Anti-CD70 antibody-drug conjugates

Abstract

The present invention relates to anti-CD70 antibodies and antibody-drug conjugates comprising at least one non-naturally encoded amino acid. Disclosed herein are anti-CD70 antibodies with one or more non-naturally encoded amino acids, as well as antibody-drug conjugates in which the anti-CD70 antibodies of the invention are conjugated to one or more toxins. Disclosed further are methods for using such non-naturally encoded amino acid-containing antibody-drug conjugates, including therapeutic, diagnostic, and other biotechnology applications.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 480,091, filed January 16, 2023, and U.S. Provisional Patent Application No. 63 / 492,848, filed March 29, 2023, each of which is incorporated by reference herein in its entirety.

[0002] (Reference to sequence listing) This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on January 14, 2024, is named AMBX_0242_00_PCT.xml, and is 14,309 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to anti-CD70 antibodies and antibody-drug conjugates comprising at least one non-naturally encoded amino acid. Disclosed herein are anti-CD70 antibodies with one or more non-naturally encoded amino acids, as well as antibody-drug conjugates in which the anti-CD70 antibodies of the invention are conjugated to one or more cytotoxic drug-linker moieties. Disclosed further are methods for using such non-natural amino acid antibody-drug conjugates, including therapeutic, diagnostic, and other biotechnology applications. [Background technology]

[0004] Cluster of differentiation 70 (CD70) is a member of the tumor necrosis factor superfamily and a ligand for CD27 (Goodwin, R.G. et al., Cell, 73:447-456 (1993); Hintzen, R.Q. et al., Int Immunol, 6:477-480 (1994)). CD70 was first identified on activated T and B lymphocytes. Ligation of CD70 and CD27 on activated lymphocytes signals the costimulation of T cells, B cells, and natural killer (NK) cells (Grewal, I. S., Expert Opin Ther Targets, 12(3):341--351(2008); Borst, J. et al., Curr Opin Immunol., 17(3):275-281(2005)), and regulates cell differentiation and T helper 1 / 2 switching (Wajant, H., Expert Opin Ther Targets, 20(8):959-973(2016)).

[0005] The primary amino acid sequence of CD70 predicts it to be a transmembrane type II protein, with its carboxyl terminus exposed extracellularly and its amino terminus found on the cytoplasmic side of the plasma membrane. Human CD70 consists of a 20-amino acid cytoplasmic domain, an 18-amino acid transmembrane domain, and a 155-amino acid extracytoplasmic domain with two potential N-linked glycosylation sites (Bowman et al., J Immunol, 152:1756-1761 (1994); Goodwin et al., Cell, 73:447-456 (1993)).

[0006] CD70 expression has been reported in different types of cancer, including lymphomas, carcinomas, and tumors of neural origin. Among malignant B cells, 71% of diffuse large B-cell lymphomas, 33% of follicle center lymphomas, 25% of mantle lymphomas, and 50% of B-CLLs have been reported to express CD70 (Lens et al., 1999, Br J Haematol, 106:491-503). CD70 has also been detected in brain tumor cells, particularly glioma cell lines, solid human gliomas, and meningiomas (Held-Feindt and Mentlein, Int J Cancer, 98:352-56 (2002); Wischlusen et al., Can Res, 62:2592-2599 (2002)). CD70 is frequently expressed in renal cell carcinoma (RCC, 87%) and non-Hodgkin's lymphoma (NHL, 77%) (Tannir, NM et al., Invest New Drugs, 32(6):1246-1257 (2014)), but is minimally expressed in normal tissues (Nakae, R. et al., Am J Obstet Gynecol., 224(2):197 (2021)).

[0007] Anti-CD70 antibodies and antibody-drug conjugates (ADCs), and methods for making and using them to treat diseases such as cancer, are disclosed in WO 2013 / 192360 A1, the entire contents of which are incorporated herein by reference in their entirety.

[0008] Multiple clinical trials evaluating anti-CD70 agents (e.g., antibodies with enhanced antibody-dependent cell-mediated cytotoxicity, ADCs, and chimeric antigen receptor (CAR) T-cell therapy) are being investigated in malignancies that exhibit high CD70 expression. Previous studies have shown that anti-CD70 monoclonal antibodies (mAbs) and anti-CD70 ADCs exhibit anti-tumor effects in xenograft models of CD70-positive malignancies, such as lymphoma, NHL, and RCC (Israel, BF et al., Mol Cancer Ther., 4(12):2037-2044 (2005); Law, CL et al., Cancer Res., 66:2328-2337 (2006); McEarchern, JA et al., Blood, 109(3):1185-92 (2007)).

[0009] Based on the results of previous clinical studies, two separate phase 1 trials of SGN-75 (an anti-CD70 mAb conjugated to maleimidocaproyl-monomethyl auristatin F (MMAF)) were conducted in patients with CD70-positive relapsed / refractory NHL or metastatic RCC, but SGN-75 showed modest efficacy against these diseases and had some intolerable side effects (Tannir, NM et al., Invest New Drugs, 32(6):1246-1257(2014)). An additional anti-CD70 ADC, SGN-CD70A (an anti-CD70 mAb conjugated to a pyrrolobenzodiazepine dimer), was introduced into a Phase 1 clinical trial (Pal, SK et al., Cancer, 125(7):1124-1132(2019)), but the Phase 1 trial of SGN-CD70A was discontinued in 2018.

[0010] Thus, there remains a need for anti-CD70 ADCs that are capable of exerting clinically useful cytotoxic, cytostatic, or immunosuppressive effects on CD70-expressing cells, and that are specifically engineered to avoid undesirable effects on non-CD70-expressing cells. Such ADCs would be useful therapeutic agents for CD70-expressing cancers or immune disorders mediated by CD70-expressing cells. The present invention provides such ADCs for use in immunology and oncology. Summary of the Invention

[0011] Disclosed herein are antibody drug conjugates (ADCs) comprising an anti-CD70 antibody attached to a drug-linker moiety via one or more unnatural amino acids, and methods for making such ADCs. Methods of treating diseases or conditions through administration of an ADC of this disclosure or a composition comprising an ADC of this disclosure are also described.

[0012] In some general aspects, the disclosure provides antibody drug conjugates (ADCs), the ADCs comprising: The following structure:

[0013] [ka] and a drug-linker group having an anti-CD70 antibody or fragment thereof comprising one or more heavy chains, wherein: at least one member of the one or more heavy chains comprises a sequence comprising SEQ ID NO:3;

[0014] [ka] represents a single bond or a double bond, # denotes connection to an anti-CD70 antibody or fragment thereof.

[0015] In some embodiments, the drug-linker group is one or more drug linker groups.

[0016] In some other general aspects, an ADC is provided, the ADC comprising: The following structure:

[0017] [ka] and one or more drug-linker groups having an anti-CD70 antibody or fragment thereof comprising one or more heavy chains, wherein at least one of the one or more heavy chains comprises an amino acid sequence that contains a first non-naturally encoded amino acid, the amino acid sequence being selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; During the ceremony, each

[0018] [ka] represents a single or double bond covalently attaching one of the one or more drug-linker groups to the anti-CD70 antibody or fragment thereof, and each # represents a site of attachment to the anti-CD70 antibody or fragment thereof.

[0019] In some embodiments, at least one of the one or more heavy chains comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the heavy chain amino acid sequence is SEQ ID NO: 3.

[0020] In some other embodiments, at least one of the one or more heavy chains comprises the amino acid sequence of SEQ ID NO:4.

[0021] In some other embodiments, at least one of the one or more heavy chains comprises the amino acid sequence of SEQ ID NO:5.

[0022] In some embodiments, the ADC further comprises one or more light chains, in some embodiments, at least one of the one or more light chains comprises an amino acid sequence that shares at least 90% identity with SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0023] In some embodiments, at least one of the one or more light chains comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, at least one of the one or more light chains is the amino acid sequence of SEQ ID NO: 2. In some embodiments, each of the one or more light chains is the amino acid sequence of SEQ ID NO: 2.

[0024] In some other embodiments, at least one of the one or more light chains comprises an amino acid sequence containing a second non-naturally encoded amino acid, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0025] In some embodiments, the anti-CD70 antibody or fragment thereof comprises two heavy chains, one heavy chain comprising a first non-naturally encoded amino acid and the other heavy chain comprising a third non-naturally encoded amino acid. In some embodiments, the one heavy chain and the other heavy chain each comprise the amino acid sequence of SEQ ID NO: 3. In some embodiments, the one heavy chain and the other heavy chain each is SEQ ID NO: 3.

[0026] In some embodiments, each

[0027] [ka] represents a double bond. In some embodiments, each

[0028] [ka] covalently attaches one of the one or more drug-linker groups to a non-naturally encoded amino acid of an anti-CD70 antibody or fragment thereof.

[0029] In some embodiments, each of the first non-naturally encoded amino acid, the second non-naturally encoded amino acid, and the third non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

[0030] In some embodiments, the ADC has formula (I):

[0031] [ka] ADC of During the ceremony, The ADC comprises one or more drug-linker groups, Ab is an anti-CD70 antibody or fragment thereof; each R is independently an unsubstituted C1-C8 alkyl; d is an integer from 1 to 10, Each of the one or more drug-linker groups has the following structure:

[0032] [ka] and where each # represents a connection site to anti-CD70 or a fragment thereof.

[0033] In some embodiments, d is 1, 2, 3, or 4.

[0034] In some embodiments, each R is methyl.

[0035] In some embodiments, the anti-CD70 antibody, or fragment thereof, comprises two heavy chains, and each heavy chain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, each heavy chain amino acid sequence is SEQ ID NO: 3.

[0036] In some embodiments, the anti-CD70 antibody, or fragment thereof, comprises two light chains, and each light chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, each light chain amino acid sequence is SEQ ID NO: 2.

[0037] In some embodiments, the anti-CD70 antibody or fragment thereof is humanized.

[0038] In some embodiments, the anti-CD70 antibody or fragment thereof is a humanized monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 3 and each light chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD70 antibody or fragment thereof is a humanized monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain amino acid sequence is SEQ ID NO: 3 and each light chain amino acid sequence is SEQ ID NO: 2.

[0039] In some embodiments, the amino acid sequence of SEQ ID NO: 3 comprises one non-naturally encoded amino acid, and the one non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

[0040] In some embodiments, d is 2.

[0041] In some embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains, two light chains, and two non-naturally encoded amino acids; each of the two heavy chains comprises the amino acid sequence of SEQ ID NO:3, wherein SEQ ID NO:3 contains one non-naturally encoded amino acid, wherein the one non-naturally encoded amino acid in SEQ ID NO:3 is para-acetyl-L-phenylalanine at position A114 (Kabat numbering) of SEQ ID NO:3; each of the light chains comprises the amino acid sequence of SEQ ID NO:2, wherein each R is methyl; the one or more Drug-Linker groups are two Drug-Linker groups; d is 2, Each of the two drug-linker groups is attached to para-acetyl-L-phenylalanine at position A114 of SEQ ID NO:3, thereby attaching each of the two drug-linker groups to the humanized anti-CD70 monoclonal antibody.

[0042] In some embodiments, each heavy chain amino acid sequence is SEQ ID NO: 3. In some embodiments, each light chain amino acid sequence is SEQ ID NO: 2.

[0043] In some embodiments, the non-naturally encoded amino acid is a single non-naturally encoded amino acid.

[0044] In some embodiments, the ADC of the disclosure is the ADC of FIG.

