Anti-CD276 antibodies, antibody-drug conjugates, and uses thereof

Abstract

An isolated anti-human B7-H3 antibody comprises two heavy chains containing hinge regions, the hinge regions comprising an amino acid sequence that enables site-specific binding of a cytotoxic drug. Each heavy chain comprises a human CH1 domain located upstream of the hinge region and linked thereto. According to the IMGT numbering scheme, the CH1 domain contains a cysteine ​​at position 142. The present invention also provides an ADC conjugated to the antibody and a cytotoxic drug. Also provided are pharmaceutical compositions comprising the antibody or ADC, as well as methods for treating cancer using the pharmaceutical compositions.

Description

Detailed Description of the Invention

[0001] CROSS REFERENCE TO RELATED APPLICATIONS: This application claims priority to International Application No. PCT / CN2020 / 130409, filed November 20, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [Background technology] CD276, also known as B7 homolog 3 (B7-H3), shares up to 30% amino acid identity with other B7 family members. [1] The B7-H3 protein is involved in the regulation of proliferation, apoptosis, invasion, cell cycle, cell differentiation, cell autophagy, and epithelial-mesenchymal transition. It is present at low levels in most normal tissues, but is overexpressed in a variety of cancers, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, glioma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, intrahepatic cholangiocarcinoma, oral squamous cell carcinoma, endometrial cancer, and melanoma. [2-6] Furthermore, B7-H3 protein is overexpressed in tumor vasculature from human lung, breast, colon, endometrial, renal, and ovarian cancers, but not in normal ovarian angiogenic blood vessels. [7-9] Therefore, B7-H3-directed therapeutics may have greater specificity for tumor vasculature than current antiangiogenic drugs, which cannot distinguish between physiological and pathological angiogenesis. Overexpression of B7-H3 on tumor cells and tumor endothelial cells (TECs) makes it an attractive target for the development of therapeutics that simultaneously destroy both cell types.

[0003] Enoblituzumab, an Fc-enhanced humanized anti-CD276 antibody, has shown activity in slowing the growth of various types of primary tumors in preclinical studies and is already in phase I clinical trials. Ivutuzumab has been studied in the treatment of refractory B7-H3-expressing tumors such as melanoma (NCT01391143), as well as other B7-H3-expressing tumors such as osteosarcoma and Ewing's sarcoma (NCT02982941). MGA271 is another B7-H3-targeting antibody that has shown potent antitumor activity in xenograft models of B7-H3-expressing renal cell and bladder cancer.

[10] The first humanized antibody identified based on its selective reactivity with human tumor cells, 8H9, was subsequently found to recognize CD276 and is also in phase I clinical trials. [11、12] Humanized 8H9 antibody can modulate the inhibitory immune properties of B7-H3 against tumor targets and impact immune checkpoint blockade

[13] .

[0004] ADCs are monoclonal antibodies conjugated with cytotoxic agents. They exploit targeting specificity to tumor cell surface proteins to deliver highly potent toxic payloads to tumors with specificity and efficacy unattainable by conventional chemotherapy. Currently, several ADCs targeting B7-H3 are in various stages of preclinical and clinical development. Multiple preclinical models have demonstrated that ADCs targeting B7-H3 can eradicate established tumors and significantly improve overall survival, suggesting the therapeutic potential of these molecules. [1] For example, exatecan derivatives (DX-8951 derivatives, DXd), used for drug conjugation as DXd-ADCs targeting B7-H3, have shown efficient antitumor effects and few side effects. Currently, multiple Phase II clinical trials are evaluating it.

[14] . 131 I-labeled anti-B7-H3 monoclonal antibody ( 131 I-4H7) is a radioactive biological agent that has therapeutic effects on human renal cell carcinoma in nude mice and is already in phase III clinical trials.

[15] .

[0005] Summary of the Invention In one aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, comprising two heavy chains and two light chains, each of which comprises: (a1) a heavy chain hinge region comprising any one of the amino acid sequences shown in SEQ ID NOs: 12-24; (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and each of which comprises (a3) ​​a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11, respectively. The antibody can specifically bind to human B7-H3 protein. In some embodiments, the amino acid sequence contained in each heavy chain hinge region is SEQ ID NO: 12. In some embodiments, each heavy chain further comprises a human CH1 domain located upstream of and linked to the hinge region, the CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme.

[0006] In another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to human B7-H3 protein, comprising two heavy chains, each heavy chain comprising (a) an amino acid sequence: -(X1)-C-(X2)-CPPCP-, where X1 is a polypeptide segment comprising 0-7 amino acid residues, each amino acid residue being independently selected from any amino acid residue other than a cysteine ​​residue, and X2 is a polypeptide segment having 2-7 amino acid residues, each amino acid residue being independently selected from any amino acid residue other than a cysteine ​​residue; and (b) a human CH1 domain located upstream of and connected to the hinge region, the human CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme, wherein the antibody specifically binds to human B7-H3 protein. In some embodiments, the amino acid sequence comprised in the hinge region is selected from the group consisting of SEQ ID NOs: 12-24, e.g., SEQ ID NO: 11, SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain, each heavy chain variable domain comprises a CDR1, CDR2 and CDR3 region containing the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, and each light chain variable domain comprises a CDR1, CDR2 and CDR3 region containing the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10.

