Anti-CD73 antibodies, antibody-drug conjugates, and uses thereof

Abstract

The isolated anti-human CD73 antibody comprises two heavy chains, each of which comprises a hinge region containing an amino acid sequence that enables site-specific conjugation 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. Furthermore, an ADC conjugated to the antibody and a cytotoxic drug is provided. Also provided are pharmaceutical compositions comprising the antibody or ADC, and 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 entire disclosure of which is incorporated herein by reference.

[0002] [Background technology] CD73 is an ecto-5'-nucleotidase (ecto-5'-NT), a glycosylphosphatidylinositol-anchored receptor expressed by immune cells, stromal cells, epithelial and endothelial cells, cancer cells, and exosomes. This enzyme catalyzes the conversion of adenosine monophosphate (AMP) to adenosine. CD73 is involved in the maintenance of endothelial integrity. [1] , which plays a role in mediating the suppressive functions of regulatory B and T lymphocytes [2] It is believed that...

[0003] In addition to their roles in healthy tissue biology, CD73 and adenosine each influence tumor biology: extracellular adenosine accumulates in tumors and inhibits cytotoxic T cells and natural killer cells. [3] A2AR is one of the adenosine receptors that is upregulated during inflammation in effector T cells, and its activation inhibits effector T cell proliferation, cytotoxic activity, and cytokine production [e.g., tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)], and interleukin-2 (IL-2)]. [4] However, A2AR activation in regulatory T (Treg) cells promotes the expansion and immune inhibitory activity of Tregs, and tumors can utilize extracellular adenosine to protect cancer cells. Reports showed that knockout mice lacking adenosine receptors rejected tumors more readily than normal mice, and knockout mice lacking CD73 showed increased antitumor immunity and reduced carcinogenicity compared to normal mice. [5-7]This further supports that tumors use CD73 to generate adenosine to inhibit antitumor immunity. Elevated CD73 expression occurs frequently in human cancers and is often associated with poor prognosis as observed in lung, colorectal, breast, ovarian, renal, and glioblastoma tumors. Clinically, elevated levels of CD73 in tumor tissues of several cancer types, including breast, ovarian, and colorectal cancer (CRC), have been associated with decreased patient survival. [8] , which highlights the important role of CD73 in tumor progression.

[0004] In recent years, antibodies and small molecule inhibitors against CD73 have progressed into preclinical and clinical trials as an attractive target for restoring antitumor immunity. [9] In many preclinical studies, inhibition of CD73 (and / or A2AR) can restore antitumor immunity, and the combination shows better efficacy. Preliminary safety data reported that the combination of BMS-986179 (anti-CD73 humanized monoclonal antibody) and nivolumab (anti-PD-1 therapy) was well tolerated by patients (NCT02754141).

[10] Future research in gastrointestinal cancers will focus on determining whether adenosine-mediated resistance to immunotherapy is present at diagnosis or develops with treatment, which would be of great merit. Early results of the combination of BMS-986179 and nivolumab are promising, with clinical benefit (partial palliative) reported in one or more patients with pancreatic and / or prostate cancer.

[10] , both of which are poorly immunogenic tumors. Preclinical studies have shown that CD73 / adenosine therapy (e.g., deletion of the A2AR) can unleash the antitumor activity of CD8+ T cells against poorly immunogenic sarcomas.

[11] AMG510 (NCT03600883), a selective inhibitor of KRAS(G12C), has recently shown promising antitumor effects, including enhancing sensitivity to ICI therapy in preclinical models.

[12] Its combination with CD73 / adenosine receptor blockade may be a promising future approach.

[0005] ADCs are monoclonal antibodies conjugated with cytotoxic agents. They exploit the targeting specificity of tumor cell surface proteins to efficiently deliver toxic payloads to tumors with specificity and potency unattainable by conventional chemotherapy. No ADCs targeting CD73 have received market approval. Yu et al. reported the first anti-CD73-ADC (Hu001-MMAE, DAR4) and demonstrated its potent cytotoxicity against tumor cells highly expressing CD73.