[0045] In some general aspects, the disclosure provides compositions comprising an ADC of the disclosure. In some embodiments, the composition comprising an ADC further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent, a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, or an immunomodulatory agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a checkpoint inhibitor, a CD70 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, a hypomethylating agent, or a platinum-based therapeutic agent, or a combination thereof. In some embodiments, the composition comprising an ADC, and optionally further comprising an additional therapeutic agent, is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0046] In some other general aspects, this disclosure provides methods of killing a cell, the method comprising contacting the cell with an effective amount of an ADC of this disclosure, or a composition comprising an effective amount of an ADC of this disclosure, or more particularly, a formulation comprising an effective amount of an ADC of this disclosure. In some embodiments, the cell is a tumor cell or a cancer cell. In some embodiments, the tumor cell or cancer cell is a renal cell carcinoma (RCC) tumor cell or cancer cell.

[0047] In some other general aspects, the disclosure provides methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an ADC of the disclosure, a composition comprising an effective amount of an ADC of the disclosure, or a formulation comprising an effective amount of an ADC of the disclosure. In some embodiments, the disease or condition is a tumor or cancer. In some embodiments, the tumor or cancer is a solid tumor. In some other embodiments, the tumor or cancer is a hematological cancer. In some embodiments, the hematological cancer is lymphoma, multiple myeloma, or leukemia. In some embodiments, the tumor or cancer is kidney cancer, brain cancer, breast cancer, Burkitt's lymphoma, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, testicular cancer, prostate cancer, colorectal cancer, esophageal cancer, bladder cancer, lung cancer, non-small cell lung cancer, urothelial carcinoma, cholangiocarcinoma, colorectal carcinoma, pancreatic cancer, renal cell carcinoma, nasopharyngeal carcinoma, mantle cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, or acute myeloid leukemia. In some embodiments, the tumor or cancer is renal cell carcinoma, brain cancer, multiple myeloma, mantle cell lymphoma, or lung cancer. In some embodiments, the tumor or cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma. In some embodiments, the tumor or cancer is a multidrug-resistant tumor or cancer. In some embodiments, a method comprises treating the subject with radiation therapy. In some embodiments, the method includes treating the subject with an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, an immunomodulatory agent, or an immunotherapeutic agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a checkpoint inhibitor, a CD70 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, a hypomethylating agent, or a platinum-based therapeutic agent, or a combination thereof. In some embodiments, the additional agent is a checkpoint inhibitor, and the checkpoint inhibitor is a PD-1 inhibitor.In some embodiments, the PD-1 inhibitor is AMP-224, atezolizumab, avelumab, BMS-936558, BMS-936559, CT-001, durvalumab, MED10680, nivolumab, PDR001, pembrolizumab, pidilizumab, and REGN2810. In some embodiments, the PD-1 inhibitor is an antibody. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the method improves or optimizes cancer cell killing. In some embodiments, the method delays tumor or cancer progression or recurrence. In some embodiments, the tumor or cancer is a CD70-expressing cancer. In some embodiments, the CD70-expressing cancer has at least about 5,000 CD70 / cell. In some embodiments, the CD70-expressing cancer has at least about 10,000 CD70 / cell. In some embodiments, the CD70-expressing cancer has at least about 15,000 CD70 / cell. In some embodiments, the subject, cancer, or tumor is resistant or refractory to previous standard therapy. In some embodiments, the subject has cancer metastasis from the same cancer or a different cancer. In some other embodiments, the disease or condition being treated is myelodysplastic syndrome. In some embodiments, the subject being treated for myelodysplastic syndrome is undergoing treatment with a hypomethylating agent or has previously undergone treatment with a hypomethylating agent.

[0048] In some embodiments, the subject being treated for a disease or condition, including a tumor or cancer, is a human subject. In some embodiments, the human subject is an adult.

[0049] In some embodiments, an effective amount of an ADC for administration to a human subject is a dose in the range of about 0.05 mg / kg to about 10 mg / kg of a human subject, or any value therebetween. In some embodiments, an effective amount of an ADC is a dose in the range of about 0.05 mg / kg to about 2 ..., about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, about 0.2 mg / kg, about 0.22 mg / kg, about 0.24 mg / kg, about 0.26 mg / kg, about 0.28 mg / kg, about 0.3 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 24 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg 2mg / kg, approx. 0.34mg / kg, approx. 0.36mg / kg, approx. 0.38mg / kg, approx. 0.4mg / kg, approx. 0.42mg / kg, approx. 0.44mg / kg, approx. 0.46mg / kg, about 0.48mg / kg, about 0.5mg / kg, about 0.52mg / kg, about 0.54mg / kg, about 0.56mg / kg, about 0.58mg / kg, about 0.6mg / kg, about 0.62mg / kg, about 0.64mg / kg, about 0.66mg / kg, about 0.68mg / kg, about 0.7mg / kg, about 0.72mg / kg, about 0.74mg / kg, about 0.7 6mg / kg, approx. 0.78mg / kg, approx. 0.8mg / kg, approx. 0.82mg / kg, approx. 0.84mg / kg, approx. 0.86mg / kg, approx. 0.88mg / kg, approx. 0.9mg / k g, about 0.92 mg / kg, about 0.94 mg / kg, about 0.96 mg / kg, about 0.98 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, or about 2 mg / kg. In some other embodiments, an effective amount of the ADC is a dose within the range of about 2 mg / kg to about 5 mg / kg for a human subject, or any value therebetween.

[0050] In some embodiments, an effective amount of an ADC is a dose of about 2 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.6 mg / kg, about 3.8 mg / kg, about 4 mg / kg, about 4.2 mg / kg, about 4.4 mg / kg, about 4.6 mg / kg, about 4.8 mg / kg, or about 5 mg / kg for a human subject. In some other embodiments, an effective amount of an ADC is a dose within the range of about 5 mg / kg to about 10 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of an ADC is a dose of about 5 mg / kg, about 5.2 mg / kg, about 5.4 mg / kg, about 5.6 mg / kg, about 5.8 mg / kg, about 6 mg / kg, about 6.2 mg / kg, about 6.4 mg / kg, about 6.6 mg / kg, about 6.8 mg / kg, about 7 mg / kg, about 7.2 mg / kg, about 7.4 mg / kg, about 7.6 mg / kg, about 7.8 mg / kg, about 8 mg / kg, about 8.2 mg / kg, about 8.4 mg / kg, about 8.6 mg / kg, about 8.8 mg / kg, about 9 mg / kg, about 9.2 mg / kg, about 9.4 mg / kg, about 9.6 mg / kg, about 9.8 mg / kg, or about 10 mg / kg for a human subject.

[0051] In some embodiments, the ADC, composition, or formulation is administered orally, intradermally, intratumorally, intravenously, or subcutaneously, hi some embodiments, the ADC, composition, or formulation is administered intravenously.

[0052] In some embodiments, administration of an effective amount of an ADC occurs in a dosing schedule. In some embodiments, the dosing schedule is once every 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, the dosing schedule is once every 2 weeks. In some embodiments, the dosing schedule is once every 3 weeks. In some embodiments, the dosing schedule is once every 4 weeks. In some embodiments, the dosing schedule is two or more times in a 3-week cycle. In some embodiments, dosing is at least once every 4 weeks for at least about 8 weeks. In some embodiments, dosing is once every 3 weeks for at least about 8 weeks.

[0053] In some other general aspects, the disclosure provides an isolated anti-CD70 antibody or fragment thereof comprising an amino acid sequence selected from the group consisting of the sequences listed in Table 1. In some embodiments, the isolated anti-CD70 antibody or fragment thereof comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the heavy chain amino acid sequence is SEQ ID NO: 3. In some embodiments, the isolated anti-CD70 antibody or fragment thereof comprises a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the light chain amino acid sequence is SEQ ID NO: 2. In some embodiments, the isolated anti-CD70 antibody or fragment thereof comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the isolated anti-CD70 antibody or fragment thereof comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the isolated anti-CD70 antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 3 and the light chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated anti-CD70 antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence is SEQ ID NO:3 and the light chain amino acid sequence is SEQ ID NO:2.

[0054] In some other general aspects, the disclosure provides nucleic acids encoding any one of SEQ ID NO:1 through SEQ ID NO:9.

[0055] In some other general aspects, the disclosure provides a vector comprising a nucleic acid encoding any one of SEQ ID NO:1-SEQ ID NO:9.

[0056] In some other general aspects, the disclosure provides use of an ADC of any one of claims 1-20, or an antibody or fragment thereof comprising an amino acid sequence listed in Table 1, in the manufacture of a medicament for the treatment of a disease or condition in a subject.

[0057] In some other aspects, the disclosure provides formulations comprising an ADC of the disclosure. In some embodiments, the ADC concentration in the formulation is in the range of about 5 mg / mL to about 25 mg / mL. In some embodiments, the formulation further comprises a buffer, sucrose, and a surfactant. In some embodiments, the formulation comprises an ADC, a histidine buffer, sucrose, and polysorbate 80. In some embodiments, the formulation has a pH in the range of about 5.4 to about 6.4. In some embodiments, the formulation has a pH in the range of about 5.2 to about 6.2. In some embodiments, the pH of the formulation is about 6 or less. In some embodiments, the pH of the formulation is less than 6.

[0058] In some embodiments, the formulation is a liquid formulation. In some embodiments, the liquid formulations of the present disclosure can be stored at room temperature. In some other embodiments, the liquid formulations can be stored frozen.

[0059] In some other embodiments, the formulation is a lyophilized pharmaceutical product. In some embodiments, the lyophilized pharmaceutical product, when reconstituted with a diluent, provides a reconstituted solution containing the ADC at a concentration in the range of about 5 mg / mL to about 25 mg / mL. In some embodiments, the reconstituted solution further comprises an L-histidine buffer at a concentration in the range of about 10 mM to about 50 mM, sucrose at a concentration in the range of about 1% (w / v) to about 20% (w / v), and polysorbate 80 at a concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the reconstituted solution has a pH in the range of about 5.4 to about 6.4. In some embodiments, the reconstituted solution has a pH in the range of about 5.2 to about 6.2. In some embodiments, the pH of the reconstituted solution is about 6 or less. In some embodiments, the pH of the reconstituted solution is less than 6. In some embodiments, the diluent is water.

[0060] In some other aspects, the present disclosure provides a formulation comprising about 5 mg / mL to about 15 mg / mL of an ADC of the present disclosure, about 15 mM to about 25 mM histidine buffer, about 5% (w / v) to about 15% (w / v) sucrose, and about 0.01% (w / v) to about 0.05% (w / v) polysorbate 80, wherein the pH of the formulation is about 5.4 to about 6.0. In some embodiments, the formulation comprises about 10 mg / mL of an ADC, about 20 mM histidine buffer, about 8% (w / v) sucrose, and about 0.02% (w / v) polysorbate 80, wherein the pH of the formulation is about 5.7.

[0061] It is understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, as such may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims.