[0007] In some embodiments, the antibodies described herein further comprise two kappa (κ) light chains, each light chain paired with one heavy chain. The two heavy chains of the antibody may be the same. The two light chains of the antibody may be the same.

[0008] In some embodiments, the CH1 domain of the antibody has the same sequence as the CH1 domain of a native human IgG2, IgG3, or IgG4 subclass antibody, or has the sequence of the CH1 domain of a native human IgG1 antibody and has the S142C mutation.

[0009] In some embodiments, each heavy chain may further comprise an Fc domain of a native human IgG2, IgG3, or IgG4 subclass antibody located downstream of and connected to the hinge region, where the Fc domain optionally comprises one or more substitutions.

[0010] In some embodiments, each heavy chain comprises a variable domain, wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, each heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0011] In some embodiments, each light chain comprises a variable domain, wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, each light chain comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0012] The antibodies of the present invention may be monoclonal antibodies, which may be of camelid, chimeric, human or humanized origin.

[0013] In another aspect, there is provided an antibody drug conjugate (ADC), or a pharma- ceutically acceptable salt thereof, in which an antibody described herein is conjugated to a cytotoxic drug and a chemical linker.

[0014] In one embodiment, an ADC may comprise (A) an isolated antibody, or antigen-binding portion thereof, and (B) a cytotoxic drug, wherein (A) the isolated antibody, or antigen-binding portion thereof, comprises (a1) a heavy chain hinge region comprising the amino acid sequence set forth in SEQ ID NO:11, (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and (a3) ​​a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, respectively.

[0015] Here, the isolated antibody, or antigen-binding portion thereof, is conjugated to a cytotoxic drug by a chemical linker.

[0016] The cytotoxic agents of the ADC include eribulin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimers, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, and thailanstatin B. A), uncialamycin, amanitins, ricin, diphtheria toxin, 131In some embodiments, the chemical linker is selected from the group consisting of 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 6-maleimidocaproyl-Val-Cit-p-aminobenzyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG, PEG ... n -Val-Cit-PAB (n = 1-20), Mal-amide-PEG n -Val-Cit-PAB (n=1-20), MC-Gly-Gly-Phe-Gly, Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridine-2-yldisulfanyl)propanoate. In one embodiment, the cytotoxic agent is eribulin. In another embodiment, the cytotoxic agent is MMAE.

[0017] In another aspect, the invention provides a pharmaceutical composition comprising an isolated antibody or antigen-binding portion thereof, or an ADC, or a pharma- ceutically acceptable salt thereof, described herein, and (b) a pharma- ceutically acceptable carrier.

[0018] In another aspect, the present invention provides a method for treating cancer in a human subject, comprising administering an effective amount of a pharmaceutical composition described herein. The cancer may be a cancer associated with overexpression of B7-H3 protein. For example, the cancer is selected from the group consisting of head and neck cancer, kidney cancer, skin cancer, colon cancer, glioblastoma, glioma, thyroid cancer, mesothelioma, melanoma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, and bladder cancer.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of the amino acid numbering scheme of the antibodies used in the present invention: (a) IMGT-based amino acid numbering scheme for human IgG1(κ), (b) numbering scheme for mutants (relative to the IgG1 hinge region).

[0020] [Figure 2] Physicochemical test results of 27B4H4L3 and BH73 antibodies. SDS-PAGE analysis of reduced (R) and non-reduced (NR) antibodies: 27B4H4L3 antibody (a) and BH73 antibody (b); Purity and yield of antibody produced in HEK293 cells (c); SEC-HPLC analysis of purified antibody, 27B4H4L3 antibody (d) and BH73 antibody (e).

[0021] [Figure 3] Conjugation spectra of ADCs prepared with 27B4H4L3 and BH73 antibodies. HIC spectra of ADCs prepared with 27B4H4L3 and BH73 antibodies. (a) 27B4H4L3-MMAE, (b) BH73-MMAE.

[0022] FIG. 4 shows the binding curves of 27B4H4L3 antibody and 27B4H4L3-MMAE with recombinant B7-H3 protein.

[0023] [Figure 5] Cytotoxicity curves of 27B4H4L3-MMAE, BH73-MMAE, and BR0102-DXd against B7-H3 expressing cancer cells: (a) U87, (b) BT-474, (c) HT-29, and (d) A431.

[0024] FIG. 6: In vivo drug efficacy of BH73-MMAE and BR0102-DXd in athymic nude mice bearing colorectal cancer HT29 xenografts.

[0025] [Mode for carrying out the invention] The present invention provides antibodies and ADCs that target B7-H3 using anti-B7-H3 antibodies and demonstrates their potent anti-tumor capabilities.