[13] CD73-ADCs have also shown high efficacy in multiple in vivo tumor models, including non-small cell lung cancer (NSCLC) and RAS-mutated gliomas. However, preliminary toxicity / pharmacokinetic studies in non-human primates have shown that these DAR4 ADCs have short half-lives, even at doses approaching the highest possible tolerated dose levels.

[0006] There is a need for anti-CD73 ADCs with improved PK profiles and better tumor-suppressing activity.

[0007] Summary of the Invention In one aspect, the invention provides an isolated antibody or antigen-binding portion thereof. In some embodiments, the antibody comprises 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 set forth in SEQ ID NOs:25-37; and (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NOs:5, 6, and 7, respectively; and each of which comprises (a3) ​​a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10, respectively. In some embodiments, the antibody comprises two heavy chains and two light chains, each of the heavy chains comprising: (b1) a heavy chain hinge region comprising any one of the amino acid sequences shown in SEQ ID NOs: 25-26; (b2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences shown in SEQ ID NOs: 11, 12, and 13, respectively; and each of the light chains comprising (b3) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences shown in SEQ ID NOs: 14, 15, and 16, respectively. The antibody can specifically bind to human CD73 protein. In some embodiments, each of the heavy chains further comprises a human CH1 domain located upstream of and linked to the hinge region, and the CH1 domain comprises a cysteine ​​at position 142 according to the IMGT numbering scheme.

[0008] In another embodiment, the antibody comprises two heavy chains, each heavy chain comprising (a) an amino acid sequence: -(X1)-C-(X2)-CPPCP-, where X1 is a polypeptide segment having 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 having 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, the human CH1 domain comprising a cysteine ​​at position 142 according to the IMGT numbering scheme, wherein the antibody specifically binds to human CD73 protein. The amino acid sequence comprised in the hinge region may be selected from the group consisting of SEQ ID NOs: 25-37, e.g., SEQ ID NO: 25 or SEQ ID NO: 26. The antibody may comprise two κ light chains, each light chain being paired with one heavy chain.

[0009] In some embodiments, the antibody CH1 domain may have the same sequence as the CH1 domain of a natural human IgG2, IgG3, or IgG4 subclass antibody. In some embodiments, the antibody CH1 domain has the sequence of the CH1 domain of a natural human IgG1 antibody and has a S142C mutation. In some embodiments, each heavy chain further comprises an Fc domain of a natural human IgG1, IgG2, IgG3, IgG4 subclass antibody, which is located downstream of and linked to the hinge region, where the Fc domain optionally includes one or more substitutions, for example, a LALA mutation.

[0010] In some embodiments of the antibodies, each heavy chain comprises a variable domain having an amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3, and each light chain comprises a variable domain having an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4.

[0011] In some embodiments of the antibodies, each heavy chain comprises any one of the amino acid sequences set forth in SEQ ID NO:17, SEQ ID NO:198, and SEQ ID NO:21, wherein each heavy chain comprises any one of the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.

[0012] In a specific embodiment, each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:17 and each light chain comprises the amino acid sequence set forth in SEQ ID NO:18.

[0013] In a specific embodiment, each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:19 and each light chain comprises the amino acid sequence set forth in SEQ ID NO:20.

[0014] In a specific embodiment, each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:21 and each light chain comprises the amino acid sequence set forth in SEQ ID NO:22.

[0015] In another aspect, the present invention provides an antibody-drug conjugate (ADC), or a pharma- ceutically acceptable salt thereof, comprising an antibody according to the present invention conjugated to a cytotoxic drug via a chemical linker. The cytotoxic drugs 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 the like. A), uncialamycin, amanitins, ricin, diphtheria toxin, 131 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 some embodiments, the cytotoxic agent is MMAE.

[0016] In another aspect, the invention provides a pharmaceutical composition comprising an isolated antibody or antigen-binding portion thereof according to the invention, or an ADC according to the invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. In another aspect, the invention provides a method of treating cancer in a human subject, comprising administering an effective amount of a pharmaceutical composition according to the invention, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, glioma, melanoma, lymphoma, leukemia, myeloma, sarcoma, or virus-associated cancer.

[0017] 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).

