within the cell. These receptors are expressed on 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. Formation of the Fc / FcγR complex recruits these effector cells to the site of the bound antigen, typically resulting in intracellular signaling events and important subsequent immune responses such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy target cells. The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR 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 FcγR recognize bound antibodies on target cells and subsequently cause phagocytosis 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 cell-independent cytotoxicity function mediated by complement, also known as complement-dependent cytotoxicity (CDC).

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

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

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

[0178] Although there are certain subclasses of human immunoglobulins that lack specific effector functions, there are no known naturally occurring immunoglobulins that lack all effector functions. Another approach would be to engineer or mutate key residues in the Fc region that are responsible for effector functions. See SCHLOTHAUER et al., 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). Indeed, several research groups have attempted such an endeavor. See, e.g., PCT Publication No. WO 2009 / 100309 (Medimmune), WO 2006 / 076594 (Xencor), WO 1999 / 58572 (Univ. Cambridge), U.S. Patent Application Publication No. 2006 / 0134709 (Macrogenics), WO 2006 / 047350 (Xencor), WO 2006 / 053301 (Xencor), U.S. Patent No. 6,737,056 (Genentech), U.S. Patent Application Publication No. 5,624,821 (Scotgen Pharmaceuticals), U.S. Patent Application Publication No. 2010 / 0166740 (Roche), and U.S. Patent No. 8,969,526 (Roche Glycart AG).

[0179] 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 important 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 significantly reduced ADCC and CDC (see ARMOUR et al., Eur. J. Immunol. 29(8)(1999)2613-2624; SHIELDS et al., J. Biol. Chem. 276(9)(2001)6591-6604).

[0180] Furthermore, IDUSOGIE et al., J.Immunol.166(2000)2571-2575, mapped the C1q binding site of RITUXAN and showed that Pro329Ala reduces the ability of rituximab to bind C1q and activate complement. Furthermore, it has been reported that the substitution of Pro329 with Ala leads to 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 to show wild-type-like binding to FcγRI and FcγRII and only slightly reduced binding to FcγRIIIA receptors (European Patent No. 1,068,241, Genentech).

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

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

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

[0184] Thus, in certain embodiments, 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 at least one amino acid residue addition, substitution or deletion to the Fc region (also referred to herein as a "wildtype polypeptide"). In certain embodiments, polypeptides comprising an Fc variant of the invention comprise 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 an Fc receptor, 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 the complement protein C1q.

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

[0186] 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, and the numbering system of the constant region is the EU index numbering system as set forth in KABAT et al., NIH Publication 91 (1991) 3242, National Technical Information Service, Springfield, Va.

[0187] 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, and the numbering system of the constant region is the EU index numbering system as set forth in KABAT et al., NIH Publication 91 (1991) 3242, National Technical Information Service, Springfield, Va.

[0188] 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, and the numbering system of the constant region is the EU index numbering system as set forth in KABAT et al., NIH Publication 91 (1991) 3242, National Technical Information Service, Springfield, Va.

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

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

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

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

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

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

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

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

[0197] In one embodiment, a polypeptide comprising an Fc variant has no effect on target binding compared to an unmodified antibody.

[0198] In one embodiment, polypeptides comprising an Fc variant of the invention exhibit inhibited FcγRI binding compared to an unmodified antibody.

[0199] In one embodiment, polypeptides comprising an Fc variant of the invention exhibit inhibited FcγRII binding compared to an unmodified antibody.

[0200] In one embodiment, polypeptides comprising an Fc variant of the invention exhibit inhibited FcγRIIIa binding compared to an unmodified antibody.

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

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

[0203] As observed, the triple mutation (L328C combined with L234A / L235A) did not affect target antigen binding, as comparable binding activity was observed when compared to the wild-type counterpart, despite the complete abrogation of FcγR binding. Antibody stability and expression also remained comparable to the wild-type antibody. Thus, the triple mutation allows for complete inhibition of Fc effector functions without compromising target specificity, antibody quality and yield.

[0204] Moreover, as described below, specific triple mutations can facilitate efficient site-specific conjugation.

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

[0206] In further embodiments, the ADCs include Her2, EGFR, Trop2, CDH3, and other TAA Mabs that further comprise triple substitutions at the following positions: L234A, L235A, L328C.

[0207] In preferred embodiments, the ADCs include Her2, EGFR, Trop2, CDH3, and other TAA Mabs that further comprise triple substitutions at the following positions: L234A, L235A, L328C, and further comprise site-specific conjugation at L328C.

[0208] By way of background, the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells, in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; NICULESCU-DUVAZ and SPRINGER (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows for targeted delivery of the drug moiety to the tumor and its intracellular accumulation, whereas systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity not only to the tumor cells to be eliminated but also to normal cells (BALDWIN et al. (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985)''Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,''in Monoclonal Antibodies'84: Biological and Clinical Applications, A. PINCHERA et al. (ed.), pp. 475-506), thereby seeking maximum efficacy with minimum toxicity. 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 for use in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (MANDLER et al., (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; MANDLER et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; MANDLER et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (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 exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

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

[0210] Additionally described herein are therapeutic agents, including but not limited to chemotherapeutic agents, useful for the generation of ADCs. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, for example, International Publication No. WO 93 / 21232, published Oct. 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. For example, 212 Bi, 131 I, 131 In, 90 Y and 186Re. Antibody and cytotoxic agent conjugates are prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins 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 chelating agent for conjugating radionucleotides to antibodies (WO 94 / 11026). Other antitumor agents that can be conjugated to the antibodies of the present invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents collectively known as LL-E33288 complexes described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Pat. No. 5,877,296).

[0211] Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, e.g., WO 93 / 21232 (published October 28, 1993).

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

[0213] For selective destruction of tumors, the antibody may contain a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 88 , Sm 53 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When the conjugates are used for detection, the conjugates may be used with radioactive atoms for scintigraphy studies, e.g., 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.