[0026] In one aspect, the invention provides an isolated antibody, or antigen-binding portion thereof, comprising two heavy chains and two light chains, Each heavy chain is (a1) a heavy chain hinge region comprising any one of the amino acid sequences shown in SEQ ID NOs: 12 to 24; (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; Each light chain is (a3) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10, respectively; An isolated antibody or antigen-binding portion thereof is provided.

[0027] The antibody can specifically bind to human B7-H3 protein. In some embodiments, the amino acid sequence contained in the heavy chain hinge region is SEQ ID NO: 12. In some embodiments, each heavy chain further comprises a human CH1 domain located upstream of and linked to the hinge region, the CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme.

[0028] Antibodies comprising this form of heavy chain preferably form an HH interchain disulfide bond between C142 (or a cysteine ​​near position 142, according to the IMGT numbering scheme further described below) of the CH1 domain and the last cysteine ​​residue of the light chain. A cysteine ​​residue in the hinge region upstream of the native CPPCP sequence forms a third HH interchain disulfide bond. The cysteine ​​at or near amino acid position 142 of the CH1 domain may be introduced by mutation or insertion of a single amino acid in the IgG1 subtype, or may consist of a native cysteine ​​residue in the CH1 domain of the IgG2, IgG3, or IgG4 subtype. Compared to the native HH disulfide bond of IgG1 (located between the amino acid paired with the cysteine ​​in the heavy chain hinge region of the IgG1 antibody and the end of the light chain), this form of HH disulfide bond is more stable and can remain intact in the reduced state during drug conjugation to the antibody. This significantly reduces the possibility of obtaining an ADC containing a light chain drug conjugate.

[0029] The IMGT numbering scheme of the immunoglobulin superfamily is used herein to simplify the numbering scheme (schematic diagram in FIG. 1a), where the VH or VL region comprises amino acid residues 1-128, respectively. Thus, the amino acid numbering of the CH1 domain is aa 129-226; the κ domain is aa 129-235; the hinge region is aa 227-241 (according to IgG1); CH2 is aa 242-351, and CH3 is aa 352-456. Based on this numbering scheme, the HL interchain disulfide bond in wild-type IgG1(κ) is formed between H(C231)-L(C235), whereas in IgG2(κ) (or IgG3(κ) or IgG4(κ)), it can be formed between H(C142)-L(C235). An IgG1 mutant in which serine at position 230 of the heavy chain is changed to cysteine ​​is named IgG1(S230C), and an IgG1 mutant in which C231 is deleted is named IgG1(Δ231). An IgG1 mutant in which one lysine is inserted after C231 is named K231.1, and an IgG1 mutant in which two amino acids KL are inserted after C231 is named K231.1 L231.2 (see FIG. 1 b, which shows some representative examples of mutations introduced in the hinge region of IgG1). For example, the hinge region of the control / reference antibody 27B4H4L3 comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 11), and clone BH73, an example of a modified antibody of the invention, comprises a modified hinge amino acid sequence containing EPPKSDCKTKTVECPPCP (SEQ ID NO: 12). Note that the control antibody 27B4H4L3 contains a human IgG1 CH1, while antibody BH73 contains a human IgG2 CH1.

[0030] In another aspect, the invention provides an isolated antibody, or antigen-binding portion thereof, comprising two heavy chains, each heavy chain comprising: (a) an amino acid sequence: -(X1)-C-(X2)-CPPCP-, where X1 is a polypeptide segment comprising 0-7 amino acid residues, each amino acid residue being independently selected from any amino acid residue that is not a cysteine ​​residue, and X2 is a polypeptide segment comprising 2-7 amino acid residues, each amino acid residue being independently selected from any amino acid residue that is not a cysteine ​​residue; and (b) a human CH1 domain located upstream of and linked to the hinge region comprising a cysteine ​​at position 142 according to the IMGT numbering scheme, wherein the antibody specifically binds to human B7-H3 protein.

[0031] In some embodiments, the amino acid sequence contained in the hinge region is selected from the group consisting of SEQ ID NOs: 11-24, e.g., SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the antibodies described herein can comprise two κ light chains, each light chain paired with one heavy chain. The two heavy chains in the antibody can be identical. The two light chains in the antibody can be identical.

[0032] In some embodiments, the CH1 domain of the antibody has a sequence identical to the sequence of the CH1 domain of a naturally occurring human IgG2, IgG3, or IgG4 subclass antibody, or has a sequence identical to the sequence of the CH1 domain of a naturally occurring human IgG1 antibody and has an S142C mutation.

[0033] In some embodiments, each heavy chain may further comprise an Fc domain of a native human IgG1, IgG2, IgG3, IgG4 subclass antibody, located downstream of and linked to the hinge region, wherein the Fc domain optionally comprises one or more substitutions.

[0034] In some embodiments, each heavy chain comprises a variable domain, wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, each heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0035] In some embodiments, each light chain comprises a variable domain, wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, each light chain comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0036] In some embodiments, each heavy chain of the antibody comprises a variable domain comprising an amino acid sequence set forth in SEQ ID NO:1, and each light chain of the antibody comprises a variable domain comprising an amino acid sequence set forth in SEQ ID NO:2.

[0037] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:5.