[0018] Figure 2: Results of production and physicochemical testing of certain antibodies according to embodiments of the invention. SDS-PAGE analysis of reduced (R) and non-reduced (NR) antibodies: M35-1 antibody (a), M35-2 antibody (b) and M45-1 antibody (c); Purity and yield of antibodies produced in HEK293 cells (d); SEC-HPLC analysis of purified antibodies: M35-1 antibody (e), M35-2 antibody (f) and M45-1 antibody (g).

[0019] Figure 3: HIC spectra of ADCs prepared with M35-1, M35-2, and M45-1 antibodies: (a) M35-1-MMAE, (b) M35-2-MMAE, and (c) M45-1-MMAE.

[0020] Figure 4: Binding curves of anti-CD73 naked antibodies and their corresponding ADCs to CD73. (a) M35-1 antibody and M35-1 MMAE; (b) M35-2 antibody and M35-2-MMAE.

[0021] [Figure 5] Internalization of anti-CD73 naked antibodies and their corresponding ADCs in CD73-expressing cancer cells. a: NCI-H1975 cells, b: NCI-H411 cells.

[0022] [Figure 6] Cytotoxicity curves of M35-1-MMAE, M35-2-MMAE, and M45-1-MMAE against CD73-expressing cancer cells. a: U87MG cells, b: NCI-H1975 cells, c: NCI-N87 cells, d: NCI-H292 cells.

[0023] FIG. 7. In vivo drug potency of M35-1-MMAE and M35-2-MMAE in athymic nude mice xenografted with U87 glioma, NCI-N87 gastric cancer, and NCI-H441 NSCLC.

[0024] [Mode for carrying out the invention] The present invention provides antibodies and ADCs that target CD73, and their use in an anti-tumor capacity.

[0025] In one aspect, the present invention provides modified anti-CD73 antibodies based on anti-CD73 antibodies, and ADCs based on these modified anti-CD73 antibodies loaded with two site-specific conjugates, i.e., each modified anti-CD73 antibody molecule is conjugated with two cytotoxic drug payloads, with a drug-to-antibody ratio ("DAR") of 2. Compared with previously reported DAR4 ADCs, the DAR2 ADCs of the present invention can provide better PK distribution, better tumor tissue penetration and potentially better tumor inhibitory activity.

[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 containing any one of the amino acid sequences shown in SEQ ID NOs: 25 to 37 (for example, SEQ ID NO: 25 or SEQ ID NO: 26); and (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, 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:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; An isolated antibody or antigen-binding portion thereof is provided.

[0027] In another 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 containing any one of the amino acid sequences shown in SEQ ID NOs: 25 to 26 (for example, SEQ ID NO: 25 or SEQ ID NO: 26); and (a2) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences set forth in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; Each light chain is (a3) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; An isolated antibody or antigen-binding portion thereof is provided.

[0028] The antibody can specifically bind to human CD73 protein. In some embodiments, the amino acid sequence contained in the heavy chain hinge region is SEQ ID NO: 25. In some embodiments, the amino acid sequence contained in the heavy chain hinge region is SEQ ID NO: 26.

[0029] 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 be a native cysteine ​​in the CH1 domain from IgG2, IgG3, or IgG4 subtypes. Compared to the native HH disulfide bond of IgG1 (located between the end of the light chain paired with a cysteine ​​in the hinge region of the IgG1 antibody heavy 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.

[0030] 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 (see FIG. 1a). 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 may 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 designated IgG1(S230C), and an IgG1 mutant in which C231 is deleted is designated IgG1(Δ231). An IgG1 mutant in which one lysine is inserted after C231 is designated K231.1, and an IgG1 mutant in which two amino acids KL are inserted after C231 is designated K231.1L231.2 (see FIG. 1b for some representative examples of mutations introduced in the hinge region of IgG1). For example, the engineered antibodies of the invention, antibodies M35-1 and M45-1, contain a modified hinge amino acid sequence comprising EPPKSCDKTHTVECPPCP (SEQ ID NO: 25), and antibody M35-2 contains a modified hinge amino acid sequence comprising EPPKSDCKTKTVECPPCP (SEQ ID NO: 26). Note that antibodies M35-1 and M45-1 contain a human IgG1 CH1 with an S142C mutation, while antibody M35-2 contains a human IgG2 CH1.