[0062] Incorporation by Reference All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, and for the purpose of describing and disclosing, for example, the chemistry, chemical syntheses, compositions, and other methodology described in the publications that might be used in connection with the invention(s) described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. [Brief explanation of the drawings]

[0063] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Figure 1] An exemplary antibody drug conjugate (ADC) of the invention is depicted containing a drug-linker (the cytotoxic tubulin inhibitor AS269) site-specifically conjugated to an anti-CD70 monoclonal antibody (one drug-linker payload per heavy chain). [Figure 2] FIG. 1 shows a graphical representation of HPLC chromatograms of unconjugated anti-CD70 mAb (DAR=0) and anti-CD70-AS269 ADC (DAR=1 and DAR=2) after 24 hours of conjugation reaction time (see Example 5). [Figure 3] FIG. 1 shows a graphical representation of anti-CD70-AS269 ADC stability in mouse plasma over 7 days of incubation at 37° C. (see Example 6). [Figure 4] FIG. 1 shows a graphic representation of intact ADC (DAR=2) and total antibody (unconjugated and conjugated species) concentrations of anti-CD70-AS269 ADC, as well as total antibody (TA) concentrations of unconjugated mAb in the blood of mice following a single dose of 1 mg / kg of anti-CD70-AS269 ADC (see Example 7). [Figure 5]FIG. 1 shows a graphic representation of the mean tumor volume in mice following a single dose of unconjugated anti-CD70 mAb or anti-CD70-AS269 ADC in a 786-O S3 renal cell carcinoma (RCC) xenograft model (see Example 8). [Figure 6] FIG. 1 shows a graphic representation of the mean tumor volume in mice treated with sunitinib (QD×35) or anti-CD70-AS269 ADC (QW×5) in a 786-O S3 RCC xenograft model (see Example 9). [Figure 7] FIG. 1 shows a graphic representation of tumor volume in mice treated with three different doses of anti-CD70-AS269 ADC (“aCD70-AS269”, QWx5) in a Caki-1 RCC xenograft model (see Example 10). [Figure 8] 1 shows dot plots of final tumor volumes from mice treated with three different doses of anti-CD70-AS269 ADC ("aCD70-AS269", QWx5), measured after Caki-1 tumor implantation (see Example 11). [Figure 9] FIG. 1 shows a graphic representation of the viability of MDR-positive 786-0 cells treated with various concentrations of anti-CD70-AS269 ADC (“aCD70-AS269”) alone or in the presence of verapamil or elacridar, or treated with anti-CD70-MMAE ADC alone or in the presence of verapamil or elacridar (see Example 12). [Figure 10] FIG. 1 shows serum Ig lambda (released by U266 cells) concentrations correlating with tumor burden in mice treated with a single dose of anti-CD70-AS269 ADC (“aCD70-AS269”) in the U266 multiple myeloma model (see Example 13). [Figure 11] FIG. 1 shows a graphic representation of survival curves in the U266 multiple myeloma mouse model after a single injection of anti-CD70-AS269 or unconjugated mAb (see Example 13). [Figure 12]FIG. 1 shows a graphic representation of CD27 signaling inhibition by anti-CD70-AS269 ADC (“aCD70-AS269”) in a CD27 reporter / Caki-1 co-culture assay (see Example 14). [Figure 13] 1 shows the affinity of anti-CD70-AS269 ADCs for human, cynomolgus monkey, rat, and mouse CD70 as measured by surface plasmon resonance. [Figure 14] 1 shows a graphic representation of anti-CD70-AS269 ADC toxicokinetic and pharmacokinetic concentration-time curves, demonstrating a clear therapeutic index (see Example 17). [Figure 15] 1 shows tumor volume measurements in the 786-OS3 / PBMC model several days after treatment with ARX305, pembrolizumab, or a combination of ARX305 and pembrolizumab. [Figure 16] 1 shows body weight measurements in the 786-OS3 / PBMC model after several days of treatment with ARX305, pembrolizumab, or a combination of ARX305 and pembrolizumab. DETAILED DESCRIPTION OF THE INVENTION

[0064] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular methodology, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0065] While various embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention, and it is intended that methods and structures within the scope of the claims and their equivalents be covered thereby.

[0066] definition Unless otherwise defined here or in the remainder of the specification below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs. Various methods, materials, and the like similar or equivalent to those described herein can be used in the practice or testing of the invention described herein.

[0068] The term "alkyl," as used herein, by itself or as part of another molecule, means, unless otherwise stated, a straight-chain or branched-chain or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and which can include divalent and polyvalent radicals, having the specified number of carbon atoms (i.e., C1-C6). 10 means 1 to 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

[0069] The term "alkylene," as used herein, by itself or as part of another molecule, means a divalent radical derived from an alkane, (-CH-) n where n can be from 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms, such as the structures -CH2CH2- and -CH2CH2CH2CH2-. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkylene" is also meant to include groups described herein as "heteroalkylene," unless otherwise specified.

[0070] The term "amino acid," as used herein, refers to naturally occurring and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but still retain the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0071] Amino acids may be referred to herein by either their names, their commonly known three letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides may be referred to by their commonly accepted single-letter codes.

[0072] The term "antibody," as used herein, refers to a protein consisting of one or more polypeptides substantially encoded by all or part of antibody genes. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Antibodies, as used herein, are meant to include full-length antibodies and antibody fragments, including naturally occurring or engineered antibodies (e.g., variants) in any organism. Thus, unless otherwise specified, descriptions and claims using the term "antibody" specifically include "antibody fragments." The term "antibody" encompasses intact, monoclonal, and polyclonal antibodies. The term "antibody" also encompasses multispecific antibodies, such as bispecific antibodies. Human antibodies are typically made from two light chains and two heavy chains, each containing a variable region and a constant region. The light chain variable region contains three CDRs, identified herein as CDRL1, CDRL2, and CDRL3, flanked by framework regions. The heavy chain variable region comprises three CDRs, identified herein as CDRH1, CDRH2 and CDRH3, flanked by framework regions.

[0073] Anti-CD70 antibodies known in the art are suitable for use with the present invention. Any known heavy chain sequence can be combined with a light chain sequence, and in some embodiments of the present invention, a non-naturally encoded amino acid is present in the antibody constant region. Accordingly, in one aspect, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising: (a) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; (b) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and (c) a non-naturally encoded amino acid in the heavy chain, the light chain, or both, wherein the antibody specifically binds to CD70. In some embodiments, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine (pAF). In another aspect, the disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising: (a) a heavy chain consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; (b) a light chain consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and (c) a non-naturally encoded amino acid in the heavy chain, the light chain, or both, wherein the antibody specifically binds to CD70. In some aspects, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine (pAF). In another aspect, the disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising: (a) a heavy chain consisting of the amino acid sequence of SEQ ID NO:3; (b) a light chain consisting of the amino acid sequence of SEQ ID NO:2; and (c) a non-naturally encoded amino acid in the heavy chain, the light chain, or both, wherein the antibody specifically binds to CD70. In some aspects, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine (pAF). Anti-CD70 (aCD70) antibodies known in the art are suitable for use in the present invention.

[0074] The term "antigen-binding fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) V L , V H , C L and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and C H1 (iv) a Fd fragment consisting of a V domain of a single arm of an antibody; L and V H Fv fragment consisting of domains, (v) V H (Ward et al., (1989) Nature 341:544-546), (vi) a dAb fragment consisting of, for example, V, with or without additional sequences (linker, framework regions, etc.) H (v) a combination of two to six isolated CDRs, with or without additional sequences (such as linkers, framework regions, etc.). Furthermore, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but using recombinant methods, V L and V HThey can be linked by a synthetic linker that allows the regions to be produced as a single polypeptide chain that pairs to form a monovalent molecule (also known as single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Furthermore, an antigen-binding fragment can be any fragment comprising: (i) a binding domain polypeptide (e.g., a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (iii) an immunoglobulin heavy chain CH2 constant region fused to the CH2 constant region. and a binding domain immunoglobulin fusion protein comprising a globulin heavy chain CH3 constant region. The hinge region may be modified by replacing one or more cysteine ​​residues with serine residues to prevent dimerization. Such binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0075] A typical antigen-binding site consists of a variable region formed by the pairing of a light immunoglobulin chain and a heavy immunoglobulin chain. The structures of antibody variable regions are highly consistent and exhibit very similar structures. These variable regions typically consist of relatively homologous framework regions (FRs) interspersed with three hypervariable regions called complementarity-determining regions (CDRs). The overall binding activity of an antigen-binding fragment is often determined by the sequences of the CDRs. The FRs often play a role in the proper three-dimensional positioning and alignment of the CDRs for optimal antigen binding.

[0076] Indeed, because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies exhibiting the characteristics of a particular naturally occurring antibody by constructing an expression vector containing CDR sequences from that particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. See. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences may differ from mature antibody gene sequences because they may not contain fully assembled variable genes formed by V(D)J joining during B-cell maturation. Germline gene sequences also contain mutations throughout the variable genes, but may differ from the sequences of high-affinity secondary repertoire antibodies, which are typically clustered in the CDRs. For example, somatic mutations are relatively rare in the amino-terminal portion of framework region 1 and the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of an antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody to regenerate an intact recombinant antibody with binding properties similar to those of the original antibody. Partial heavy and light chain sequences spanning the CDR regions are typically sufficient for this purpose. Partial sequences are used to determine which germline variable segments and joining gene segments contributed to the recombinant antibody variable genes. The germline sequences are then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized to create a completely synthetic variable region clone.This process has certain advantages, such as the elimination or inclusion of certain restriction sites or the optimization of certain codons.

[0077] Of course, all or part of the framework regions of the antibodies described herein may be used in conjunction with the CDRs to optimize antibody affinity, specificity, or any other desired property.

[0078] The term "antibody fragment," as used herein, refers to any form of an antibody other than the full-length form. Antibody fragments, as used herein, include smaller components of a full-length antibody and engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is the single-chain Fv (scFv), which consists of the variable regions of immunoglobulin heavy and light chains covalently connected by a peptide linker (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules.

[0079] The term "antibody drug conjugate" or "ADC," as used herein, refers to an antibody molecule, or fragment thereof, covalently linked to one or more biologically active molecules. The biologically active molecules may be conjugated to the antibody via a linker, polymer, or other covalent bond.

[0080] As used herein, the terms "ARX305," "anti-CD70-AS269," or "anti-CD70-AS269 ADC" refer to an ADC containing (i) a humanized monoclonal antibody (mAb, subclass IgG1) with specificity for human CD70 and (2) the drug-linker AS269 (see Figure 1). The humanized mAb with specificity for human CD70 comprises two heavy chains and two light chains. The amino acid sequence of each heavy chain is SEQ ID NO:3, which contains one unnatural amino acid, para-acetyl-L-phenylalanine (pAF), that is genetically encoded and biosynthetically incorporated at amino acid position 114 (Kabat numbering) of each heavy chain, and the amino acid sequence of each light chain is SEQ ID NO:2. AS269 is site-specifically conjugated via an oxime bond to pAF incorporated at amino acid position 114 (Kabat numbering) of each anti-CD70 mAb heavy chain (one drug-linker per heavy chain), thereby providing an ARX305 drug-to-antibody ratio (DAR) of about 2. A composition containing the ARX305 ADC (e.g., an ARX305 pharmaceutical formulation including a reconstitution solution) may have a DAR of about 1.9 (see Example 19).

[0081] The term "bifunctional polymer" (also referred to as "bifunctional linker"), as used herein, refers to a polymer containing two functional groups that can specifically react with other moieties to form covalent or non-covalent linkages. Such moieties may include, but are not limited to, natural or unnatural amino acids, or side chain groups on peptides containing such natural or unnatural amino acids. The other moieties that may be linked to the bifunctional linker or bifunctional polymer may be the same or different moieties. By way of example only, a bifunctional linker may have a functional group that is reactive with a group on a first peptide and another functional group that is reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules are known for attaching various compounds to peptides. See European Patent Application No. 188,256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789 (incorporated herein by reference in their entireties). A "multifunctional polymer," also referred to as a "multifunctional linker," refers to a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, side groups on natural or unnatural amino acids, or peptides containing such natural or unnatural amino acids (including, but not limited to, amino acid side groups), to form covalent or noncovalent linkages. Bifunctional or multifunctional polymers may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and the molecule or compound to which it is attached.