[0038] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. An isolated antibody that specifically binds to an antigen is substantially free of antibodies that do not bind to the antigen.

[0039] The term "monoclonal antibody" as used herein refers to a preparation of a population of antibody molecules of substantially homogeneous molecular composition, where the individual antibodies in the population are identical except for possible naturally occurring mutations which may be present in very small amounts.

[0040] An antibody or molecule that "specifically binds to human B7-H3" refers to an antibody or polypeptide molecule that binds to human B7-H3 protein but does not substantially bind to non-human B7-H3 protein.

[0041] Those skilled in the art use various methods / systems to define the CDRs (complementarity determining regions) of said antibodies. These systems and / or definitions have been developed and improved over the years and include Kabat, Chothia, IMGT, AbM and Contact. The commonly used Kabat definition is based on sequence variability. The Chothia definition is based on the location of the structural ring region. The IMGT system is based on sequence variability and location within the variable domain structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. One exemplary system is a combination of Kabat and Chothia.

[0042] DNA encoding amino acid sequence mutants of the starting polypeptide can be prepared by various methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared DNA encoding the polypeptide. Mutants of recombinant antibodies can also be constructed by restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. Mutagenic primers code for cysteine ​​codon substitutions. Standard mutagenesis techniques can be used to generate DNA encoding such mutant engineered antibodies.

[0043] In another aspect, the invention provides a nucleic acid molecule encoding an antibody or an antigen-binding portion of any antibody described herein. Host cells (e.g., CHO cells, lymphocytic cells, human embryonic kidney cells or microorganisms, such as fungi, e.g., E. coli and yeast) harboring an expression vector containing the nucleic acid molecule can be used to produce the antibodies of the invention, preferably monoclonal antibodies. In one embodiment, DNA encoding a partial or full-length antibody of the invention can be obtained by standard molecular biology techniques, and the gene is operably linked to transcriptional and translational regulatory sequences by insertion into one or more expression vectors. The term "operatively linked" indicates that the antibody gene is linked to a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Such regulatory sequences are, for example, as described by Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include those derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, such as the adenovirus major late promoter (AdMLP) and polyoma promoter and / or enhancer. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or the β-globin promoter. Furthermore, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which includes sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0044] DNA encoding the antibody can be inserted into an expression vector. The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The DNA encoding the antibody can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody encoded by the DNA. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0045] In another aspect, there is provided an antibody-drug conjugate (ADC), or a pharma- ceutically acceptable salt thereof, in some embodiments, the ADC comprises an antibody of the invention conjugated to a cytotoxic drug via a chemical linker described in the invention.

[0046] In some embodiments, the antibody portion of the ADC comprises: (a1) a heavy chain hinge region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 12-24, (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, (a3) ​​a heavy chain further comprising a human CH1 domain located upstream of and linked to the hinge region, the CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme, and (a4) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively. In one embodiment, the antibody portion of the ADC is a BH73 antibody.

[0047] In some embodiments, an ADC comprises: (A) an isolated antibody, or antigen-binding portion thereof; and (B) a cytotoxic agent, wherein the (A) isolated antibody, or antigen-binding portion thereof, comprises: (a1) a heavy chain hinge region comprising the amino acid sequence set forth in SEQ ID NO:11; and (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; and (a3) ​​a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; wherein the isolated antibody, or antigen-binding portion thereof, is conjugated to a cytotoxic drug by a chemical linker. In one embodiment, the antibody portion of the ADC is 27B4H4L3.

[0048] In some embodiments, the cytotoxic agent is selected from the group consisting of eribulin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin E, auristatin F, maytansines DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, anthracyclines, calicheamicin, dolastatin 10, duocarmycins, doxorubicin, tylanstatin A, uncialamicin, amanitin, ricin, diphtheria toxin, 131 In certain embodiments, the cytotoxic agent in the ADC is selected from the group consisting of ERIBULIN, IL, interleukins, tumor necrosis factors, chemokines, irinotecan (SN38), exotecan, and nanoparticles. In certain embodiments, the cytotoxic agent in the ADC is ERIBULIN. In other embodiments, the cytotoxic agent in the ADC is MMAE.

[0049] The chemical linker linking the antibody moiety and the cytotoxic agent can be cleavable or non-cleavable. In some embodiments, the linker comprises a PEG spacer, where n is 1-20 (i.e., having 1-20 repeating units (CH2CHO)). In some embodiments, the chemical linker further comprises a linker segment linked to the PEG spacer. In some embodiments, the chemical linker comprises a linker segment but does not comprise a PEG spacer. In some embodiments, the chemical linker is 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (ala-phe), p-aminobenzoxyloxycarbonyl (PAB), 6-maleimidocaproyl-Val-Cit-p-aminobenzoxyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG n -Val-Cit-PAB (n = 1-20), Mal-amide-PEG n -Val-Cit-PAB (n=1-20), MC-Gly-Gly-Phe-Gly, Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, perfluorophenyl 3-(pyridin-2-yldisulfanyl)propanoate, and combinations thereof.