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

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

[0216] The antibody-drug conjugate compound comprises an antibody unit covalently linked to at least one drug unit. The drug unit can be covalently linked to the antibody unit directly or via a linker unit (-LU-). In addition, the drug unit is conjugated at the site-specific position of L328C.

[0217] In some embodiments, the antibody drug conjugate compound has the formula: L-(LU-D) p (I) or a pharma- ceutically acceptable salt or solvate thereof; L is an antibody unit, for example a Her2, EGFR, Trop2, CDH3, or another TAA MAb of the invention, which MAb comprises a triple mutation at the following positions: L234A, L235A and L328C, (LU-D) is the Linker unit-Drug unit moiety, wherein LU- is a linker unit, -D is a drug entity having cytostatic or cytotoxic activity against a target cell, p ranges from 1 to 20 or alternatively from 1 to 50. Additionally, the drug unit moiety is conjugated at a site-specific position on the MAb at L328C.

[0218] In some embodiments, the antibody drug conjugate compound has the formula: L-(A a -W w -Y y -D) p (II) or a pharma- ceutically acceptable salt or solvate thereof; L is an antibody unit, for example a Her2, EGFR, Trop2, CDH3 or another TAA MAb, the MAb comprising a triple mutation at the following positions: L234A, L235A and L328C, -A a -W w -Y y - is a linker unit (LU), wherein -A- is a stretcher unit, a is 0 or 1 or 2 or 3, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12, -Y- is a self-immolative spacer unit; y is 0, 1 or 2, -D is a drug entity having cytostatic or cytotoxic activity against a target cell, p is an integer from 1 to 20, or alternatively an integer from 1 to 50. Additionally, the drug unit moiety is conjugated at a site-specific position on the MAb at L328C.

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

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

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

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

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

[0224] Generally speaking, bioconjugation strategies involve covalently linking a protein or peptide (biologic) to a small molecule, carbohydrate, oligonucleotide, synthetic polymer, or another protein / peptide. This approach can be crucial to create differentiation and drive product development in the highly competitive biologics market. These strategies were the basis for the development of highly successful conjugate vaccines such as Prevnar 13, Menactra, Menomune, and HibTITER. Four (4) of them were created by conjugating bacterial polysaccharides to immunogenic carrier proteins. Similarly, bioconjugation to half-life extending polymeric carriers (such as polyethylene glycol (PEG)) has been applied to create currently marketed drugs (e.g., certodilumab (Cimzia), pegfilgrastim (Neulasta), and pegvisomant (Somavert)) with longer duration of action than their non-conjugated counterparts and dosing regimens that promote patient compliance.

[0225] As known in the art, the choice of linker and conjugation chemistry for making first generation ADCs was dictated by the limitations of working with proteins. Thus, linkers were 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).

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

[0227] It is important to note that beyond the number of drugs per antibody molecule, another level of heterogeneity occurs in the conjugation sites. Thus, ADCs with specific drug-to-antibody ratios (DARs) generated by cysteine ​​conjugation are still heterogeneous mixtures of conjugates with different sites of conjugation. However, since there are only eight sites available for cysteine ​​conjugation (compared to the maximum of 80 available for lysine-directed chemistry), it is fair to say that the cysteine ​​conjugation approach offers greater control over the conjugation sites, facilitating better characterization. A controlled reduction-alkylation strategy has been successfully used to produce the approved ADCETRIS (Seattle Genetics, Bothell, WA) product, along with several other ADCs currently undergoing clinical trials.

[0228] The researchers studied the in vivo efficacy of ADCs targeting CD30+ tumor cells with two, four, and eight monomethylauristatin E (MMAE) toxins per antibody molecule and demonstrated that the stoichiometry of drug loading significantly affected the drug's pharmacokinetics (PK), efficacy, and toxicity. Hamblett et al. found that in their system, ADCs with four drugs per antibody were more potent than ADCs with two drugs, but had comparable efficacy and better tolerability than ADCs with eight drugs / antibody. The results showed that, in general, ADCs with higher drug loading had greater clearance, higher efficacy, and increased toxicity. This implied that each ADC had an optimal drug loading with the right balance of efficacy and toxicity.

[0229] His seminal work established the concept that DAR is a key design parameter for ADCs. It became clear that chemical conjugation to natural cysteine ​​or lysine residues is not optimal because they produce heterogeneous ADC mixtures. The heterogeneity comes from differences in DAR and conjugation site, resulting in ADC subpopulations that may have lower potency, higher toxicity, and different configuration and PK properties. Furthermore, for such non-selective conjugation methods, analytical characterization and control of batch-to-batch variation during manufacturing remains a major challenge. To overcome these limitations, the concept of site-specific conjugation has evolved, which aims to first produce homogeneous ADCs and control the DAR and conjugation site.

[0230] Several site-specific conjugation techniques have been demonstrated that optimize payload placement using a variety of techniques (see, e.g., ThioMab (Genentech), Seattle Genetics, and Ambrx, Inc.).

[0231] The reported ThioMab study revealed another fundamental concept. Not only is ADC uniformity key to improved biophysical and therapeutic properties, but the actual site of conjugation on the antibody backbone also has a significant impact on the in vivo behavior of the ADC molecule. SHEN et al. generated multiple uniform TDC conjugates with a HER2-targeting antibody using an MMAE payload, where engineered cysteines for conjugation were located in either the light chain (LC), heavy chain (HC) or Fc region of the antibody. Although all conjugates showed comparable in vitro potency, the authors report significant differences in their in vivo efficacy and PK properties.

[0232] The LC conjugates showed the greatest efficacy when studied in mouse xenograft models, where the HC conjugates were intermediate and the Fc conjugates had little or no activity. Mouse PK studies showed a similar trend, with the LC conjugates showing the greatest stability and lowest clearance, followed by the HC conjugates, with the Fc conjugate ADC being cleared the fastest and providing the lowest ADC exposure. These results were attributed to differences in the local microenvironment and solvent accessibility contributing to the differential stability of the linker systems at different sites.