[0082] The term "bioavailability," as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at a site of action or in the systemic circulation. An increase in bioavailability refers to increasing the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at a site of action or in the systemic circulation. By way of example, an increase in bioavailability may be demonstrated as an increase in the concentration of a substance or its active moiety in the blood when compared to other substances or active moieties. Non-limiting examples of methods for assessing increased bioavailability are provided in Examples 21-25. This method may be used to assess the bioavailability of any polypeptide.

[0083] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active agent" refer to any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction associated with an organism, including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, biologically active molecules include, but are not limited to, any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance the physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroidal agents, microbial toxins, and the like.

[0084] The term "modulating biological activity," as used herein, refers to increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, or enhancing or decreasing the substrate selectivity of a polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-naturally occurring polypeptide with that of the naturally occurring polypeptide.

[0085] The term "biosynthetically," as used herein, refers to any method that utilizes a translation system (cellular or non-cellular) that includes the use of at least one of the following components: polynucleotides, codons, tRNAs, and ribosomal. In a non-limiting example, an unnatural amino acid can be "biosynthetically incorporated" into an unnatural amino acid polypeptide using the methods and techniques described in U.S. Patent No. 7,083,970 and U.S. Patent Application Publication No. 2021 / 0017527, the entire contents of each of which are incorporated herein by reference in their entirety.

[0086] The term "carbonyl" as used herein refers to a -C(O)- moiety. Non-limiting examples of groups containing a carbonyl include ketones, aldehydes, carboxylic acids, and esters. Furthermore, the carbonyl can be part of a straight-chain, branched-chain, or cyclic molecule.

[0087] The term "chemically cleavable group" (also called "chemically labile"), as used herein, refers to a group that is destroyed or cleaved upon exposure to an acid, base, oxidizing agent, reducing agent, chemical initiator, or radical initiator.

[0088] The term "chemiluminescent group," as used herein, refers to a group that emits light as a result of a chemical reaction without the addition of heat. By way of example only, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with an oxidizing agent such as hydrogen peroxide (HO) in the presence of a base and a metal catalyst to produce an excited-state product (3-aminophthalate, 3-APA).

[0089] The term "chromophore," as used herein, refers to a molecule that absorbs light in the visible, UV, or IR wavelengths.

[0090] The term "comparison window," as used herein, refers to any one segment of contiguous positions used to compare a sequence to a reference sequence of the same number of contiguous positions after the two sequences have been optimally aligned. Such contiguous positions include, but are not limited to, groups of about 20 to about 600 contiguous units, including about 50 to about 200 contiguous units and about 100 to about 150 contiguous units. By way of example only, such sequences include polypeptides and polypeptides containing unnatural amino acids, where the contiguous units include, but are not limited to, natural and unnatural amino acids. Further, by way of example only, such sequences include polynucleotides having corresponding contiguous nucleotide units. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, but is not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0091] By way of example, algorithms that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1997) Nuc. Acids Res. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M of 5, N of -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Natl. Acad. Sci. USA 89:10915), alignment (B) of 50, an expectation (E) of 10, M of 5, N of -4, and a comparison of both strands. The BLAST algorithm is typically run with the "low complexity" filter turned off.

[0092] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

[0093] The term "conservatively modified variants," as used herein, applies to both natural and non-natural amino acids and natural and non-natural nucleic acid sequences, as well as combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to those natural and non-natural nucleic acids that encode identical or essentially identical natural and non-natural amino acid sequences, or, if the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, to essentially identical sequences. By way of example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Thus, by way of example, every natural or non-natural nucleic acid sequence herein that encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. Those skilled in the art will recognize that each codon (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) in a natural or non-natural nucleic acid can be modified to obtain a functionally identical molecule. Accordingly, each silent variation of natural and non-natural nucleic acids that encode natural and non-natural polypeptides is implicit in each described sequence.

[0094] With respect to amino acid sequences, individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter, add, or delete a single natural and unnatural amino acid, or a small percentage of natural and unnatural amino acids in the encoded sequence, are "conservatively modified variants" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural and unnatural amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0095] Conservative substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman & Co., 2002). nd edition, December 1993).

[0096] As used herein, the term "cytotoxic" refers to a compound that is harmful to cells.

[0097] The term "diamine," as used herein, refers to a group / molecule containing at least two amine functional groups, including, but not limited to, hydrazine, amidine, imine, 1,1-diamine, 1,2-diamine, 1,3-diamine, and 1,4-diamine groups. Furthermore, such groups can be part of a linear, branched, or cyclic molecule.

[0098] The term "drug" as used herein refers to any substance used in the prevention, diagnosis, mitigation, treatment, or cure of a disease or condition.

[0099] The term "effective amount," as used herein, refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, the agent or compound being administered can include, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-amino acid polypeptides can be administered for prophylactic, augmentative, and / or therapeutic treatment. An appropriate "effective" amount in any individual case can be determined using techniques, such as dose escalation studies.

[0100] The terms "enhance" or "enhancing," as used herein, mean to increase or prolong, in either potency or duration, a desired effect. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong, in either potency or duration, the effect of the therapeutic agent during the treatment of a disease, disorder, or condition. An "enhancing-effective amount," as used herein, refers to an amount sufficient to enhance the effect of a therapeutic agent in treating a disease, disorder, or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0101] The term "eukaryote," as used herein, refers to organisms belonging to the phylogenetic domain Eukarya, including, but not limited to, animals (including, but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including, but not limited to, monocotyledons, dicotyledons, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

[0102] The terms "functional group," "active moiety," "activating group," "leaving group," "reactive site," "chemically reactive group," and "chemically reactive moiety," as used herein, refer to the portion or unit of a molecule at which a chemical reaction occurs. These terms are somewhat synonymous in the chemical arts and are used herein to refer to the portion of a molecule that performs a function or activity and is reactive with other molecules.

[0103] The term "identical," as used herein, refers to two or more sequences or subsequences that are the same. Additionally, the term "substantially identical," as used herein, refers to two or more sequences that have a percentage of contiguous units that are the same when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the contiguous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over the designated region. Such percentages describe the "percent identity" of two or more sequences. Sequence identity may exist over a region that is at least about 75-100 contiguous units in length, over a region that is about 50 contiguous units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical if the amino acid residues are the same, but the amino acid residues may be about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, Two or more polypeptide sequences are "substantially identical" if they are about 95% identical or more than about 75-100 amino acids in length, over a region that is about 50 amino acids in length, or, if not specified, over the entire sequence of the peptide sequence. Additionally, by way of example only, two or more polynucleotide sequences are identical if the nucleic acid residues are the same, whereas two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a particular region. Identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, if not specified, over the entire sequence of the polynucleotide sequence.

[0104] In the case of sequence comparison, typically one sequence serves as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0105] The term "immunogenicity," as used herein, refers to the antibody response to administration of a therapeutic agent. Immunogenicity to a therapeutic non-natural amino acid polypeptide can be obtained using quantitative and qualitative assays to detect anti-non-natural amino acid polypeptide antibodies in biological fluids. Such assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), luminescent immunoassays (LIA), and fluorescent immunoassays (FIA). Analysis of immunogenicity to a therapeutic non-natural amino acid polypeptide involves comparing the antibody response upon administration of the therapeutic non-natural amino acid polypeptide with the antibody response upon administration of the therapeutic natural amino acid polypeptide.

[0106] The term "isolated," as used herein, refers to the separation and removal of a component of interest from components that are not of interest. An isolated material can be in either a dry or semi-dry state, or in a solution, including, but not limited to, an aqueous solution. An isolated component can be in a homogeneous state, or the isolated component can be part of a pharmaceutical composition that includes additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity can be determined using analytical chemistry techniques, including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. Additionally, a component of interest is described herein as being substantially purified if it is isolated and is the predominant species present in a preparation. The term "purified," as used herein, can refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% or more pure. By way of example only, a nucleic acid or protein is "isolated" if it is free from at least some of the cellular components with which it is naturally associated, or if the nucleic acid or protein is concentrated to a level greater than its in vivo or in vitro production. Also, by way of example, a gene is isolated when it is separated from open reading frames that flank it and encode proteins other than the gene of interest.

[0107] The term "label," as used herein, refers to a substance that is incorporated into a compound and is readily detectable so that its physical distribution can be detected and / or monitored.

[0108] The term "linkage" or "attachment moiety," as used herein, refers to a bond or chemical moiety formed from a chemical reaction between a functional group of one group, such as a linker of the present disclosure, and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, while such chemical moieties can include, but are not limited to, esters, carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, oximes, and oligonucleotide linkages. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water for extended periods of time, perhaps even indefinitely, at useful pH values, including, but not limited to, physiological conditions. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or aqueous solutions, including, for example, blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers can include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on the biologically active agent; such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of an amine with an aldehyde, phosphate ester linkages formed by the reaction of an alcohol with a phosphate group, hydrazone linkages which are the reaction product of a hydrazide with an aldehyde, acetal linkages which are the reaction product of an aldehyde with an alcohol, orthoester linkages which are the reaction product of a formate with an alcohol, peptide linkages formed by an amine group at the terminus of a polymer, such as, but not limited to, PEG, and oligonucleotide linkages formed by a phosphoramidite group at the terminus of a polymer, such as, but not limited to, a 5' hydroxyl group of an oligonucleotide.

[0109] The term "linker," as used herein, refers to any polyvalent group that connects or can connect a first group to at least one other group. Typically, a linker is a divalent or trivalent organic moiety that connects a drug or payload (first group) to a biologically active agent (second group), for example, via a linking or attachment moiety, or connects a drug or payload (first group) to a reactive moiety (second group) that can react with a biologically active agent. The linker can be susceptible to cleavage (cleavable linker), such as acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions in which the drug or payload and at least one other group remain active. Alternatively, the linker can be substantially resistant to cleavage (e.g., a stable linker or a non-cleavable linker).

[0110] In some embodiments, the linker is a divalent or trivalent group comprising or consisting of at least one moiety, each at least one moiety independently selected from a bond, an unsubstituted alkylene, a substituted alkylene, -(alkylene-O) n -, optionally substituted arylene, -O-, -C(O)-, -C(S)-, -N(R W )-, -S(O) 0-2 -, methine (-CH)-, amino acid, peptide, disulfide (-SS-), and phosphate-containing moiety, and combinations thereof, wherein each R W are independently H, C1-C8 alkyl, or a bond, and each phosphate-containing moiety is independently selected from the group consisting of phosphate ester, pyrophosphate ester, triphosphate ester, tetraphosphate ester, phosphonate, diphosphonate, phosphoramidate, pyrophosphoramidate, triphosphoramidate, tetraphosphoramidate, phosphorothioate, and diphosphorothioate. Unless otherwise expressly indicated, no orientation of the linker is implied by the direction in which the formula of the linker group is written. By way of example, the formula -C(O)CH2CH2- is *represents both -C(O)CH2CH2- and -CH2CH2C(O)-. In another example, the formula -C(O)CH2CH2- * -C(O)CH2CH2- and -C(O)CH2CH2- * and wherein: * indicates a point of attachment, e.g., attachment to a drug or payload. In some embodiments, when a selected moiety occurs more than once in the same linker, the two or more occurrences are not adjacent. In some embodiments, the linker is not a bond.