[0050] In the present invention, pharma- ceutically acceptable salts of ADCs include acid addition salts of inorganic acids, carboxylic acids, and sulfonic acids, such as the salts of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid, and benzoic acid.

[0051] Pharmaceutically acceptable salts of drugs conjugated to the antibodies of the present invention further include salts of conventional bases, such as alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and ammonia or ammonium salts derived from organic amines containing 1 to 16 carbon atoms, where the organic amines are, for example, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzamide, N-methylpiperidine, N-methylmorpholine, arginine, lysine, 1,2-ethylenediamine.

[0052] ADC, as used herein, refers to a molecule comprising a drug molecule and an antibody (or antigen-binding portion thereof), where the drug and antibody (or antigen-binding portion thereof) are covalently linked by a linker. An "ADC formulation" herein refers to a collection or population of ADC molecules, the structures of which may vary with different attachment sites of the chemical linker and the antibody (or antigen-binding portion thereof). In some embodiments, the chemical linker is conjugated primarily or predominantly (e.g., ≥80%, ≥85%, ≥90%, ≥95%, or ≥98%) to cysteines on the heavy chain, resulting in an ADC formulation with substantially no light chains to be conjugated. In some embodiments, the chemical linker is conjugated to the antibody primarily through cysteines in the hinge region of the heavy chain of the antibody. In some embodiments, ADC molecules with a drug-to-antibody ratio (DAR) of 2 account for at least 60%, at least 70%, at least 80%, at least 85%, or at least 90% of the total amount of ADC molecules.

[0053] In another aspect, the present invention provides pharmaceutical compositions comprising one or more antibodies, ADCs, or pharma- ceutically acceptable salts thereof of the present invention and a pharma- ceutically acceptable carrier. As used herein, "pharma- ceutically acceptable carrier" includes pharma- ceutical acceptable carriers, excipients, or stabilizers. These include, but are not limited to, physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending aids, solubilizing agents, colorants, flavoring agents, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and the like. The selection of a suitable carrier is within the knowledge of one skilled in the art.

[0054] The composition may comprise one or more additional pharma- ceutical active ingredients, such as another antibody, a drug, e.g., a cytotoxic or antitumor drug, etc. The pharmaceutical compositions of the invention may also be administered in combination therapy with, for example, another anti-cancer drug, another anti-inflammatory drug, etc.

[0055] The pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, epidermal, and other routes of administration. Depending on the route of administration, the active ingredient may be coated with a material or packed into a material or structure to protect it from acids and other natural conditions that may inactivate it. The term "parenteral administration" as used in the present invention refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present invention may be administered via parenteral routes, such as topical, epidermal, or mucosal routes of administration, such as, for example, intranasal, oral, intravaginal, intrarectal, sublingual, or topical.

[0056] In another aspect, the present invention provides a method for treating cancer in a human subject, comprising administering an effective amount of the pharmaceutical composition of the present invention. The cancer can be a cancer associated with overexpression of B7-H3 protein. For example, the cancer can be selected from the group consisting of head and neck cancer, kidney cancer, skin cancer, colon cancer, glioblastoma, glioma, thyroid cancer, mesothelioma, melanoma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, and bladder cancer. The cancer can be a metastatic cancer, a refractory cancer, or a recurrent cancer.

[0057] When the composition is administered to a subject, the dosage can be adjusted to provide an optimal desired response (e.g., a therapeutic response). A single bolus or divided doses can be administered depending on the subject, the disease being treated, etc. A dosage unit form as used herein refers to a physically separated unit suitable as a unitary dosage for the subject to be treated. Each unit dosage contains a predetermined amount of active ingredient calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. A sustained release formulation can be used, in which case it needs to be administered less frequently.

[0058] For administration of a pharma- ceutically acceptable salt of an antibody or ADC thereof of the invention, the dosage range may be about 0.0001-100 mg / kg, more usually 0.01-10 mg / kg body weight, depending on the subject's body weight. For example, dosages may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or within the range of 1-10 mg / kg. Suitable treatment regimens may be once a week, once every two weeks, once every three weeks, once every four weeks, once a month, etc. Illustrative administration regimens for anti-B7-H3 antibodies of the invention may include intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight.

[0059] A "therapeutically effective amount" of an antibody or ADC of the invention, or a pharma- ceutical acceptable salt thereof, preferably reduces the severity of disease symptoms, increases the frequency and / or duration of disease symptom-free periods, or prevents or reduces the potential for injury or disability due to the disease, or inhibits or slows the progression of the disease. For example, in treating a tumor-bearing subject, a "therapeutically effective amount" of the antibody composition may inhibit tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, as compared to an untreated subject. Working Example 1. Generation of 27B4H43L3 (reference) and BH73 antibodies Briefly, Balb / C female mice (6-8 weeks old) were immunized with recombinant human B7-H3 antigen, and splenic lymphocytes from the highest titer mice electrofused with SP2 / 0 myeloma cells were used. Positive binders were screened by B7-H3 ELISA, and clone selection was performed by multiple rounds of subcloning and amplification. Clone 27B4 was identified as one of the best binders and selected for gene cloning. It was humanized by CDR grafting, confirmed for B7-H3 binding activity, and prepared in huIgG1 format, humanized clone 27B4H43L3. The heavy and light chain sequences of the reference antibody are as shown in SEQ ID NO:3 and SEQ ID NO:5, respectively.