[0031] In some embodiments, each heavy chain further comprises a human CH1 domain located upstream of and linked to the hinge region, said CH1 domain comprising a cysteine ​​located at position 142 according to the IMGT numbering scheme.

[0032] In another aspect, the invention provides an isolated antibody, or antigen-binding portion thereof, comprising two heavy chains, each heavy chain comprising: (a) the amino acid sequence: -(X1)-C-(X2)-CPPCP-, where X1 is a polypeptide segment comprising 0 to 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 to 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 a hinge region that contains a cysteine ​​at position 142 according to the IMGT numbering scheme, wherein the antibody specifically binds to a human CD73 protein.

[0033] In some embodiments, the amino acid sequence contained in the hinge region is selected from the group consisting of SEQ ID NOs: 25-37, e.g., SEQ ID NO: 25 or SEQ ID NO: 26. 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.

[0034] 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 having an S142C mutation.

[0035] In some embodiments, each heavy chain may comprise an Fc domain of a native human IgG1, IgG2, IgG3, IgG4 subclass antibody, which is located downstream of and linked to the hinge region, where the Fc domain optionally comprises one or more substitutions, e.g., a LALA mutation.

[0036] 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 or SEQ ID NO:3, and each light chain comprises a variable domain, wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.

[0037] In some embodiments, each heavy chain comprises an amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21; and each light chain comprises an amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.

[0038] In some embodiments, each heavy chain comprises an amino acid sequence set forth in SEQ ID NO:17 and each light chain comprises an amino acid sequence set forth in SEQ ID NO:18.

[0039] In some embodiments, each heavy chain comprises an amino acid sequence set forth in SEQ ID NO:19 and each light chain comprises an amino acid sequence set forth in SEQ ID NO:20.

[0040] In some embodiments, each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:21 and each light chain comprises the amino acid sequence set forth in SEQ ID NO:22.

[0041] 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.

[0042] 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.

[0043] An antibody or molecule that "specifically binds to human CD73" refers to an antibody or polypeptide molecule that binds to human CD73 protein but does not substantially bind to non-human CD73 protein.

[0044] 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.

[0045] 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 E. coli and fungi such as 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 adenovirus, such as the adenovirus major late promoter (AdMLP) and polyoma promoter and / or enhancer derived from cytomegalovirus (CMV), simian virus 40 (SV40). 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 contains 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.

[0046] 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).

[0047] 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.

[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 another embodiment, the cytotoxic agent in the ADC is selected from the group consisting of MMAE, an interleukin, a tumor necrosis factor, a chemokine, an irinotecan (SN38), an exotecan, and a nanoparticle.

[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 to 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, and 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, toluenesulfonic 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 the antibody-drug conjugates of the 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, and 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 conjugated. In some embodiments, the chemical linker is conjugated to the antibody primarily through cysteines in the hinge region of the antibody's heavy chain. 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 of ordinary skill 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 herein. The cancer can be a cancer associated with overexpression or upregulation of human CD73 protein. For example, the cancer can be selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, glioma, melanoma, lymphoma, leukemia, myeloma, sarcoma, or virus-related cancer. The cancer can be metastatic cancer, refractory cancer, or 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 the anti-EGFR 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" or "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 a disease, or inhibits or delays the progression of a 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.

[0060] Working Example 1. Engineered M35-1, M45-1, and M35-2 Antibodies Engineered antibodies to generate ADCs with high DAR2 ratios were designed with the specifications shown in Table 1.

[0061] [Table 1]

[0062] 2. Expression and purification of M35-1, M35-2 and M45-1 antibodies For expression of M35-1, M35-2 and M45-1 antibodies, codon optimization and gene synthesis were performed to improve production in HEK293 cells. Full-length heavy and light chain DNA was cloned into separate pcDNA3 plasmids. The paired plasmids were transiently transfected into HEK293 cells and purified with one step of Protein A to prepare sufficient protein for testing. Antibodies produced in this form had good detection, sufficient yields and could be purified to high purity with one step Protein A purification process (Figure 2).