[0111] In some embodiments, the linker is a divalent group connecting a first group and a second group, hi some other embodiments, the linker is a trivalent moiety connecting a first group, a second group, and a third group.

[0112] In some embodiments, a linker connects at least a first group and a second group, where the first group is a drug or payload and the second group is a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein comprises at least one non-naturally encoded amino acid. In some embodiments, the linker connects the drug or payload to the non-naturally encoded amino acid of the biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein is an antibody. Thus, an antibody connected to a drug or payload via a linker can be an antibody-drug conjugate (ADC), such as an ADC of the present disclosure.

[0113] In some other embodiments, the linker connects at least a first group and a second group, where the first group is a drug or payload and the second group is a reactive moiety. In some embodiments, the second group is a reactive moiety that can react with a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein comprises at least one non-naturally encoded amino acid. Thus, in some embodiments, the reactive moiety can react with a non-naturally encoded amino acid of the biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein is an antibody.

[0114] In some embodiments, a first linker is connected to a second linker, and the combined linker (composite linker) connects at least a first group and a second group. The composite linkers of the present disclosure can include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more linker groups. In a non-limiting example, a first linker group, a second linker group, and a third linker group can be combined together to provide a composite linker that can connect a first group (e.g., a drug or payload) to at least one other group, such as a reactive moiety and / or a biologically active polypeptide or protein (e.g., an antibody). In some embodiments, the biologically active polypeptide or protein (e.g., an antibody) contains at least one non-naturally encoded amino acid.

[0115] In some embodiments, the linker is linear. In other embodiments, the linker is branched.

[0116] The term "medium," as used herein, refers to any culture medium used to grow and harvest cells and / or products expressed and / or secreted by such cells. Such "medium" includes, but is not limited to, a solution, solid, semi-solid, or rigid support that can support or contain any host cell (e.g., including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells) and cellular contents. Such "medium" includes, but is not limited to, the medium in which host cells from which a polypeptide has been secreted have been grown, including medium before or after a growth step. Such "medium" also includes, but is not limited to, buffers or reagents containing host cell lysates (e.g., polypeptides produced intracellularly) and host cells are lysed or disrupted to release the polypeptide.

[0117] The term "metabolite," as used herein, refers to a derivative of a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" refers to a biologically active derivative of a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, is metabolized.

[0118] The term "metabolized," as used herein, refers to the totality of processes by which a particular substance is transformed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information regarding metabolism can be found in The Pharmacological Basis of Therapeutics, 9 th Edition, McGraw-Hill (1996). By way of example only, metabolic products of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be identified by administering the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides to a host and analyzing tissue samples from the host, or by incubating the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides with hepatocytes in vitro and analyzing the resulting compounds.

[0119] As used herein, the term "metal chelator" refers to a molecule that forms a metal complex with a metal ion. For example, such a molecule may form two or more coordinate bonds with a central metal ion, forming a ring structure.

[0120] The term "modified," as used herein, refers to the presence of an alteration to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such an alteration, or modification, can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or by co- or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. The phrase "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under consideration is optionally modified, i.e., the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under consideration can be modified or unmodified.

[0121] The term "adjusted serum half-life," as used herein, refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to its unmodified form. Exemplary modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. For example, serum half-life is measured by taking blood samples at various time points after administration of a biologically active molecule or a modified biologically active molecule and determining the concentration of the molecule in each sample. Correlation of serum concentration with time allows for calculation of serum half-life. For example, an adjusted serum half-life may be an increased serum half-life, which may enable improved dosing regimens or avoid toxic effects. Such an increase in serum may be at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold. Methods for assessing serum half-life are well known in the art and may be used to assess the serum half-life of the antibodies and antibody-drug conjugates of the present invention.

[0122] The term "modulated therapeutic half-life," as used herein, refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule compared to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. By way of example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. By way of example, increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the unmodified molecule, or, by way of example only, increased or decreased degradation of the molecule by enzymes such as proteases. Methods for assessing therapeutic half-life are well known in the art and can be used to assess the therapeutic half-life of the antibodies and antibody-drug conjugates of the present invention.

[0123] The term "nearly stoichiometric," as used herein, refers to a molar ratio of the compounds participating in a chemical reaction of about 0.75 to about 1.5.

[0124] The term "non-eukaryote" as used herein refers to an organism that is not a eukaryote. By way of example, a non-eukaryote may belong to the phylogenetic domain Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), or the phylogenetic domain Archaea (including, but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1).

[0125] As used herein, the term "unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated versions thereof. The term "unnatural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of non-naturally encoded naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Additionally, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur in nature and that may be obtained synthetically or by modification of an unnatural amino acid.

[0126] The term "nucleic acid," as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof, in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized similarly to naturally occurring nucleotides, (ii) oligonucleotide analogs, including, but not limited to, PNAs (peptide nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.), and (iii) conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) as well as complementary sequences and sequences explicitly indicated. By way of example, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0127] The term "oxidizing agent," as used herein, refers to a compound or material capable of removing electrons from a compound being oxidized. Exemplary oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

[0128] The term "pharmaceutically acceptable," as used herein, refers to a material, including but not limited to, salts, carriers, or diluents, that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or adversely interacting with any of the components of the composition in which it is contained.

[0129] The term "photoaffinity label," as used herein, refers to a label having a group that, upon exposure to light, forms a linkage with a molecule for which the label has an affinity. By way of example only, such a linkage can be covalent or non-covalent.

[0130] The term "photocleavable group," as used herein, refers to a group that is cleaved upon exposure to light.

[0131] The term "photocrosslinker," as used herein, refers to a compound containing two or more functional groups that become reactive upon exposure to light and form covalent or non-covalent links with two or more monomeric or polymeric molecules.

[0132] The term "photoisomerizable moiety," as used herein, refers to a group that changes from one isomer to another upon irradiation with light.

[0133] The term "polyalkylene glycol," as used herein, refers to linear or branched polymeric polyether polyols. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed in commercial supplier catalogs, such as, for example, Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).

[0134] The term "polymer," as used herein, refers to a molecule composed of repeating subunits, including, but not limited to, a polypeptide, a polynucleotide, or a polysaccharide or polyalkylene glycol.

[0135] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply not only to naturally occurring amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are unnatural amino acids. Additionally, such "polypeptide," "peptide," and "protein" include amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0136] The term "post-translationally modified," as used herein, refers to any modification of a natural or unnatural amino acid that occurs after such amino acid is translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[0137] The term "prodrug" or "pharmaceutically acceptable prodrug," as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, does not abolish the biological activity or properties of the drug, and is relatively non-toxic; i.e., the material can be administered to an individual without causing undesired biological effects or adversely interacting with any of the components of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active or more active species through some process, such as conversion by a metabolic pathway. Some prodrugs have chemical groups present on the prodrug that make it less active and / or confer solubility or some other property on the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is generated. Prodrugs are converted to active drugs in the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemical properties, such as better solubility, enhanced delivery properties, such as specific targeting of particular cells, tissues, organs, or ligands, and improved therapeutic value of the drug. Advantages of such prodrugs include, but are not limited to, (i) ease of administration compared to the parent drug, (ii) the prodrug may be bioavailable by oral administration, whereas the parent drug is not, and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include derivatives of active drugs that are pharmacologically inactive or have reduced activity. Prodrugs can be designed to regulate the amount of a drug or biologically active molecule that reaches a desired site of action through manipulation of drug properties, such as physicochemical, biopharmaceutical, or pharmacokinetic properties. A non-limiting example of a prodrug is a non-natural amino acid polypeptide that is administered as an ester ("prodrug") to facilitate transport across cell membranes, where water solubility is detrimental to mobility, but is then metabolically hydrolyzed to the active entity, a carboxylic acid, once inside the cell, where water solubility is beneficial. Prodrugs can be designed as reversible drug derivatives for use as modifiers to enhance drug transport to site-specific tissues.

[0138] The term "prophylactically effective amount," as used herein, refers to an amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide applied prophylactically to a patient that will relieve to some extent one or more of the symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amount may depend on the patient's state of health, weight, etc. It is well considered to be within the skill of the art to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, dose escalation clinical trials.

[0139] The term "protected," as used herein, refers to the presence of a "protecting group" or moiety that prevents reaction of a chemically reactive functional group under certain reaction conditions. The protecting group can vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or hydrazide, the protecting group can be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), (ii) if the chemically reactive group is a thiol, the protecting group can be orthopyridyl disulfide, and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic acid or propionic acid, or a hydroxyl group, the protecting group can be benzyl or an alkyl group, such as methyl, ethyl, or tert-butyl.

[0140] By way of example only, blocking / protecting groups may be selected from:

[0141] [ka]

[0142] Further protecting groups include, but are not limited to, photolabile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3 rdEd., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0143] The term "reactive compound," as used herein, refers to a compound that is reactive toward another atom, molecule, or compound under appropriate conditions.

[0144] The term "recombinant host cell" (also referred to as "host cell"), as used herein, refers to a cell that contains an exogenous polynucleotide; methods used to insert the exogenous polynucleotide into the cell include, but are not limited to, direct uptake, transduction, F-mating, or other methods well known in the art for generating recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrative vectors, including, but not limited to, plasmids, or may be integrated into the host genome.

[0145] The term "redox active agent," as used herein, refers to a molecule that oxidizes or reduces another molecule, whereby the redox active agent is reduced or oxidized. Examples of redox active agents include ferrocene, quinone, Ru, 2+ / 3+ Complex, Co 2+ / 3+ complexes, and Os 2+ / 3+ These include, but are not limited to, complexes.

[0146] The term "reducing agent," as used herein, refers to a compound or material capable of adding electrons to a compound being reduced. Examples of reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents can be used, by way of example only, to maintain sulfhydryl groups in a reduced state and to reduce intramolecular or intermolecular disulfide bonds.

[0147] The term "saccharides," as used herein, refers to a range of carbohydrates, including but not limited to sugars, monosaccharides, oligosaccharides, and polysaccharides.

[0148] The term "safety" or "safety profile," as used herein, refers to side effects that may be associated with the administration of a drug relative to the number of times the drug is administered. By way of example, a drug that is administered multiple times with mild or no side effects is said to have a good safety profile.

[0149] The phrases "selectively hybridize" or "specifically hybridize," as used herein, refer to the binding, duplex formation, or hybridization of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture, including, but not limited to, whole cell or library DNA or RNA.

[0150] The terms "standard of care," "best practice," "standard of medical care," or "standard of therapy," as used herein, refer to treatments accepted by medical experts as appropriate treatments for a particular type of disease and widely used by health care workers (see, e.g., https: / / www.cancer.gov / publications / dictionary / cancer-terms / def / standard-of-care).

[0151] The term "stoichiometric," as used herein, refers to a molar ratio of compounds participating in a chemical reaction of about 0.9 to about 1.1.

[0152] The term "stoichiometric-like," as used herein, refers to a chemical reaction that becomes stoichiometric or near-stoichiometric in the presence of a change in reaction conditions or additives. Such changes in reaction conditions include, but are not limited to, an increase in temperature or a change in pH. Such additives include, but are not limited to, promoters.

[0153] The term "subject," as used herein, refers to an animal that has been the object of treatment, observation, or experiment. By way of example only, a subject may be a mammal, including, but not limited to, a human.