[0060] The BH73 antibody, an example of a modified antibody of the present invention, was prepared with the variable sequence of the 27B4H4L3 antibody, the CH1 domain of IgG2, the hinge sequence containing EPPKSDCKTKTVECPPCP (SEQ ID NO: 12), and the IgG1 Fc containing LALA mutations (SEQ ID NO: L234A, L235A). The purpose is to generate an ADC containing two drug payloads at specific sites (site-specific binding). The heavy and light chain sequences of the BH73 clone are as shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. 2. Expression and purification of 27B4H4L3 and BH73 antibodies For the expression of 27B4H4L3 and BH73 antibodies, codon optimization and gene synthesis were performed to improve production in HEK293 cells. Full-length heavy and light chain DNAs were cloned into separate pcDNA3 plasmids. The paired plasmids were transiently transfected into HEK293 cells, and sufficient protein for testing was prepared by one-step protein A purification. Antibodies produced in this format had good detection, sufficient yields, and could be purified to high purity by a one-step protein A purification process (Figure 2). 3. Generation of ADCs by conjugation of 27B4H4L3 and BH73 antibodies To generate the conjugated drug, TCEP was added to a PBS solution containing purified antibody at neutral pH. Under mild reduction conditions (TCEP:mAb=1-3, neutral pH, room temperature for less than 240 min), the interchain disulfide bonds of the antibody were partially reduced. A drug-linker (MC-Val-Cit-PAB-MMAE) was added to the DMA to react with the antibody to obtain the desired drug-to-antibody ratio (DAR). To characterize the ADC, hydrophobic interaction chromatography (HIC) was used to evaluate the drug distribution and drug-to-antibody molar ratio in the ADC, and a representative HIC chromatogram of the drug in the ADC is shown in Figure 3. Under these mild reduction / conjugation conditions, the ADC prepared with 27B4H4L3 contained a main peak of 27B4H4L3 conjugated to 2, 4, and 6 drug molecules (DAR2, DAR4, and DAR6, respectively). The minor peaks were 27B4H4L3 (DAR0) and 27B4H4L3 bound to eight drug molecules (DAR8) (Figure 3a). The ADCs prepared with the BH73 antibody consisted mainly of DAR2 ADC species, with the minor peaks being BH73 (DAR0) and BH73 bound to four drug molecules (DAR4) (Figure 3b). 4. Measurement of B7-H3 binding activity of antibodies and their corresponding ADCs ELISA measurements were used to evaluate and compare the B7-H3 binding ability of comparative antibodies (27B4H4L3 and BH73) and their corresponding ADCs (27B4H4L3-MMAE, BH73-MMAE). Human B7-H3 protein (1000ng / mL) was coated onto a 96-well plate, and the wells were incubated overnight at 4°C. The diluted samples were then transferred to the B7-H3-coated well plate and incubated at room temperature for 1.5 hours. HRP-labeled goat anti-human IgG Fc antibody (Sigma, A0170) was used as the detection reagent, and the colorimetric reaction was performed using TMB, and the absorbance of the plate at 450 / 650 nm was read on a microplate reader (Molecular Devices, SpectraMax 190), and data analysis was performed using a four-parameter logistic model in the form of a dose-response curve.

[0061] The results of FIG. 4 and Table 1 show that BH73 modified with 27B4H4L3 has the same EC 50 and ADCs prepared with 27B4H4L3 or BH73 antibodies (27B4H4L3-MMAE and BH73-MMAE) showed similar binding activity to B7-H3 compared to the corresponding naked antibodies, suggesting that payload conjugation does not affect the target binding ability of the ADCs.

[0062] [Table 1]

[0063] 5. Measurement of antibody and ADC binding to B7-H3-expressing cells Screening of B7-H3 expressing cells was performed using FACS of 27B4H4L3 antibody and MFI of 27B4H4L3 against the cells shown in Table 2. FACS measurements were used to test and compare the binding ability of 27B4H4L3 antibody, BH73 antibody, 27B4H4L3-MMAE and BH73-Eribulin to B7-H3 expressing cells. For the measurements, the target cells and samples were incubated at 4°C for 1 hour. After washing, the samples were incubated with secondary antibody (goat pAb anti-human IgG (FITC) (Abcam, ab97224)). FACS was performed on a flow cytometer (BD, Accuri C6 Plus) and the mean fluorescence intensity (MFI) was recorded. The binding ability of different antibody constructs and ADCs was compared using three cell lines (U87MG, BT-474 and HT-29) representing high and low B7-H3 expressing cells. The BH73 antibody showed similar MFI levels as the 27B4H4L3 antibody in all cells tested, and the different forms of ADC (27B4H4L3-MMAE and BH73-MMAE) also showed similar MFI levels (Table 3), indicating that their B7-H3 binding activity was not affected by the process and conjugate.