[0063] 3. Generation of ADCs by conjugation of M35-1, M35-2 and M45-1 antibodies To generate drug conjugates, TCEP was added to a PBS solution containing purified antibody at neutral pH. Mild reducing conditions (TCEP:mAb=1.1-2.4, neutral pH, room temperature for less than 240 min) partially reduced the interchain disulfide bonds of the antibody. 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 ADCs, hydrophobic interaction chromatography (HIC) was used to assess the drug distribution and drug-to-antibody molar ratio in the ADCs, and a representative HIC chromatogram is shown in Figure 3. Under these mild reduction / conjugation conditions, ADCs prepared with all three engineered antibodies (M35-1, M35-2, and M45-1) were composed primarily of DAR2 ADC species, with minor peaks being naked antibody (DAR0) and DAR4 ADC species (Figures 3a, b, and c).

[0064] 4. Measurement of CD73 binding activity of antibodies and their corresponding ADCs ELISA measurements were used to evaluate and compare the CD73 binding ability of antibodies and ADCs. Human CD73 protein was coated onto 96-well plates and the plates were incubated overnight at 4°C. Samples were gradient diluted to a total of eight dilutions. The diluted samples were then transferred to the CD73-coated plates 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 colorimetric reaction was performed using TMB. The absorbance of the plates was read at 450 / 650 nm with 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.

[0065] The results in Figure 4 and Table 2 indicate that ADCs prepared with M35-1 or M35-2 antibodies (M35-1-MMAE and M35-2-MMAE) have similar binding activity to CD73 compared to the corresponding naked antibodies, suggesting that payload conjugation does not affect the target binding ability of the ADCs.

[0066] [Table 2]

[0067] 5. Measuring antibody and ADC binding to CD73-expressing cells FACS measurements were used to test and compare the binding ability of antibodies and ADCs to CD73-expressing cells. For the measurements, target cells and samples were incubated for 1 hour at 4°C. After washing with PBS containing 0.5% BSA, samples and secondary antibodies (goat pAb anti-human IgG (FITC) (Abcam, ab97224)) were incubated for 0.5 hours at 4°C. FACS was performed on a flow cytometer (BD, Accuri C6 Plus) and the mean fluorescence intensity (MFI) was recorded. The MFI of M35-1 and M35-2 against multiple CD73-expressing cells is shown in Table 3. The binding ability of different antibody constructs and ADCs was compared using four cell lines (NCI-H1975, NCI-H292, NCI-H441 and PC9) representing high and low CD73 expressing cells. The M35-1 and M35-2 antibodies and their corresponding ADCs (M35-1-MMAE, M35-2-MMAE) showed similar MFI levels on all cells tested (Table 4), indicating that CD73 binding activity was not affected by conjugation.

[0068] [Table 3]

[0069] [Table 4]

[0070] 6. Internalization of antibodies and ADCs into CD73-expressing cells A FACS-based internalization assay was used to detect and compare the internalization of naked antibodies and their corresponding ADCs into NCI-H1975 and NCI-H441 (high and low CD73 expressing cells, respectively). The detection started with a tube of antibody or ADC (10000ng / mL) mixed with target cells, cooled at 4°C for 1 hour, and then transferred to 37°C. The internalization measurement time of antibody-target or ADC-target complex was 4 hours. As shown in Figure 5, both the antibody and ADC could be efficiently internalized into NCI-H1975 and NCI-H441.

[0071] 7. Cytotoxicity of ADCs against CD73-expressing cells The cytotoxicity of the ADCs (M35-1-MMAE, M35-2-MMAE, or M45-1-MMAE) was evaluated and compared to multiple cell lines with different CD73 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 cultured overnight (16-20 h) at 37 °C and 5% CO2. Gradient dilutions of the ADC samples were transferred to the cell plates and the assay plates were cultured for a defined period (3-7 days depending on the cell line) to achieve optimal killing. Data analysis was performed using a 4-parameter logistic model dose-response curve. As shown in Figure 6, M35-1-MMAE, M35-2-MMAE, and M45-1-MMAE exerted similar cytotoxicity against U87MG cells, which highly express CD73, while M35-2-MMAE appeared to be more potent against NCI-H1975 cells than M35-1-MMAE. The difference in efficacy appeared to be greater in NCI-N87 and NCI-H292 cells, which have low expression of CD73.