[0233] Although several additional site-specific bioconjugation methods aimed at delivering uniform ADCs compared to first-generation lysine and cysteine ​​conjugation have been reported, only a subset of these techniques offer greater versatility in finding the optimal conjugation site for a given antibody-payload combination. These include conjugation with unnatural amino acids (nnAAs) introduced by genetic code modification (Ambrx, LaJolla, CA; Sutro Biopharma, South San Francisco, CA; Allozyne, acquired by Mediimmune, Seattle, WA), transglutaminase (TG)-mediated conjugation to engineered glutamine tags (Pfizer, New York, NY), and conjugation with aldehyde-tagged antibodies generated by co-expressed formylglycine generating enzyme (FGE) (Redwood Bioscience, acquired by Catalent Pharma Solutions, Emeryville, CA).

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

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

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

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

[0238] In a further embodiment, a site-specific conjugation approach by conversion of Leu-328 to Cys in the Fc domain of the Trop2 antibody allows for controlled conjugation without affecting target binding. Moreover, it can be shown that neither the expression level nor the stability of the antibody is compromised by the introduction of L328C.

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

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

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

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

[0243] In addition, L328C also shows the added advantage of reducing Fc effector function. Safety liability associated with infusion reactions caused by therapeutic monoclonal antibodies and their interaction with FcγR has been reported. The ability to partially silence Fc effector function without compromising antibody stability is an attractive feature of L328C. Potential mitigation strategies for improved clinical safety profile can be further developed in possible combination with other variances previously shown to reduce Fc effector function, either for L328C-mediated conjugation or as naked antibodies.

[0244] VI.) Linker Units Typically, an antibody-drug conjugate compound comprises 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 yet other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

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

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

[0247] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid labile linkers (e.g., oximes, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, for example, 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.)

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

[0249] In yet other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation. (See PCT Publication WO 2012 / 166560 (Ambrx, Inc.), which is incorporated by reference in its entirety.)

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

[0251] Various exemplary linkers that can be used with the compositions and methods of the present invention are described in International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649 and U.S. Patent Application Publication No. 2006 / 0024317 (each of which is incorporated by reference in its entirety for all purposes).

[0252] VII.) Stretcher Unit The Stretcher unit (A) can link 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 can be present on Her2, EGFR, Trop2, CDH3, or other TAA MAbs naturally or through chemical engineering include, but are not limited to, keto, aldehyde, sulfhydryl, amino, hydroxyl, anomeric hydroxyl groups of carbohydrates, and carboxyl. Suitable functional groups are keto, aldehyde, sulfhydryl, and amino. In one example, a keto group is present on a non-natural amino acid (nnAA) that is incorporated into the Mab of the invention. In a further example, an aldehyde group is present on an nnAA that is incorporated into the Mab of the invention. In another example, a sulfhydryl group can be generated by reduction of an intramolecular disulfide bond of Her2, EGFR, Trop2, CDH3, or other TAA MAb. In another embodiment, sulfhydryl groups can be generated by reaction of amino groups on lysine moieties of Her2, EGFR, Trop2, CDH3, or other TAA MAbs with 2-iminothiolane (Traut's reagent) or other sulfhydryl generating reagents. In certain embodiments, the Her2, EGFR, Trop2, CDH3, or other TAA MAbs are recombinant antibodies and are engineered to carry one or more lysines. In certain other embodiments, the recombinant Her2, EGFR, Trop2, CDH3, or other TAA MAbs are engineered to have additional sulfhydryl groups, e.g., additional cysteines.

[0253] In a preferred embodiment, the Her2, EGFR, Trop2, CDH3 or other TAA MAb comprises Fc modifications at L234A, L235A, and further comprises another modification at L328C, and further comprises site-specific conjugation at L328C.

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

[0255] VIII.) Amino Acid Units The Amino Acid unit (-W-), when present, 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 absent, and links the antibody unit to the Drug unit if the Stretcher unit and Spacer unit are absent.

[0256] In certain embodiments, the amino acid units can comprise natural amino acids, hi other embodiments, the amino acid units can comprise unnatural amino acids.

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

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

[0259] IX.) Spacer Units The spacer unit (-Y-), when present, links the amino acid unit to the drug unit if the amino acid unit is present. Alternatively, the spacer unit links the stretcher unit to the drug unit if the amino acid unit is absent. The spacer unit also links the drug unit to the antibody unit if both the amino acid unit and the stretcher unit are absent. Spacer units are of two general types: non-self-immolative or self-immolative. Examples of possible spacers of the present invention are known in the art. See TOKI et al., 2002, J. Org. Chem. 67:1866-1872 and Nature Biotechnology 21(7):778-784).

[0260] Other examples of self-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 ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon amide bond hydrolysis can 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 amides (AMSBERRY et al., 1990, J. Org. Chem. 55:5867). The exclusion 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.

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

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

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

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

[0265] 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 endonucleases. Determination of any of these effects on cancer cells indicates that the ADC is useful for treating cancer.

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

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

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

[0269] Commercially available photometric methods for quantitative in vitro determination of DNA fragmentation are available. 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).

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

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

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

[0273] In vivo assays that evaluate the promotion of apoptosis are useful for evaluating therapeutic compositions. In one embodiment, xenografts from mice bearing tumors treated with a therapeutic composition can be examined for the presence of apoptotic foci and compared to mice bearing untreated control xenografts. The extent to which apoptotic foci are found in the tumors of treated mice provides an indication of the therapeutic effectiveness of the composition.