[0154] The term "substantially purified," as used herein, refers to a component of interest that may be substantially or essentially free from other components that normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" if a preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. By way of example only, natural or non-natural amino acid polypeptides may be purified from native cells, or, in the case of recombinantly produced natural or non-natural amino acid polypeptides, from host cells. By way of example, a preparation of natural or non-natural amino acid polypeptides may be "substantially purified" if the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating material. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide can be present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cells.By way of example, a "substantially purified" natural or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more as determined by suitable methods, including but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0155] The term "therapeutically effective amount," as used herein, refers to the amount of a composition containing at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide or antibody-drug conjugate that, when administered to a patient already suffering from a disease, condition, or disorder, is sufficient to cure or at least partially arrest, or alleviate to some extent, one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0156] The term "toxic moiety" or "toxic group," as used herein, refers to a compound that can cause harm, interference, or death. Toxic moieties include NCA1, auristatins, DNA minor groove binders, DNA minor groove alkylators, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, vinca alkaloids, AFP, monomethyl auristatin E (MMAF), monomethyl auristatin E (MMAE), AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, and rhizoxin. , cyanomorpholinodoxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide, etc.), baccatin and its derivatives, antitubulin agents, cryptophycin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colcimide, estramustine cemadoxin, cemadotin, discodermolide, maytansine, eleutherobin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, cytarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercapto topramine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracyclines, gentamicin, cephalothin, vancomycin, telavancin, daptomycin,azithromycin, erythromycin, roxithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral agents (e.g., abacavir, acyclovir, ampligen, cidofovir, delavirdine, etc.), methicone, didanosine, efavirenz, entecavir, phosphonet, ganciclovir, ibacitabine, immunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valacyclovir, zanamivir, etc.), daunorubicin hydrochloride, daunomycin, rubidomycin, cerubicin, idarubicin, doxorubicin, epirubicin and morpholino derivatives, fenoxazolidinone cyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrroloindoledione (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphite, "Taxanes" include, but are not limited to, cyclosporine ...Bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; antifolate antineoplastic agents such as pemetrexed (ALIMTA®, Eli Lilly), aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (its adzecine derivatives) cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembite, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as the enediyne antibiotics calicheamicin, calicheamicin gamma II, and calicheamicin omega II; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chloazolomycins. bromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin cin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimethoprim, thiazolinone ... methotrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such asFolinic acid; Aceglatone; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanol; Nitraeline; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Kreskin (Polysaccharide-K); Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triazicoside triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verulaculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophor-free, albumin, nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs,Examples include, but are not limited to, cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0157] The terms "treat," "treating," or "treatment," as used herein, include alleviating, attenuating, or ameliorating disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of a symptom, inhibiting a disease or condition, e.g., halting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating conditions caused by a disease or condition, or halting the symptoms of a disease or condition. The terms "treat," "treating," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatment.

[0158] The compounds provided herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the above compounds) include isotopically labeled compounds, which are identical to those recited in the various formulas and structures provided herein except for the fact that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O.17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, such as 3 H and 14 Incorporation of radioactive isotopes such as 3C are useful in drug and / or substrate tissue distribution assays. 2 Substitution with isotopes such as H may afford certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0159] Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for making such compounds) possess asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by well-known methods, such as chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture to a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers, and converting the individual diastereomers to the corresponding pure enantiomers (e.g., by hydrolysis). All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered part of the compositions described herein.

[0160] In further or additional embodiments, the compounds described herein (including but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for producing the compounds) are used in the form of prodrugs. In further or additional embodiments, the compounds described herein (including but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for producing the compounds) are metabolized upon administration to an organism in need to produce a metabolite, which is then used to bring about a desired effect, including a desired therapeutic effect. In further or additional embodiments, the compounds are active metabolites of the non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.

[0161] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. In certain embodiments, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides can exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides presented herein. Furthermore, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein can exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

[0162] Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for making the compounds) can exist in several tautomeric forms. All such tautomers are considered to be part of the compositions described herein. Also, for example, all enol-keto forms of any of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for making the compounds) are considered to be part of the compositions described herein.

[0163] Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing any of the above compounds) may be acidic and may form salts with pharmaceutically acceptable cations. Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the above compounds) may be basic and may therefore form salts with pharmaceutically acceptable anions. All such salts, including disalts, are within the scope of the compositions described herein and can be prepared by conventional methods. For example, salts can be prepared by contacting acidic and basic entities in aqueous, non-aqueous, or partially aqueous media. Salts are recovered using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[0164] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein can be formed when acidic protons present in the parent non-natural amino acid polypeptide are replaced by metal ions, such as alkali metal ions, alkaline earth ions, or aluminum ions, or coordinate with an organic base. Additionally, salt forms of the disclosed non-natural amino acid polypeptides can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable acid addition salts (which are a type of pharmaceutically acceptable salt) by reacting the free base form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable base addition salts (which are a type of pharmaceutically acceptable salt) by reacting the free acid form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

[0165] Types of pharmaceutically acceptable salts include, but are not limited to, the following: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo[2.2 (2) acid addition salts formed with oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced with a metal ion, e.g., an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0166] The corresponding counterions of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be analyzed and identified using a variety of methods, including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectrometry, mass spectrometry, or any combination thereof. Additionally, the therapeutic activity of such pharmaceutically acceptable salts of non-natural amino acid polypeptides can be tested using the techniques and methods described in Examples 87-91.

[0167] Reference to a salt should be understood to include its solvent addition forms or crystalline forms, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystalline packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, optical and electrical properties, stability, and solubility. Various factors, such as the recrystallization solvent, crystallization rate, and storage temperature, may result in the predominance of a single crystalline form.

[0168] Screening and characterization of pharmaceutically acceptable salts, polymorphs, and / or solvates of non-natural amino acid polypeptides can be accomplished using a variety of techniques, including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermochemical decomposition or thermophysical processes, including, but not limited to, polymorphic transformations, and such methods are used to analyze relationships between polymorphic forms, determine weight loss, find glass transition temperatures, or for excipient compatibility studies. Such methods include, but are not limited to, differential scanning calorimetry (DSC), modulated differential scanning calorimetry (MDCS), thermogravimetric analysis (TGA), and thermogravimetric and infrared analysis (TG / IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopic techniques include, but are not limited to, polarized light microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray (EDX), environmental scanning electron microscopy (in a gas or water vapor atmosphere) with EDX, IR microscopy, and Raman microscopy.

[0169] Introduction Antibody-based therapeutics have emerged as an important component of therapy for an increasing number of human malignancies in fields such as oncology, immunology, inflammatory and infectious diseases. In most cases, the basis for their therapeutic function is the high degree of specificity and affinity that antibody-based drugs have for their target antigens. Arming monoclonal antibodies with drugs, toxins, or radionuclides is yet another strategy by which monoclonal antibodies can induce therapeutic effects. By combining the exquisite targeting specificity of antibodies with the tumor-killing power of toxic effector molecules, immunoconjugates enable highly sensitive discrimination between targets and normal tissues, thereby resulting in fewer side effects than most conventional chemotherapeutic agents. The toxins utilized can be specifically, stably, and irreversibly conjugated to unique sites on the antibody. This unique conjugation process allows for precise control of the location of the toxin on the antibody as well as the number of toxins conjugated to each antibody. Both of these features are important for controlling the biophysical characteristics and toxicity associated with ADCs. (See, for example, Jackson et al., 2014, Tian et al., 2014).

[0170] Currently, ADCs are advancing the field of cancer therapy, with several ADCs targeting various drugs either approved or undergoing clinical trials. However, ADCs face challenges due to their lack of therapeutic index and toxicity. Linker technology for conjugating cytotoxic drugs to antibodies affects the stability of ADCs in the systemic circulation. Release of free drug in the circulation instead of within antigen-expressing cancer cells can lead to loss of ADC efficacy, insufficient killing of immunogenic cancer cells, and increased toxicity. Therefore, there is a need to design stable linkers for drug design and antibody conjugation.

[0171] Cluster of differentiation 70 (CD70) is frequently expressed in various malignancies, including clear cell renal carcinoma (RCC), with a reported incidence ranging from 70% to 100%, but is minimally expressed in normal tissues. This disclosure provides next-generation ADCs using a technology platform in which a CD70-specific monoclonal antibody is conjugated to AS269, a potent cytotoxic tubulin inhibitor. The site-specificity, high homogeneity, and stable covalent conjugation of the ADC result in enhanced stability and pharmacokinetics, which may contribute to increased targeted delivery of the payload to tumor cells at lower systemic toxicity and lower effective doses compared to other CD70 ADCs. The ADCs of this disclosure are designed to inhibit the growth of CD70-overexpressing cells through multiple sequential steps, including binding to CD70 on the surface of cancer cells, rapid internalization, transport to lysosomes, and metabolism within the lysosome to release pAF-AS269, which binds to microtubules and induces cancer cell cycle arrest and cell death.

[0172] Thus, in some aspects, the present disclosure provides next-generation site-specific anti-CD70 ADCs comprising humanized CD70-targeting monoclonal antibodies (mAbs) conjugated to cytotoxic tubulin inhibitors using a non-natural amino acid incorporation technology platform. ADCs synthesized in this manner can be homogeneous and highly stable, thereby delivering drugs to target tumor cells with higher efficiency, maximizing on-target efficacy, and minimizing off-target toxicity, resulting in a broader therapeutic window.

[0173] In some embodiments, the ADC contains a short, non-cleavable hydroxylamine-PEG 4 linker attached to the N-terminus of monomethyl auristatin F (MMAF) to generate the cytotoxic payload, the cytotoxic tubulin inhibitor linker derivative AS269. MMAF is a highly potent synthetic auristatin derivative that inhibits cell proliferation by disrupting tubulin polymerization. In some cases, the ADC contains two AS269 cytotoxic payloads site-specifically attached to an anti-CD70 antibody containing an unnatural amino acid. In some cases, the ADC can exhibit anti-tumor activity by optimizing the number and position of the payloads and the chemical bond conjugating the payloads to the antibody. In some cases, AS269 is a generally non-cell-permeable tubulin inhibitor specifically designed to form a highly stable covalent bond with the antibody and kill tumor cells only upon entering the cell when assisted by a conjugated targeting antibody, thereby limiting off-target effects on healthy tissues. In some cases, AS269 exhibits limited permeability through cell membranes that are not conjugated to a targeting antibody, thereby reducing off-target toxicity. In some cases, AS269 is a poor substrate for multidrug resistance (MDR) pumps, thereby retaining and concentrating the drug within cancer cells, which may result in more potent killing of cancer cells. Combining the unique features of AS269 with optimized payload number and positioning and chemical linkages that conjugate the payload to the antibody with a drug-to-antibody ratio (DAR) of 2 can result in in vivo stability, enhanced potency, and reduced payload exposure in serum, which may contribute to the observed antitumor activity and tolerability profile of the ADC. In some cases, the ADC comprises a structure synthesized according to Figure 1. In some cases, the ADC comprises AS269 as a payload and an anti-CD70 antibody comprising one or more unnatural amino acids as disclosed herein.In some embodiments, the AS269 payload is specifically and stably conjugated to the unnatural amino acid pAF on a unique site in the heavy chain of the mAb (one payload per heavy chain), and the anti-CD70 antibody comprises a heavy chain having the corresponding amino acid sequence SEQ ID NO:3 with the unnatural amino acid pAF at position A114 (Kabat numbering), and a light chain sequence having the corresponding amino acid sequence SEQ ID NO:2.