[0072] 8. In vivo efficacy of M35-1-MMAE and M35-2-MMAE in athymic nude mice bearing anti-glioma cancer xenografts.

[0073] The antitumor potential of M35-1-MMAE and M35-2-MMAE was evaluated in human U87 glioma, NCI-H1975 NSCLC, and NCI-N87 gastric cancer xenograft models established in nude mice. Cancer cells were implanted into the backs of athymic nude mice. Tumor-bearing mice received a single intravenous treatment with either control (vehicle), M35-1, or M35-2 ADC. Tumor volumes were measured at various time points after treatment. The results shown in Figure 7 indicated that treatment with anti-CD73 high DAR2 ADCs could not only dramatically inhibit tumor growth in all tested models, but also induce tumor rejection in CD73-high expressing tumor models (U87 and NCI-H1975 models).

[0074] References [1] Saze Z, Schuler PJ, et al., Blood, 2013; 122:9-18 [2] Jalkanen S, Salmi M. Arterioscler Thromb Vasc Biol, 2008; 28:18-26 [3] Willingham SB, Ho PY, et al., Cancer Immunol Res. 2018, 6:1136-1149 [4] Lappas CM, Rieger JM, et al., J Immunol. 2005, 174:1073-80 [5] Ohta A, Gorelik E, Prasad SJ, et al., Proc Natl Acad Sci USA. 2006; 103:13132-13127 [6] Waickman AT, Alme A, Senaldi L, et al., Cancer Immunol Immunother. 2012; 61:917-926 [7] Stagg J, Beavis PA, Divisekera U, et al., Cancer Res. 2012; 72:2190-2196 [8] Beavis PA, Milenkovski N, Henderson MA, et al., Cancer Immunol Res. 2015; 3:506-517 [9] Perrot I, Michaud HA, Giraudon-Paoli M, et al., Cell Rep. 2019, 27:2411-2425

[10] Siu LL, Burris H, Le DT, et al., Cancer Res. 2018,78

[11] Kjaergaard J, Hatfield S, Jones G, et al., J Immunol. 2018, 201:782-791

[12] Canon J, Rex K, Saiki AY, et al., Nature 2019, 575:217-223.

[13] Jin R, Liu L, Xing Y, et al., Molecular Cancer Therapeutics, 2020.

[0075] All patent and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0076] 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.

[0077] Sequence Listing M35-1 and M35-2 VH: QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYYIYWVRQAPGQRLEWMGWIYPGNLNIKYNEKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCARDDNYAWFAYWGQGTLVTVSS (SEQ ID NO: 1).

[0078] M35-1 and M35-2 VL: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYWTNTRHTGVPSRFSGSGSGTDHTLTISSLQPEDFATYYCQQHYSTPFTFGQGTKLEIK (SEQ ID NO:2).

[0079] M45-1 VH: QVQLVQSGAEVKKPGASVKVSCKASGYTLTSYWMHWVRQAPGQGLEWMGEINPSQGRSNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRGVSGNYFDYWGQGTLVTVSS (sequence number 3).

[0080] M45-1 VL: DIQMTQSPSSLSASVGDRVTITCKASQDINTYLSWFQQKPGKSPKSLIYRSNILVSGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKLEIK (SEQ ID NO:4).

[0081] CDR1 of VH of M35-1 and M35-2: NYYIY (sequence number 5).

[0082] CDR2 of VH of M35-1 and M35-2: WIYPGNLNIKYNEKFKG (sequence number 6).

[0083] CDR3 of VH of M35-1 and M35-2: DDNYAWFAY (sequence number 7).

[0084] CDR1 of VL of M35-1 and M35-2: KASQDVSTAVA (sequence number 8).

[0085] CDR2 of VL of M35-1 and M35-2: WTNTRHT (sequence number 9).