[0274] The therapeutic composition used to carry out the above-mentioned method can be formulated into a pharmaceutical composition that includes 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 is generally non-reactive with the patient's immune system. Examples include any of a number of standard pharmaceutical carriers, such as, but not limited to, sterile phosphate buffered saline solution, bacteriostatic water, and the like (see generally Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

[0275] The therapeutic formulation can be solubilized and administered via any route that can deliver 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, orthotopic, etc. A preferred formulation for intravenous injection contains the therapeutic composition in a solution of preserved bacteriostatic water, sterile non-preserved water, and / or diluted in a polyvinyl chloride or polyethylene bag containing 0.9% sterile sodium chloride for injection, USP. The therapeutic protein preparation can be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted with bacteriostatic water (e.g., containing benzyl alcohol preservative) or sterile water before injection.

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

[0277] In one embodiment, a pharmaceutical composition of the invention may comprise two or more ADCs of the invention with modification of Her2, EGFR, Trop2, CDH3, or other TAA MAb. For example, the invention includes pharmaceutical compositions comprising the ADCs of the invention, where the Her2, EGFR, Trop2, CDH3, or other TAA MAb is an antibody with a partially or completely deleted C-terminal lysine, an antibody with an N-terminal post-translational modification, an antibody lacking a heavy chain C-terminal lysine and with an N-terminal post-translational modification, and / or an antibody with a heavy chain C-terminal lysine and no N-terminal post-translational modification.

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

[0279] XII.) Treatment of cancer(s) expressing Her2, EGFR, Trop2, CDH3 and TAA The identification of Her2, EGFR, Trop2, CDH3, or other TAAs as a protein(s) that is normally expressed in a limited set of tissues or cells, but is also expressed in cancers, such as those listed in Table I, opens up a number of therapeutic approaches for the treatment of such cancers.

[0280] Of note, targeted antitumor therapy has been useful even when targeted proteins are expressed in normal tissues or cells, even in vital normal organ tissues.Vital organs are organs that are necessary to sustain 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 prostate.

[0281] Expression of target protein in normal tissues (even vital normal tissues) does not eliminate the usefulness of the targeting agent for the protein as a therapeutic agent for certain tumors in which the protein is also overexpressed. For example, expression in vital organs is not harmful in itself. Furthermore, organs that are considered non-essential, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some vital organs are immune privileged and are not affected by normal organ expression. Immune privileged organs are organs that are protected from blood by blood-organ barriers and therefore cannot be accessed by immunotherapy. Examples of immune privileged organs are the brain and testes.

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

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

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

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

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

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

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

[0289] When mammal is subjected to further chemotherapy, the above-mentioned chemotherapy agent can be used.In addition, growth factor inhibitor, biological response modifier, antihormonal treatment, selective estrogen receptor modulator (SERM), angiogenesis inhibitor and antiandrogen agent can be used.For example, antihormonal agent, such as antiestrogenic agent, such as Nolvadex (tamoxifen) or antiandrogen agent, such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-'-(trifluoromethyl)propionanilide) can be used.

[0290] The above-mentioned therapeutic approach can be combined with any one of a wide variety of surgical, chemotherapy or radiotherapy regimens.The therapeutic approach of the present invention can allow the use of low doses of chemotherapy (or other therapies) and / or less frequent administration, which is advantageous for all patients, especially those who do not tolerate the toxicity of chemotherapy agents well.

[0291] XIV.) Kits / Manufactured Products For use in the laboratory, prognostic, preventive, diagnostic and therapeutic applications described herein, kits are within the scope of the present invention. Such kits can include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, each of which contains one of the separate elements used in the method, along with a label or insert that includes instructions for use, such as those described herein. For example, the container(s) can include a Her2, EGFR, Trop2, CDH3 or other TAA MAb of the present disclosure or several Her2, EGFR, Trop2, CDH3 or other TAA MAbs. The kit can include a container that includes a drug unit. The kit can include all or a portion of a Her2, EGFR, Trop2, CDH3, or other TAA ADC, and / or a diagnostic assay for detecting cancer and / or other immunological disorders.

[0292] Kits of the invention typically include the containers described above, as well as buffers, diluents, filters, needles, syringes; carrier, packaging, container, vial and / or tube labels listing the contents and / or instructions for use, and package inserts containing instructions for use, and will include one or more other containers associated therewith that contain materials desirable from a commercial and user standpoint.

[0293] A label can be present on or with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic use, such as prognostic, preventive, diagnostic or laboratory use, and can also indicate instructions for either in vivo or in vitro use, such as those described herein. The instructions and / or other information can also be included in an insert(s) or label(s) included on or with the kit. The label can be on or associated with the container. The label can be on the container if letters, numbers or other characters forming the label are molded or etched into the container itself, or the label can be associated with the container if present in a receptacle or carrier that holds the container, for example, as a package insert. The label can indicate that the composition is used to diagnose, treat, prevent or prognose a condition, such as cancer or other immunological disorder.

[0294] The terms "kit" and "article of manufacture" may be used synonymously.

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

[0296] The container may alternatively hold a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be a Her2, EGFR, Trop2, CDH3, or other TAA Mab or ADC of the disclosure.

[0297] The article of manufacture may further include a second container containing a pharma- ceutically 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 standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and package inserts with indications and / or instructions for use.

[0298] Exemplary embodiments Among the embodiments provided are the following: 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 Fcγ receptor binding and antibody effector function.

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

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

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

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

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

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

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

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

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

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

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

[0310] 13) The antibody of claim 1, wherein the antibody comprises an anti-glypican 3 antibody.

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

[0312] 15) The antibody described in claim 1, wherein the antibody comprises a Nectin-4 antibody.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0341] 44) The ADC of claim 38, wherein the TAA antibody is as set forth in Table IV.

[0342] 45) The ADC of claim 38, further comprising a Stretcher unit.

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

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

[0345] 48) An article of manufacture comprising the antibody of claim 1.

[0346] 49) An article of manufacture comprising the ADC of claim 38.

[0347] 50) A pharmaceutical composition comprising a therapeutically effective amount of the ADC of claim 38 and a pharma- ceutical acceptable excipient.

[0348] 51) A pharmaceutical composition comprising a therapeutically effective amount of the antibody of claim 1 and a pharma- ceutically acceptable excipient.