[0174] Unnatural amino acids The present disclosure provides antibodies, antibody fragments, or variants comprising at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into an antibody may allow for the application of conjugation chemistries involving specific chemical reactions with one or more non-naturally encoded amino acids, but which do not react with the 20 commonly occurring amino acids.

[0175] The selection of non-naturally encoded amino acid sites was based on surface exposure / site accessibility within the antibody, with hydrophobic or neutral amino acid sites selected to maintain charge on the antibody. Methods for introducing non-naturally encoded amino acids into sites in proteins are described, for example, in WO 2018223108, WO 2010 / 011735, and WO 2005 / 074650. The present disclosure employs such methodologies and techniques. The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group on the side chain of the unnatural amino acid has at least one characteristic and / or activity and / or reactivity that is orthogonal to the chemical reactivity of the 20 common, genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) or that is at least orthogonal to the chemical reactivity of a naturally occurring amino acid present in a polypeptide that includes the unnatural amino acid; and (2) the introduced unnatural amino acid is orthogonal to the chemical reactivity of the 20 common, genetically encoded amino acids. (3) the unnatural amino acid can be stably incorporated into a polypeptide, preferably with stability comparable to that of naturally occurring amino acids or under typical physiological conditions, and more preferably, such incorporation can occur via an in vivo system, and (4) the unnatural amino acid comprises an oxime functional group or a functional group that can be converted to an oxime group by reaction with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide containing the unnatural amino acid (unless, of course, such destruction of biological properties is the goal of the modification / conversion), or where the conversion can occur under aqueous conditions at a pH of about 4 to about 8, or where the reactive site on the unnatural amino acid is an electrophilic site. Any number of unnatural amino acids can be introduced into a polypeptide.The unnatural amino acid can also contain a protected or masked oxime, or a protected or masked group that can be converted to an oxime group after deprotection of the protected group or unmasking of the masked group. The unnatural amino acid can also contain a protected or masked carbonyl group that can be converted to a carbonyl group after deprotection of the protected group or unmasking of the masked group, thereby available to react with a hydroxylamine or aminooxy group to form an oxime group. Oxime-based unnatural amino acids can be synthesized by methods well known in the art (see, e.g., WO 2013 / 185117 and WO 2005 / 074650), including reacting a carbonyl-containing unnatural amino acid with a hydroxylamine- or aminooxy-containing reagent.

[0176] In some embodiments, the selection of a non-naturally encoded amino acid site is based on surface exposure. For example, one possible site is an amino acid with a solvent-accessible surface area ratio of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. In some embodiments, one possible site is an amino acid with a solvent-accessible surface area ratio of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% or more. The solvent-accessible surface area can be calculated using the DSSP program [Biopolymers, 22, 2577-2637 (1983)] using the crystal structure data file of an antibody or antibody fragment deposited in the Protein Data Bank (PDB).

[0177] The ratio of the solvent-accessible surface area of ​​the amino acid residue of interest can be calculated by dividing the solvent-accessible surface area of ​​the antibody structure calculated above by the solvent-accessible surface area of ​​alanine-X-alanine (where X represents the amino acid residue of interest). In this regard, two or more PDB files may exist for a protein of one species, any one of which can be used in the present invention.

[0178] Alternatively, the solvent accessibility of an amino acid can be determined by a solvent accessibility test in which a functional group (i.e., a thiol, amino, or carbonyl group) on the amino acid is functionalized when treated with an electrophile or a nucleophile, for example. Based on the results of the test, the functional group (i.e., a thiol, amino, or carbonyl group) can be said to be at least 50% solvent accessible if, for example, at least 50% of the functional group is functionalized in the test. In some embodiments, the non-naturally encoded amino acid site is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% solvent accessible. Examples of solvent accessibility tests include, but are not limited to, propargylation of surface thiol groups or α-bromopyruvate reacting with surface thiol groups.

[0179] In some embodiments, antibodies comprising one or more non-naturally encoded amino acids are disclosed herein. The one or more non-natural amino acids can be encoded by a codon that does not encode one of the 20 natural amino acids. The one or more non-natural amino acids can be encoded by a nonsense codon (stop codon). The stop codon can be an amber codon. The amber codon can comprise a UAG sequence. The stop codon can be an ochre codon. The ochre codon can comprise a UAA sequence. The stop codon can be an opal or umber codon. The opal or umber codon can comprise a UGA sequence. The one or more non-natural amino acids can be encoded by a four-base codon.

[0180] Unnatural amino acids of the disclosure include: 1) substituted phenylalanine and tyrosine analogs, such as 4-amino-L-phenylalanine, 4-acetyl-L-phenylalanine (pAF), 4-azido-L-phenylalanine, 4-nitro-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-isopropyl-L-phenylalanine, 3-nitro-L-tyrosine, O-methyl-L-tyrosine, and O-phosphotyrosine; 2) amino acids that can be photocrosslinked, such as amino acids with an aryl azide or benzophenone group, such as 4-azidophenylalanine or 4-benzoylphenylalanine; 3) amino acids with unique chemical reactivity, such as 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-2-propyn-1-yl-L-tyrosine, N-(ethylthio)thiocarbonyl-L-phenylalanine, and p-(3-oxobutyric acid). 4) heavy atom-containing amino acids, for example, 4-iodo-L-phenylalanine or 4-bromo-L-phenylalanine, for phasing in X-ray crystallography, for example, 4-iodo-L-phenylalanine or 4-bromo-L-phenylalanine, 5) redox-active amino acids, for example, 3,4-dihydroxy-L-phenylalanine, 6) fluorinated amino acids, for example, 2-fluorophenylalanine (for example, 2-fluoro-L-phenylalanine), 3-fluorophenylalanine (for example, 3-fluoro-L-phenylalanine) or 4-fluorophenylalanine (for example, 4-fluoro-L-phenylalanine), 7) fluorescent amino acids, for example, amino acids containing naphthyl, dansyl or 7-aminocoumarin side chains, 8) photocleavable or photoisomerizable amino acids, for example, amino acids containing azobenzyl or nitrobenzyl, for example, cysteine, serine or tyrosine containing azobenzyl or nitrobenzyl, 9) β-amino acids (for example, β 2 or β 3amino acids), 10) homo-amino acids, for example, homoglutamine (e.g., beta-homoglutamine) or homophenylalanine (e.g., beta-homophenylalanine), 11) proline or pyruvate derivatives, 12) 3-substituted alanine derivatives, 14) glycine derivatives, 15) linear core amino acids, 16) diamino acids, 17) D-amino acids, 18) N-methyl amino acids, 19) phosphotyrosine mimetics, for example, carboxymethylphenylalanine (pCMF) (e.g., 4-carboxymethyl-L-phenylalanine), 20) 2-aminooctanoic acid, and 21) amino acids containing a sugar moiety, for example, N-acetyl-L-glucosaminyl-L-serine, beta-N-acetylglucosamine-O-serine, N-acetyl-L-galactosaminyl-L-serine, alpha-N-acetylgalactosamine-O-serine, O-(3-O-galactosamine), alpha-N-acetylgalactosamine-O-threonine, 3-O-(N-acetyl-beta-D-glucosaminyl)-L-serine, N-acetyl-L-glucosaminyl-L-threonine, alpha-N-acetylgalactosamine-O-threonine, 3-O-(N-acetyl-beta-D-glucosaminyl)-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, N4-(β-N-acetyl-D-glucosaminyl)-L-asparagine, and O-(mannosyl)-L-serine; amino acids in which the naturally occurring N- or O-linkage between the amino acid and sugar is replaced by a covalent linkage not commonly found in nature, including, but not limited to, an alkene, oxime, thioether, amide, etc.; or amino acids containing a sugar not commonly found in naturally occurring polypeptides, such as 2-deoxy-glucose, 2-deoxy-galactose, etc.Specific examples of unnatural amino acids include p-acetylphenylalanine (4-acetylphenylalanine), 4-acetyl-L-phenylalanine, also referred to herein as p-acetyl-L-phenylalanine (pAF), 4-boronophenylalanine (pBoF) (e.g., 4-borono-L-phenylalanine, 4-propargyloxyphenylalanine (pPrF) (e.g., 4-propargyloxy-L-phenylalanine), O-methyltyrosine (e.g., O-methyl-L-tyrosine), 3-(2-naphthyl)alanine (NapA) (e.g., 3-(2-naphthyl)-L-alanine), 3-methylphenylalanine (e.g., 3-methyl-L-phenylalanine), O-allyltyrosine (e.g., O-allyl-L-tyrosine), O-isopropyltyrosine (e.g., O-isopropyl-L-tyrosine), dopamine ( For example, L-dopa), 4-isopropylphenylalanine (for example, 4-isopropyl-L-phenylalanine), 4-azidophenylalanine (pAz) (for example, 4-azido-L-phenylalanine), 4-benzoylphenylalanine (pBpF) (for example, 4-benzoyl-L-phenylalanine), O-phosphoserine (for example, O-phospho-L-serine), O-phosphotyrosine (for example, O-phospho-L-tyrosine), 4-iodophenylalanine (pIF) (for example, 4-iodo-L-phenylalanine), 4-bromophenylalanine (for example, 4-bromo-L-phenylalanine), 4-aminophenylalanine (for example, 4-amino-L-phenylalanine), 4-cyanophenylalanine (pCNF) (for example, 4-cyano-L-phenylalanine), (8-hydroxyquinolin-3-yl)alanine (HQ Examples of suitable hydroxyquinolin-3-yl (A) amino acids include, but are not limited to, (8-hydroxyquinolin-3-yl)-L-alanine, (2,2-bipyridin-5-yl)-alanine (BipyA) amino acids, and (2,2-bipyridin-5-yl)-alanine (BipyB) amino acids.Additional unnatural amino acids are disclosed in Liu et al. (2010) Annu Rev Biochem, 79:413-44, Wang et al. (2005) Angew Chem Int Ed, 44:34-66, and published International Publication Nos. 2012 / 166560, 2012 / 166559, 2011 / 028195, 2010 / 037062, 2008 / 083346, 2008 / 077079, 2007 / 094916, 2007 / 079130, 2007 / 070659, and 2007 / 059312, the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the one or more unnatural amino acids can be p-acetylphenylalanine. In some more particular embodiments, the one or more unnatural amino acids can be p-acetyl-L-phenylalanine (pAF).

[0181] In some embodiments, the one or more unnatural amino acids are 4-acetylphenylalanine, 3-O-(N-acetyl-beta-D-glucosaminyl)threonine, N4-(β-N-acetyl-D-glucosaminyl)asparagine, O-allyltyrosine, alpha-N-acetylgalactosamine-O-serine, alpha-N-acetylgalactosamine-O-threonine, 2-aminooctanoic acid, 2-aminophenylalanine, 3-aminophenylalanine, 4-aminophenylalanine, 2-aminotyrosine, 3-allyl Aminotyrosine, 4-azidophenylalanine, 4-benzoylphenylalanine, (2,2-bipyridin-5yl)alanine, 3-boronophenylalanine, 4-boronophenylalanine, 4-bromophenylalanine, p-carboxymethylphenylalanine, 4-carboxyphenylalanine, p-cyanophenylalanine, 3,4-dihydroxyphenylalanine, 4-ethynylphenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, O-(3 -OD-Galactosyl-N-acetyl-beta-D-galactosaminyl)serine, homoglutamine, (8-hydroxyquinolin-3-yl)alanine, 4-iodophenylalanine, 4-isopropylphenylalanine, Oi-propyltyrosine, 3-isopropyltyrosine, O-mannopyranosylserine, 2-methoxyphenylalanine, 3-methoxyphenylalanine, 4-methoxyphenylalanine, 3-methylphenylalanine, O-methyltyrosine, 3-(2-naphthyl)alanine, 5-nitrohistidine 4-nitrophenylalanine, 4-nitrohistidine, 4-nitroleucine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrophenylalanine, 4-nitrotryptophan, 5-nitrotryptophan, 6-nitrotryptophan, 7-nitrotryptophan, 2-nitrotyrosine, 3-nitrotyrosine, O-phosphoserine, O-phosphotyrosine, 4-propargyloxyphenylalanine, O-2-propyn-1-yltyrosine, 4-sulfophenylalanine, and O-sulfotyrosine.