[0064] [Table 2]

[0065] [Table 3]

[0066] 6. Cytotoxicity of ADCs against B7-H3-expressing cells The cytotoxicity of ADCs with antibody moieties containing 27B4H4L3 or BH73 was evaluated and compared to multiple cell lines with different B7-H3 expression levels in an in vitro cytotoxicity assay. To perform the assay, target cells were seeded in 96-well flat-bottom tissue culture plates at optimized cell density for each cell line and incubated overnight at 37°C and 5% CO2. Gradient dilutions of ADC samples were transferred to cell plates and assay well plates were incubated for a predefined time (3-7 days depending on cell line) to achieve optimal killing. Data analysis was performed using a dose-response curve of a four-parameter logistic model. As shown in Figure 5, 27B4H4L3-MMAE and BH73-MMAE exerted cytotoxicity against cancer cells highly expressing BH-73. 7. In vivo animal model and treatment The in vivo antitumor activity of ADCs containing the antibody moiety of BH73 was evaluated and compared with BR0102-DXd ADC (WO_2020_130125_A1). An HT29 colorectal cancer xenograft model was established in nude mice. HT29 cells were implanted into the backs of athymic nude mice. Tumor-bearing mice were treated with vehicle (control group) or ADC solution via a single tail vein injection. The dose of ADC was 20 mg / kg. The results shown in Figure 6 indicate that in the HT29 model, BH73-MMAE treatment is effective in tumor inhibition, whereas BR0102-DXd has a poor therapeutic effect at the same dose level.

[0067] References: [1].Seaman S, Stevens J, et al., Cancer Cell, 2007, 11, 539-554. [2].Brunner A, Hinterholzer, S, et al., Gynecol. Oncol., 2012, 124,105-111. [3].Huang C, Zhou L, et al., Oncol. Rep., 2016, 35, 2183-2190. [4].Arigami T, Narita N, et al., Ann. Surg., 2010, 252, 1044-1051. [5].Chen JT, Chen CH, et al., Proc. Natl. Acad. Sci., 2015, 112, 13057-13062. [6].Crispen PL, Sheinin Y, et al., Clin. Cancer Res., 2008, 14, 5150-5157. [7].Brunner A, Hinterholzer S, et al., Gynecol. Oncol., 2012, 124, 105-111. [8].Qin X, Zhang H, et al., Onco. Targets Ther., 2013, 6, 1667-1673. [9].Zang X, Sullivan PS, et al., Mod. Pathol., 2010, 23, 1104-1112.

[10] .Loo D, Alderson RF, et al., Clin. Cancer Res., 2012, 18, 3834-3845.

[11] .Ahmed M, Cheng M, et al., J. Biol. Chem., 2015, 290, 30018-30029.

[12] .Modak S, Kramer K, et al., Cancer Res., 2001, 61, 4048-4054.

[13] .Chen ZZ, Wang LP, et al., Curr. Pharm. Biotechno., 2016, 17, 1324-32.

[14] .Ogitani Y, Abe Y, et al., Bioorg. Med. Chem. Lett., 2016, 26, 5069-72.

[15] .Wang G, Wu Z, et al., Oncotarget, 2016, 7, 24888-98. All patent and non-patent literature mentioned herein is hereby incorporated by reference in its entirety.

[0068] While the invention has been described in conjunction with one or more embodiments, it should be understood that the invention is not limited to those embodiments, and that the description is intended to cover all alternatives, modifications, and equivalents that may be included within the spirit and scope of the appended claims.

[0069] Sequence Listing VH of 27B4H4L3 and BH73: EVQLVQSGAEVKKPGESLKISCKASGYTFTTYWIGWVRQMPGKGLEWMGIIYPGDGGSRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGNYISFPMYYWGQGTLVTVSS (SEQ ID NO: 1).

[0070] VL of 27B4H4L3 and BH73: EIVMTQSPATLSVSPGERATLSCKASRGVSTALAWYQQKPGQAPRLLIYWASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQHYDTPYTFGQGTKLEIK (SEQ ID NO: 2).

[0071] Heavy chain of 27B4H4L3: EVQLVQSGAEVKKPGESLKISCKASGYTFTTYWIGWVRQMPGKGLEWMGIIYPGDGGSRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGNYISFPMYYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 3).

[0072] BH73 heavy chain: EVQLVQSGAEVKKPGESLKISCKASGYTFTTYWIGWVRQMPGKGLEWMGIIYPGDGGSRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGNYISFPMYYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPPKSDCKTKTV ECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 4).

[0073] VL-CL of 27B4H4L3 and BH73 (human Kappa): EIVMTQSPATLSVSPGERATLSCKASRGVSTALAWYQQKPGQAPRLLIYWASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQHYDTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 5).

[0074] Heavy chain CDR1 of BH73 (and 27B4H4L3): GYTFTTYW (sequence number 6).

[0075] Heavy chain CDR2 of BH73 (and 27B4H4L3): IYPGDGGS (sequence number 7).

[0076] Heavy chain CDR3 of BH73 (and 27B4H4L3): ARGGNYISFPMYY (sequence number 8).