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

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

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

[0352] 55) A method for treating a disease in an individual, comprising: (i) a therapeutically effective amount of the antibody of claim 1 is administered to said individual, wherein the disease comprises cells expressing a cancer described in Table V.

[0353] 56) Antibody-boron-conjugate (ABC), (i) an antibody composition comprising a triple mutation, the triple mutation comprising a L234A modification, a L235A modification and a L328C modification, the triple mutation modifying an antibody effector function; and (ii) a linker; and (iii) an antibody-boron-conjugate (ABC) comprising a Drug unit, said Drug unit comprising a borylation composition, and said Drug unit is specifically conjugated at site L328C.

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

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

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

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

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

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

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

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

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

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

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

[0365] 68) The antibody of claim 56, wherein the antibody comprises an anti-glypican 3 antibody.

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

[0367] 70) The antibody described in claim 56, wherein the antibody comprises a Nectin-4 antibody.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0396] Example 1: Methods for generating antibodies. Anti-Her2, anti-EGFR, anti-Trop2 and anti-CDH3 MAbs were generated by obtaining amino acid sequences and performing codon optimization on the corresponding nucleotide sequences. (See Table VI-Antibody Sequences 1-30). Gene fragments were synthesized and cloned into the restriction enzyme sites of dual HC and LC expression vector cassettes for stable expression. For transient constructs, separate expression vectors for HC and LC were generated and co-transfected at a HC to LC ratio of 1 to 3. Fc variants were constructed by either site-directed mutagenesis or partial gene fragment synthesis followed by subcloning using techniques known in the art. Stable or transient expression vectors were constructed and purified with an endotoxin-free DNA purification kit prior to transfection. Stably transfected Chinese Hamster Ovary (CHO) cells underwent a selection and recovery process for the generation of stable pools expressing recombinant antibodies and Fc variants. For antibody generation, a fed-batch production process with a typical culture period of 8-12 days was used for stably transfected pools, and for transient expression, transfected cells were cultured for 6-8 days post-transfection before being harvested. Protein A affinity purification was subsequently performed on the harvested cell culture fluid, and the purified material was 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.

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

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

[0399] Example 3: Inhibition of FcγRI binding is observed for triple mutant MAbs. FcγRI binding was assessed using enzyme-linked immunoassay (ELISA). Briefly, 96-well ELISA plates were coated with recombinant human soluble ECD of CD64 / FCGRIA (Sino Biological, Inc. Beijing, China) in 1×PBS (pH 7.4) coating buffer. Plates were then washed and blocked with ELISA blocking buffer. Plates were incubated at room temperature for two (2) hours with test samples (i.e., trastuzumab, panitumumab, cetuximab, nimotuzumab, sacituzumab, or anti-CDH3 (5836) wild type or Fc variants) at concentrations ranging from 0.005 to 80 ug / ml (or 0.002 to 30 ug / ml) serially diluted 4-fold in 1×PBS containing 1% BSA, 0.05% Tween®-20. After washing to remove unbound antibody, bound antibody was detected with a horseradish peroxidase (HRP)-conjugated goat anti-human Fc-specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells. Color developed in the wells 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 binding of the antibody over the range of test concentrations was analyzed using one-site binding nonlinear regression analysis using GraphPad Prism.

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

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

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

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

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

[0405] Example 6: Inhibition of FcRn binding is substantially unaffected by Fc variant and / or triple mutant MAbs. FcRn binding was assessed using enzyme-linked immunoassay (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. The plates were then washed and blocked with ELISA blocking buffer. The plates were incubated for 2 hours at room temperature with test samples (i.e., trastuzumab, panitumumab, cetuximab or nimotuzumab, sacituzumab, or wild-type or Fc variants of anti-CDH3 (5836)) at concentrations ranging from 0.04 to 90ug / ml (or 0.04 to 30ug / ml) serially diluted 3-fold in 1x PBS (pH 6.0) containing 1% BSA, 0.05% Tween®-20. After washing to remove unbound antibody, bound antibody was detected with a horseradish peroxidase (HRP)-conjugated goat anti-human Fc-specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). After washing to remove unbound detection antibody, a slow kinetic substrate solution of tetramethylbenzidine (TMB) was added to the wells. Color developed in the wells was proportional to the amount of test sample. The optical density (OD) of the color was measured at 650 nm and used to determine the amount of test sample bound to FcRn. Detectable binding of the antibody over the range of test concentrations was analyzed using one-site binding nonlinear regression analysis using GraphPad Prism.

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

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

[0408] The results, summarized in Figure 29, show the effect of introducing single or triple mutations into the Fc domain on binding to FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA (F158 and V158 variants) and FcyRIIIB, as well as the effect on FcRn binding. For FcγRI binding, the K observed for the triple mutants was 0.01, 0.02, and 0.03. D The substantial decrease in the value is mainly due to the antibody dissociation rate (k d ) (Figure 29). The rapid dissociation of the triple mutant from its antigen is also shown in the chromatogram (Figure 30). The association rate (k on ) was observed. Due to the undetectable binding of the triple mutant, no significant reduction in K was observed for other Fcγ receptors. d It was not possible to determine the K value for FcRn (Figure 29). In contrast, the K value observed for FcRn was DValues ​​were not impaired for the triple mutant, implying that the reduced binding affinity for the triple mutant is Fc gamma receptor isoform specific.

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

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

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

[0412] The results show that reduced ADCC was observed with the triple mutant and significantly reduced Fc variants for each mAb compared to wild type (Figure 31, Figure 32, Figure 33, and Figure 34).