[0086] M35-1 and M35-2 V L CDR3: QQHYSTPFT (Sequence number 10).

[0087] CDR1 of VH of M45-1: SYWMH (sequence number 11).

[0088] CDR2 of VH of M45-1: EINPSQGRSNYNEKFKS (sequence number 12).

[0089] CDR3 of VH of M45-1: RGVSGNYFDY (sequence number 13).

[0090] CDR1 of M45-1 VL: KASQDINTYLS (sequence number 14).

[0091] CDR2 of M45-1 VL: RSNILVSG (sequence number 15).

[0092] CDR3 of VL of M45-1: LQYDEFPYT (sequence number 16).

[0093] Heavy chain of M35-1:

[0094]

number

[0095] M35-1 light chain: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYWTNTRHTGVPSRFSGSGSGTDHTLTISSLQPEDFATYYCQQHYSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 18).

[0096] Heavy chain of M35-2:

[0097]

number

[0098] M35-2 light chain: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYWTNTRHTGVPSRFSGSGSGTDHTLTISSLQPEDFATYYCQQHYSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 20).

[0099] M45-1 Heavy Chain:

[0100]

number

[0101] M45-1 light chain: DIQMTQSPSSLSASVGDRVTITCKASQDINTYLSWFQQKPGKSPKSLIYRSNILVSGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 22).

[0102] CH1 of M35-1 and M45-1 (IgG1S142C format): ASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (sequence number 23).

[0103] CH1 of M35-2 (hereafter IgG2 format): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (sequence number 24).

[0104] Hinge sequences of the heavy chains of M35-1 and M45-1: EPPKSCDKTHTVECPPCP (sequence number 25).

[0105] M35-2 heavy chain hinge sequence: EPPKSDCKTKTVECPPCP (sequence number 26).

[0106] Other examples of heavy chain hinge sequences: CKTHTCPPCP (SEQ ID NO:27) DKTCHTCPPCP (SEQ ID NO:28) ERKSCVECPPCP (SEQ ID NO: 29) EPKSDCKTHTCPPCP (SEQ ID NO:30) EPKSDKCTHTCPPCP (SEQ ID NO:31) EPKSDCKTHTVECPPCP (SEQ ID NO:32) EPKSDCKTVECPPCP (SEQ ID NO:33) EPKSDKCTHTVECPPCP (SEQ ID NO:34) EPPPKSCDKTHTVECPPCP (SEQ ID NO: 35) EPPPPKSCDKTHTVECPPCP (SEQ ID NO:36) EPPKSDCKTKTVECPPCP (sequence number 37). [Brief description of the drawings]

[0107] [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] Results of production and physicochemical testing of certain antibodies according to embodiments of the invention: SDS-PAGE analysis of reduced (R) and non-reduced (NR) antibodies: M35-1 antibody (a), M35-2 antibody (b) and M45-1 antibody (c); Purity and yield of antibodies produced in HEK293 cells (d); SEC-HPLC analysis of purified antibodies: M35-1 antibody (e), M35-2 antibody (f) and M45-1 antibody (g). [Diagram 3] HIC spectra of ADCs prepared with M35-1, M35-2, and M45-1 antibodies: (a) M35-1-MMAE, (b) M35-2-MMAE, and (c) M45-1-MMAE. [Figure 4] Binding curves of anti-CD73 naked antibodies and their corresponding ADCs to CD73: (a) M35-1 antibody and M35-1 MMAE; (b) M35-2 antibody and M35-2-MMAE. [Diagram 5] Internalization of anti-CD73 naked antibodies and their corresponding ADCs in CD73-expressing cancer cells. a: NCI-H1975 cells, b: NCI-H411 cells. [Figure 6]CD73 cytotoxicity curves of M35-1-MMAE, M35-2-MMAE, and M45-1-MMAE against CD73-expressing cancer cells: a: U87MG cells, b: NCI-H1975 cells, c: NCI-N87 cells, and d: NCI-H292 cells. [Figure 7] In vivo drug potency of M35-1-MMAE and M35-2-MMAE in athymic nude mice xenografted with U87 glioma, NCI-N87 gastric carcinoma and NCI-H441 NSCLC.