[0077] Light chain CDR1 of BH73 (and 27B4H4L3): RGVSTA (sequence number 9).

[0078] Light chain CDR2 of BH73 (and 27B4H4L3): WAS.

[0079] Light chain CDR3 of BH73 (and 27B4H4L3): QQHYDTPYT (sequence number 10).

[0080] 27B4H4L3 heavy chain hinge sequence: EPKSCDKTHTCPPCP (sequence number 11).

[0081] BH73 heavy chain hinge sequence: EPPKSDCKTKTVECPPCP (sequence number 12).

[0082] Other examples of heavy chain hinge sequences: CKTHTCPPCP (SEQ ID NO:13); DKTCHTCPPCP (SEQ ID NO:14); ERKSCVECPPCP (SEQ ID NO: 15); EPKSDCKTHTCPPCP (SEQ ID NO:16); EPKSDKCTHTCPPCP (SEQ ID NO:17); EPKSDCKTHTVECPPCP (SEQ ID NO:18); EPKSDCKTVECPPCP (SEQ ID NO:19); EPKSDKCTHTVECPPCP (SEQ ID NO:20); EPPKSCDKTHTVECPPCP (SEQ ID NO:21); EPPKSDCKTHTVECPPCP (SEQ ID NO:22); EPPPKSCDKTHTVECPPCP (SEQ ID NO:23); EPPPPKSCDKTHTVECPPCP (sequence number 24). [Brief description of the drawings]

[0083] [Figure 1] Schematic diagram of amino acid numbering schemes for antibodies used in the present invention: (a) IMGT-based amino acid numbering scheme for human IgG1(κ), (b) Numbering scheme for mutants (relative to the IgG1 hinge region). [Diagram 2] Physicochemical test results of 27B4H4L3 and BH73 antibodies. SDS-PAGE analysis of reduced (R) and non-reduced (NR) antibodies: 27B4H4L3 antibody (a) and BH73 antibody (b); Purity and yield of antibody produced in HEK293 cells (c); SEC-HPLC analysis of purified antibody, 27B4H4L3 antibody (d) and BH73 antibody (e). [Diagram 3]Conjugation spectra of ADCs prepared with 27B4H4L3 and BH73 antibodies. HIC spectra of ADCs prepared with 27B4H4L3 and BH73 antibodies. (a) 27B4H4L3-MMAE, (b) BH73-MMAE. [Figure 4] Binding curves of 27B4H4L3 antibody and 27B4H4L3-MMAE to recombinant B7-H3 protein. [Diagram 5] Cytotoxicity curves of 27B4H4L3-MMAE, BH73-MMAE, and BR0102-DXd against B7-H3 expressing cancer cells: (a) U87, (b) BT-474, (c) HT-29, and (d) A431. [Figure 6] In vivo drug efficacy of BH73-MMAE and BR0102-DXd in athymic nude mice bearing colorectal cancer HT29 xenografts.

Claims

1. 1. An isolated antibody, or antigen-binding portion thereof, comprising two heavy chains and two light chains, each heavy chain comprises a heavy chain hinge region containing the amino acid sequence set forth in SEQ ID NO:12; each of the heavy chains comprises the amino acid sequence set forth in SEQ ID NO:4; each of the light chains comprises the amino acid sequence set forth in SEQ ID NO:5; the antibody specifically binds to human B7-H3 protein; An isolated antibody or antigen-binding portion thereof.

2. 13. An antibody-drug conjugate (ADC), or a pharma- ceutically acceptable salt thereof, comprising the antibody of claim 1 conjugated to a cytotoxic drug by a chemical linker.

3. The cytotoxic drug is selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin E, auristatin F, maytansines DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, anthracyclines, calicheamicin, dolastatin 10, duocarmycins, doxorubicin, tylanstatin A, uncialamicin, amanitin, ricin, diphtheria toxin, １３１ 3. The ADC or a pharma- ceutically acceptable salt thereof of claim 2, wherein the ADC is selected from the group consisting of: I, interleukins, tumor necrosis factors, chemokines, irinotecan (SN38), exatecan, and eribulin.

4. The chemical linker may be 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit), alanine-phenylalanine (ala-phe), p-aminobenzoxyloxycarbonyl (PAB), 6-maleimidocaproyl-Val-Cit-p-aminobenzoxyloxycarbonyl (MC-Val-Cit-PAB), Mal-PEG ｎ -Val-Cit-PAB (n=1-20), Mal-amide-PEG ｎ 4. The ADC or a pharma- ceutically acceptable salt thereof according to claim 2 or 3, wherein the ADC or a pharma- ceutically acceptable salt thereof is selected from the group consisting of -Val-Cit-PAB (n=1 to 20), MC-Gly-Gly-Phe-Gly, Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridin-2-yldisulfanyl)propanoate.

5. 10. A pharmaceutical composition comprising the isolated antibody or antigen-binding portion thereof of claim 1, or the ADC or a pharma- ceutically acceptable salt thereof of any one of claims 2 or 3, and a pharma- ceutically acceptable carrier.

Images