[0413] Example 10: CDC analysis of Fc variants using flow cytometry. Complement-dependent cytotoxicity (CDC) was determined by lactate dehydrogenase (LDH) release assay using baby rabbit serum as a complement source and human cancer cells as target cells. Briefly, target cells (20 × 10e per well) were incubated with 100 mM NaCl and 100 mM NaCl. 3) were dispensed into 96-well U-bottom plates and pre-incubated with Fc variants for 30 min on ice. Diluted complement was then added and further incubated at 37°C (5% CO2, humidified atmosphere) for 4 h. Assays were performed in triplicate with or without antibodies. Maximum release was prepared with target cells lysed with lysis solution. LDH activity of the supernatant was measured using a non-radioactive cytotoxicity assay kit (Promega cat#G1781). Released LDH activity, indicative of cell death, was determined by optical density reading at 490 nm suing a spectrophotometer (Cytation1 Biotek). Percentage cytotoxicity was calculated according to the formula: Cytotoxicity (%) = 100 x (experimental release - spontaneous release) / (maximum release - spontaneous release). Data are shown as % cytotoxicity bar graphs generated using GraphPad Prism software.

[0414] The results show that reduced CDC was observed with the Fc variants, with the triple mutant being significantly reduced, for each MAb (Figure 35, Figure 36, Figure 37, and Figure 38).

[0415] Example 11: C1q binding of Fc variants using ELISA. C1q binding was assessed using enzyme-linked immunoassay (ELISA). Briefly, 96-well ELISA plates were coated with 1 ug / ml test samples (i.e., wild-type or Fc variants of trastuzumab, panitumumab, sacituzumab and anti-CDH3-5836), 60 ul / well in 1×PBS (pH 7.4) coating buffer. Plates were then washed and blocked with ELISA blocking buffer (PBS containing 1% BSA). Plates were incubated with diluted human C1q (sigma Cat#C1740 lot#SCC6462) at concentrations ranging from 0.625 to 40 ug / ml, serially diluted 2-fold in 1×PBS containing 1% BSA, 0.05% Tween®-20 for two (2) hours at room temperature. After washing the plate, rabbit anti-hC1q (Dako A0136 lot#20047640) was added at 5ug / ml in 1% BSA in PBS and 0.05% Tween® (PBS-T), 60 ul / well, and incubated for 1.0 hour at room temperature. After washing to remove unbound antibody, bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit HRP-specific polyclonal antibody (Jackson ImmunoResearch, cat#111-036-046 West Grove, PA). A slow kinetic substrate solution of tetramethylbenzidine (TMB) was added and the optical density (OD) of the color was measured at 650 nm. Data representing detectable binding of the antibody over the range of test concentrations was analyzed by one-site binding nonlinear regression analysis using GraphPad Prism software.

[0416] The results show that reduced C1q binding was observed with the Fc variants, with the triple mutant being significantly reduced for each MAb (Figure 39, Figure 40, Figure 41, and Figure 42).

[0417] Example 12: Generation and characterization of site-specific ADCs Site-specific thiol generation The hinge disulfides of the Fc variants were reduced with DTT in PBS for fifteen (15) minutes at room temperature and the antibodies were purified from excess reducing agent by desalting into pH 6.5 buffer using a PD-10 column. The predicted free thiols were confirmed by Ellman's 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 is monitored by Ellman's test. When the thiol-to-antibody ratio reaches approximately 2.0, the maleimide-payload is added.

[0418] Preparation of conjugates To generate the site-specific ADCs of the invention, antibody conjugates using a proprietary linker(s) denoted LOL1 were prepared by adding a maleimide-linker to the activated site-specific antibody. The process of activation (i.e., liberation of a free thiol) is described above (generation of the site-specific thiol). A solution of the linker in DMSO 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.

[0419] Conjugate evaluation To validate the ability to generate site-specific ADCs using either the single mutant (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 for isolating cysteine-based conjugates and was used to confirm that the payload was primarily or solely conjugated to the heavy chain and only at a single position within the heavy chain, and thus the product was site-specific. Intact analysis by mass spectrometry was used to confirm that the conjugated heavy chain had the correct expected mass (i.e., HC+payload), while peptide mapping confirmed that the payload was anchored to a specific position within the heavy chain. Here, intact mass analysis was utilized to demonstrate that the triple mutant LOL-1-conjugated anti-HER2 mAb was composed of a single conjugated heavy chain and an unconjugated light chain (Figure 44). Peptide mapping of both the single mutant and triple mutant vcMMAE-conjugated anti-EGFR mAb No. 1, carrying either the single or triple mutation, revealed that the ACPAPIEK peptide within the heavy chain was both the site of the L328C mutation and the site of conjugation (Figure 54, Figure 55).

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

[0421] Generally, each Cys-based ADC carries a finite number of payloads that need to be known. It is generally accepted that ADCs utilizing vcMMAE as a payload should carry anywhere from two (2) to four (4) payloads to be efficient in tumor killing. Furthermore, their configurations are distributions of payloads ranging from 0 (unconjugated) to 8 (fully conjugated). Antibodies engineered to carry a defined number (typically two per mAb) of payloads (i.e., site-specific ADCs lacking distribution and high payload vs. mAb species) have been shown to be advantageous in clinical efficacy, safety or stability.

[0422] Therefore, we use analytical methods, in which reversed-phase chromatographic assay is the main technique, to separate the ADC configurations resulting from the conjugation of vcMMAE. Each species (i.e., heavy chain, light chain, and conjugate species) is identified based on both retention time and UV250 / 280 ratio, and DAR (drug-to-mAb ratio) is calculated. Furthermore, we show that the payload is mainly or completely tethered to the heavy chain, and the heavy chain with a single payload is the major species, which is a key attribute of site-specific conjugation.