Claims

1. 1. 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 containing any one of the amino acid sequences set forth in SEQ ID NOs: 25 to 26, 32, and 34 to 36; and (a2) a variable domain containing the amino acid sequence set forth in SEQ ID NO:1; Each light chain is (a3) a variable domain containing the amino acid sequence set forth in SEQ ID NO:2; and the antibody specifically binds to human CD73 protein; An isolated antibody or antigen-binding portion thereof.

2. 1. An isolated antibody, or antigen-binding portion thereof, comprising two heavy chains and two light chains, Each heavy chain is (b1) a heavy chain hinge region containing any one of the amino acid sequences set forth in SEQ ID NOs: 25 to 26; and (b2) a variable domain containing the amino acid sequence set forth in SEQ ID NO:3; Each light chain is (b3) a variable domain containing the amino acid sequence set forth in SEQ ID NO:4; and the antibody specifically binds to human CD73 protein; An isolated antibody or antigen-binding portion thereof.

3. 3. The isolated antibody or antigen-binding portion thereof of claim 1 or 2, wherein each heavy chain further comprises a human CH1 domain located upstream of and linked to the hinge region, and wherein the CH1 domain comprises a cysteine ​​at position 142 according to the IMGT numbering scheme.

4. The antibody or antigen-binding portion thereof according to claim 3, wherein the amino acid sequence contained in the heavy chain hinge region is SEQ ID NO:

25.

5. The antibody or antigen-binding portion thereof according to claim 3, wherein the amino acid sequence contained in the heavy chain hinge region is SEQ ID NO:

26.

6. The antibody or antigen-binding portion thereof according to any one of claims 3 to 5, wherein the CH1 domain of the antibody has the same sequence as the CH1 domain of a native human IgG2, IgG3, or IgG4 subclass antibody.

7. The antibody or antigen-binding portion thereof according to any one of claims 3 to 5, wherein the CH1 domain of said antibody has the sequence of the CH1 domain of a native human IgG1 antibody and carries the S142C mutation.

8. 6. The antibody or antigen-binding portion thereof of any one of claims 3 to 5, wherein each heavy chain comprises an Fc domain of a native human IgG1, IgG2, IgG3, IgG4 subclass antibody, said Fc domain being located downstream of and linked to said hinge region, wherein said Fc domain optionally comprises one or more substitutions. (a) each of the heavy chains comprises the amino acid sequence set forth in SEQ ID NO:17 and each of the light chains comprises the amino acid sequence set forth in SEQ ID NO:18; or (b) the antibody or antigen-binding portion thereof of claim 1, wherein each of the heavy chains comprises an amino acid sequence set forth in SEQ ID NO: 19 and each of the light chains comprises an amino acid sequence set forth in SEQ ID NO:

20.

10. An antibody or antigen-binding portion thereof described in claim 2, wherein each heavy chain comprises an amino acid sequence shown in SEQ ID NO:21 and each light chain comprises an amino acid sequence shown in SEQ ID NO:

22.

11. An antibody-drug conjugate (ADC) or a pharma- ceutically acceptable salt thereof, comprising the antibody of any one of claims 1 to 10 conjugated to a cytotoxic drug by a chemical linker.

12. The cytotoxic drug 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, duocarmycin, doxorubicin, tylanstatin A, uncialamycin, amanitin, ricin, diphtheria toxin, １３１ 12. The ADC or pharma- ceutically acceptable salt thereof of claim 11, wherein the ADC is selected from the group consisting of: I, interleukins, tumor necrosis factors, chemokines, irinotecan (SN38), and exotecan.

13. 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 13. The ADC or a pharma- ceutically acceptable salt thereof according to claim 11 or 12, wherein the ADC 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.

14. 13. The ADC or a pharma- ceutically acceptable salt thereof of claim 11 or 12, wherein the cytotoxic agent is MMAE.

15. A pharmaceutical composition comprising the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 10, or the ADC or a pharma- ceutically acceptable salt thereof of any one of claims 11 to 14, and a pharma- ceutically acceptable carrier.

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