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

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

[0425] The results show that the LOL1-conjugated anti-HER2 antibody with triple mutation resulted in a clear retention shift, suggesting that only one positional isomer was formed, highly indicative of site-specific conjugation (Figure 43). Further confirmation by intact mass analysis showed that the above LOL1-conjugated triple mutant anti-HER2 antibody was site-specific (Figure 44). Only unconjugated light chain (LC) with an average mass of 23440 Daltons (Da) was detected (Figure 44). The peak at 23462 Da represents a sodium adduct that is typically formed in LC-MS systems. For the heavy chain (HC), a conjugated species is found with an average mass of 49487 Da (reflecting a c-terminal lysine deletion (-K) typically observed in Chinese Hamster Ovary (CHO) cell culture systems utilized for antibody production) and a product of 49613 Da (reflecting a HC with an intact unprocessed c-terminal lysine (+K)). No unconjugated HC species or HCs with a second conjugation site were found (Figure 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 conjugates could only be formed with the engineered Cys-328 and not with any of the hinge region cysteines.

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

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

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

[0429] Figure 47 shows the reversed-phase column chromatography profile of the L234A, L235A, L328C triple mutant of anti-Trop2 Mab conjugated with vcMMAE. It is shown that more than 70% of the site-specific conjugation occurs on the Cys328 heavy chain. Figure 48 shows the reversed-phase column chromatography profile for the L234A, L235A, L328C variant of anti-EGFR Mab conjugated with vcMMAE. It is shown that DAR1 is the predominant species with more than 80%-90% of the site-specific conjugation occurring on the Cys328 heavy chain.

[0430] Figure 49 shows peak assignments and DAR calculations based on the RP-HPLC data provided in Figure 49. Unconjugated Mab (control) was used to identify both retention times and UV250 / 280 ratios for both heavy and light chains. Note that these parameters are required for both peak assignments and DAR determination. DAR was calculated as follows:

number

[0431] FIG. 50 shows a summary of analytical attributes including mean DAR, percent monomer peak as determined by SEC, and percent DAR1 species for vcMMAE- or LOL1-conjugated mAbs with single or triple Fc variants.

[0432] Peptide mapping To confirm that the payload was anchored to a specific location within the heavy chain, peptide mapping analysis was performed using the following protocol.

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

[0434] (ii) LC-MS: After sample preparation, 5.0 μL of sample was injected into a final 3000 nanoLC equipped with a 75 μm × 2 cm trap column packed with C18 3 μm bulk resin (Acclaim PepMap 100, Thermo Scientific) and a 75 μm × 15 cm analytical column containing C18 2 μm resin (Acclaim PepMap RSLC, Thermo Scientific). The nanoLC gradient was 3–35% solvent B (A = H2O with 0.1% formic acid, B = acetonitrile with 0.1% formic acid) over 40 min, 35%–85% solvent B in 5 min, with 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. 3 × 10 6 All spectra (m / z 350–2000) were acquired in profile mode with a resolution of 70,000 at m / z 200 using an automated gain control (AGC) target of 1000. The 15 ions of highest abundance were subjected to fragmentation by higher energy collisional dissociation (HCD) with a normalized collision energy of 25. MS / MS spectra were acquired in center-of-mass mode with a resolution of 17,500 at m / z 200. The AGC target for fragment ions was set to 2 × 10 with a maximum injection time of 50 ms. 4 Charge states 1, 7, 8, and unassigned were excluded from the tandem MS experiment. Dynamic exclusion was set to 45.0 sec.

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

[0436] A list of the antibody fragment and peptide map confidence scores for the single variants of vcMMAE-conjugated anti-EGFR antibody No. 1 is shown in Table VII. A list of the antibody fragment and peptide map confidence scores for the triple variants of vcMMAE-conjugated anti-EGFR antibody No. 1 is shown in Table VIII.

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

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

[0439] Example 14: Human clinical trials for the treatment of human cancers using Her2, EGFR, TROP2, CDH3, or other TAA triple mutant MAbs and site-specific conjugate ADCs

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

[0441] I.) Adjuvant Therapy: In adjuvant therapy, patients are treated with Her2, EGFR, Trop2, CDH3, or other TAA ADC in combination with chemotherapy or pharmaceutical or biologic or combination thereof. Primary cancer target is treated under standard protocol with addition of Her2, EGFR, Trop2, CDH3, or other TAA ADC. Protocol design addresses efficacy as assessed by the following examples, including but not limited to reduction of tumor burden of primary or metastatic lesions, extension of progression-free survival, overall survival, improvement of patient health, disease stabilization, and ability to reduce regular doses of standard chemotherapy and other biologic agents. These dose reductions allow additional and / or long-term treatment by reducing dose-related toxicity of chemotherapy or biologic agents.

[0442] II.) Monotherapy: In relation to the use of Her2, EGFR, Trop2, CDH3, or other TAA ADCs in tumor monotherapy, the Her2, EGFR, Trop2, CDH3, or other TAA ADCs are administered to patients without chemotherapy, pharmaceutical, or biological agents. In one embodiment, monotherapy is performed clinically in advanced cancer patients with widespread metastatic disease. Protocol design addresses efficacy as assessed by the examples below, including, but not limited to, reduction in tumor burden in primary or metastatic lesions, prolonged progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce regular doses of standard chemotherapy and other biological agents.

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

[0444] Clinical Development Plan (CDP) The CDP is then developed to treat cancer(s) and / or immune disorders (see Tables I and V) using the Her2, EGFR, Trop2, CDH3, or other TAA ADCs of the present disclosure. The trial will first demonstrate safety and then confirm efficacy in repeat administration. The trial will be open-label comparing standard chemotherapy with Her2, EGFR, Trop2, CDH3, or other TAA ADCs added to standard treatment. As will be appreciated, one non-limiting criterion that can be utilized in connection with patient enrollment is the concentration of Her2, EGFR, Trop2, CDH3, or other TAA ADCs in the tumor as determined by standard detection methods known in the art.

[0445] The present invention should not be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any functional equivalents are within the scope of the present invention. Various modifications to the models, methods, and lifecycle methodologies 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 also intended to be within the scope of the present invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention.

[0446] [Table 1]

[0447] [Table 2]

[0448] [Table 3]

[0449] [Table 4]

[0450] [Table 5]

[0451] Table VI Antibody Sequences Anti-HER2 Ab heavy chain wild type EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) Anti-HER2 Ab heavy chain L328C EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2) Anti-HER2 Ab heavy chain L234A, L235A EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3) Anti-HER2 Ab heavy chain L234A, L235A, L328C EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4) Anti-HER2 Ab light chain wild type DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 5) Anti-EGFR Ab No.1 heavy chain wild type QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6) Anti-EGFR Ab No.1 Heavy Chain L328C QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7) Anti-EGFR Ab No.1 heavy chain L234A, L235A QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) Anti-EGFR Ab No.1 heavy chain L234A, L235A, L328C QVQLQESGPGLVKPSETLSLTTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTM VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) Anti-EGFR Ab No.1 light chain wild type DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) Anti-EGFR Ab No.2 heavy chain wild type QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) Anti-EGFR Ab No.2 Heavy Chain L328C QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12) Anti-EGFR Ab No. 2 heavy chain L234A, L235A QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13) Anti-EGFR Ab No. 2 heavy chain L234A, L235A, L328C QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) Anti-EGFR Ab No.2 light chain wild type DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15) Anti-EGFR Ab No.3 heavy chain wild type QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16) Anti-EGFR Ab No. 3 Heavy Chain L328C QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17) Anti-EGFR Ab No. 3 heavy chain L234A, L235A QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) Anti-EGFR Ab No. 3 heavy chain L234A, L235A, L328C QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19) Anti-EGFR Ab No.3 light chain wild type DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20) Anti-Trop2 Ab heavy chain wild type QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21) Anti-Trop2 Ab heavy chain L328C QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) Anti-Trop2 Ab heavy chain L234A, L235A QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) Anti-Trop2 Ab heavy chain L234A, L235A, L328C QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24) Anti-Trop2 Ab light chain wild type DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25) Anti-CDH3 Ab heavy chain wild type QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26) Anti-CDH3 Ab heavy chain L328C QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27) Anti-CDH3 Ab heavy chain L234A, L235A QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28) Anti-CDH3 Ab heavy chain L234A, L235A, L328C QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKACPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29) Anti-CDH3 Ab light chain wild type DIQMTQSPSSLSASVGDRVTITCRASQTISNTLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLSWFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30)

[0452] [Table 7-1] [Table 7-2] [Table 7-3]

[0453]

Table 8-1

Table 8-2

Table 8-3

Claims

1. An antibody comprising a triple amino acid mutation, the triple amino acid mutation comprising a L234A modification, a L235A modification and a L328C modification, wherein the position numbering of the amino acid mutations is according to the EU index as set forth in Kabat, the triple amino acid mutation inhibits binding to FcγRI, FcγRIIA and FcγRIIIA to reduce antibody-dependent cellular cytotoxicity (ADCC), and the triple amino acid mutation further reduces C1q binding to reduce complement dependent cytotoxicity (CDC).

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

3. The antibody of claim 1 , wherein the antibody comprises an anti-Her2 antibody.

4. The antibody of claim 1 , wherein the antibody comprises an anti-Trop2 antibody.

5. The antibody of claim 1 , wherein the antibody comprises an anti-CDH3 antibody.

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

7. 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, the triple amino acid mutation comprising a L234A modification, a L235A modification and a L328C modification, wherein the position numbering of the amino acid mutations is according to the EU index as set forth in Kabat, and wherein the triple amino acid mutation inhibits binding to FcγRI, FcγRIIA and FcγRIIIA, thereby reducing antibody-dependent cellular cytotoxicity (ADCC), and the triple amino acid mutation further reduces C1q binding, thereby reducing complement-dependent cytotoxicity (CDC); (ii) a linker; and (iii) a Drug unit, wherein said Drug unit is specifically conjugated at site L328C.

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

10. The ADC of claim 8 , wherein the antibody comprises an anti-Her2 antibody.

11. 9. The ADC of claim 8, wherein the antibody comprises an anti-Trop2 antibody.

12. The ADC of claim 8, wherein the antibody comprises an anti-CDH3 antibody.

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

14. 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.

15. 9. The ADC of claim 8 further comprising a Stretcher unit.

16. The ADC of claim 8, further comprising a spacer unit.

17. 9. The ADC of claim 8, further comprising an amino acid unit.

18. An article of manufacture comprising the antibody of claim 1.

19. 9. An article of manufacture comprising the ADC of claim 8.

20. 10. A pharmaceutical composition comprising a therapeutically effective amount of the ADC of claim 8 and a pharma- ceutically acceptable excipient.

21. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of claim 1 and a pharma- ceutically acceptable excipient.

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

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

24. 23. The ABC of claim 22, wherein the antibody comprises an anti-Her2 antibody.

25. 23. The ABC of claim 22, wherein the antibody comprises an anti-Trop2 antibody.

26. 23. The ABC of claim 22, wherein the antibody comprises an anti-CDH3 antibody.

27. 23. The ABC of claim 22, wherein the antibody comprises an anti-tumor associated antigen (anti-TAA) antibody.

28. 28. The ABC 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. 23. The ABC of claim 22, further comprising a stretcher unit.

30. 23. The ABC of claim 22 further comprising a spacer unit.

31. 23. The ABC of claim 22, further comprising an amino acid unit.

32. 23. A pharmaceutical composition comprising a therapeutically effective amount of the ABC of claim 22 and a pharma- ceutically acceptable excipient.

33. 23. A composition for treating cancer in an individual, said composition comprising the ABC of claim 22, wherein said cancer is selected from the group consisting of acute myeloid leukemia, adrenocortical carcinoma, adult T-cell leukemia / lymphoma, B-cell Hodgkin's lymphoma, bladder cancer, breast cancer, clear cell renal cell carcinoma, colorectal cancer, gastric cancer, head and neck cancer, multiple myeloma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, reproductive cancer, cervical cancer, esophageal cancer and glioma.

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