[0182] In some further embodiments, the one or more unnatural amino acids are 4-acetyl-L-phenylalanine (para-acetyl-L-phenylalanine (pAF)), 3-O-(N-acetyl-β-D-glucosaminyl)-L-threonine, N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine, O-allyl-L-tyrosine, alpha-N-acetylgalactosamine-OL-serine, alpha-N-acetylgalactosamine-OL-threonine, 2-aminooctanoic acid, 2-amino-L-phenylalanine ... , 3-amino-L-phenylalanine, 4-amino-L-phenylalanine, 2-amino-L-tyrosine, 3-amino-L-tyrosine, 4-azido-L-phenylalanine, 4-benzoyl-L-phenylalanine, (2,2-bipyridin-5-yl)-L-alanine, 3-borono-L-phenylalanine, 4-borono-L-phenylalanine, 4-bromo-L-phenylalanine, p-carboxymethyl-L-phenylalanine, 4-carboxy-L-phenylalanine, p-cyano-L-phenylalanine, 3,4-Dihydroxy-L-phenylalanine (L-DOPA), 4-ethynyl-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine, O-(3-OD-galactosyl-N-acetyl-beta-D-galactosaminyl)-L-serine, L-homoglutamine, (8-hydroxyquinolin-3-yl)-L-alanine, 4-iodo-L-phenylalanine, 4-isopropyl-L-phenylalanine, Oi-propyl-L-tyrosine, 3-isopropyl-L-tyrosine, O-mannopyranosyl-L-serine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-methoxy-L-phenylalanine, 3-methyl-L-phenylalanine The amino acid sequence is selected from the group consisting of 5-nitro-L-alanine, O-methyl-L-tyrosine, 3-(2-naphthyl)-L-alanine, 5-nitro-L-histidine, 4-nitro-L-histidine, 4-nitro-L-leucine, 2-nitro-L-phenylalanine, 3-nitro-L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-L-tryptophan, 5-nitro-L-tryptophan, 6-nitro-L-tryptophan, 7-nitro-L-tryptophan, 2-nitro-L-tyrosine, 3-nitro-L-tyrosine, O-phospho-L-serine, O-phospho-L-tyrosine, 4-propargyloxy-L-phenylalanine, O-2-propyn-1-yl-L-tyrosine, 4-sulfo-L-phenylalanine and O-sulfo-L-tyrosine. In some embodiments, one or more unnatural amino acids can be p-acetyl-L-phenylalanine (pAF). Thus, in some embodiments, each and every one of the one or more unnatural amino acids is pAF.

[0183] In certain embodiments of the present disclosure, antibodies with at least one unnatural amino acid comprise at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises the attachment of a molecule, including, but not limited to, a biologically active agent, e.g., a drug, including a small molecule drug, or any other desired compound or substance, comprising a second reactive group, to at least one unnatural amino acid comprising a first reactive group, using chemical methodologies known to those of skill in the art to be suitable for the particular reactive group. For example, the first reactive group is a keto moiety (including, but not limited to, the unnatural amino acid p-acetyl-phenylalanine, or more specifically, p-acetyl-L-phenylalanine (pAF)), and the second reactive group is an aminooxy moiety. In another example, the first reactive group is an azido moiety (including, but not limited to, the unnatural amino acid p-azido-L-phenylalanine), and the second reactive group is an alkynyl moiety. Certain embodiments of modified antibody polypeptides of the disclosure use at least one unnatural amino acid (including, but not limited to, an unnatural amino acid comprising a keto functionality) that comprises at least one post-translational modification, wherein the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification occurs in vivo in a eukaryotic cell or a non-eukaryotic cell. In other embodiments, the post-translational modification occurs in vitro. In further embodiments, the post-translational modification occurs in vitro and in vivo.

[0184] In some embodiments, the unnatural amino acids can be modified to incorporate a chemical group. In some embodiments, the unnatural amino acids can be modified to incorporate a ketone group. One or more of the unnatural amino acids can include at least one carbonyl.

[0185] In some embodiments disclosed herein, unnatural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. In some embodiments, unnatural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. Methods for incorporating unnatural amino acids into molecules, such as proteins, polypeptides, or peptides, are disclosed in U.S. Patent Nos. 7,332,571, 7,928,163, 7,696,312, 8,008,456, 8,048,988, 8,809,511, 8,859,802, 8,791,231, 8,476,411, or 9,637,411 (each of which is incorporated by reference herein in its entirety), and in the Examples herein. One or more unnatural amino acids can be incorporated by methods well known in the art. For example, cell-based or cell-free systems can be used, and auxotrophic strains can also be used in place of engineered tRNAs and synthetases. In certain embodiments, orthogonal tRNA synthetases are disclosed in, for example, WO2002085923(A2), WO2002086075(A2), WO2004035743(A2), WO2007021297(A1), WO2006068802(A2), and WO2006069246(A2), the contents of each of which are incorporated herein by reference in their entirety. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include modifying one or more amino acid residues in the antibody or antibody fragment or variant. Modifying one or more amino acid residues in the antibody, antibody fragment, or variant may involve mutating one or more nucleotides in the nucleotide sequence encoding the antibody, antibody fragment, or variant. Mutating one or more nucleotides in the nucleotide sequence encoding the antibody, antibody fragment, or variant may involve changing a codon encoding an amino acid to a nonsense codon.Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include modifying one or more amino acid residues in the antibody or antibody fragment or variant to generate one or more amber codons in the antibody or antibody fragment or variant. One or more unnatural amino acids can be incorporated into the antibody or antibody fragment or variant in response to an amber codon. One or more unnatural amino acids can be site-specifically incorporated into the antibody or antibody fragment or variant. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include one or more genetically encoded unnatural amino acids with orthogonal chemical reactivity to the standard 20 amino acids for site-specific modification of a biologically active molecule or targeting agent. Incorporating one or more unnatural amino acids can include using a tRNA / aminoacyl-tRNA synthetase pair to site-specifically incorporate one or more unnatural amino acids into a defined site in a biologically active molecule or targeting agent in response to one or more amber nonsense codons. Additional methods for incorporating unnatural amino acids include, but are not limited to, those disclosed in Chatterjee et al., A Versatile Platform for Single- and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli, Biochemistry, 2013; Kazane et al., J Am Chem Soc, 135(1):340-6, 2013; Kim et al., J Am Chem Soc, 134(24):9918-21, 2012; Johnson et al., Nat Chem Biol, 7(11):779-86, 2011; and Hutchins et al., J Mol Biol, 406(4):595-603, 2011. One or more unnatural amino acids can be produced through selective reaction of one or more natural amino acids. The selective reaction can be mediated by one or more enzymes.In a non-limiting example, selective reaction of one or more cysteines with a formylglycine generating enzyme (FGE) can produce one or more formylglycines, as described in Rabuka et al., Nature Protocols 7:1052-1067, 2012. One or more unnatural amino acids can be involved in a linker-forming chemical reaction. The linker-forming chemical reaction can include a bioorthogonal reaction. The linker-forming chemical reaction can include click chemistry. See, e.g., WO 2006 / 050262, incorporated herein by reference in its entirety.

[0186] Any position in an antibody or antibody fragment is suitable for selection for incorporating an unnatural amino acid, and selection can be based on rational design or by random selection without any or particular desired goal. Selection of desired sites can be based on generating a non-natural amino acid polypeptide (which can be further modified or left unmodified) with any desired property or activity, including, but not limited to, receptor binding modulator, receptor activity modulator, modulator of binding with a binding partner, modulator of binding partner activity, modulator of binding partner structure, dimer or multimer formation, no change in activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide, such as solubility, aggregation, or stability. Alternatively, sites identified as important for biological activity may also be good candidates for substitution with an unnatural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative is to simply make serial substitutions with an unnatural amino acid at each position along the polypeptide chain and observe the effect on the activity of the polypeptide. Any means, technique, or method for selecting the location of a non-natural amino acid substitution into any polypeptide is suitable for use in the methods, techniques, and compositions described herein.

[0187] The structure and activity of naturally occurring variants of a polypeptide, including deletions, can also be investigated to determine regions of the protein that are likely to tolerate substitution with an unnatural amino acid. Once residues that are likely to be intolerant to substitution with an unnatural amino acid have been eliminated, the impact of proposed substitutions at each remaining position can be investigated using methods including, but not limited to, the three-dimensional structure of the relevant polypeptide and any associated ligands or binding proteins. X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Data Bank (PDB, see the World Wide Web at rcsb.org), a centralized database containing three-dimensional structural data for large protein and nucleic acid molecules, and can be used to identify amino acid positions that can be substituted with an unnatural amino acid. In addition, if three-dimensional structural data is not available, models can be generated that explore the secondary and tertiary structure of the polypeptide. Thus, the identity of amino acid positions that can be substituted with an unnatural amino acid can be readily obtained.

[0188] Examples of sites for incorporation of unnatural amino acids include, but are not limited to, regions for binding to binding proteins or ligands that are excluded from potential receptor binding regions or that may be fully or partially solvent exposed, have minimal or no hydrogen bonding interactions with nearby residues, be minimally exposed to nearby reactive residues, and / or be in highly malleable regions as predicted by the three-dimensional crystal structure of a particular polypeptide with its associated receptor, ligand, or binding protein.

[0189] A wide variety of unnatural amino acids can be substituted into or incorporated into a given position in a polypeptide. By way of example, a particular unnatural amino acid can be selected for incorporation based on preference for conservative substitutions, based on examination of the three-dimensional crystal structure of the polypeptide and its associated ligand, receptor, and / or binding protein.

[0190] The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42:6735-6746 (2003), and the synthesis of p-acetyl-L-phenylalanine is described in WO 2015 / 153761(A2), the entire contents of each of which are incorporated herein by reference in their entirety. Other carbonyl-containing amino acids can be prepared similarly.

[0191] Anti-CD70 antibody The present invention provides novel ADCs comprising antibodies, antibody fragments, or variants thereof, engineered to incorporate one or more non-naturally encoded amino acids at any desired position in the heavy and / or light chain amino acid sequence. Additionally, the present invention provides ADCs comprising one or more antibodies, antibody fragments, or variants thereof, engineered to incorporate one or more non-naturally encoded amino acids site-specifically into the heavy and / or light chain amino acid sequence, conjugated to a drug or payload via a phosphate-based linker. In some embodiments, the antibody, antibody fragment, or variant thereof binds to a tumor-associated CD70 antigen. In some embodiments, the present invention provides anti-CD70 ADCs comprising antibodies, antibody fragments, or variants thereof, engineered to incorporate one or more non-naturally encoded amino acids at any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-CD70 ADCs comprising one or more antibodies, antibody fragments, or variants thereof, engineered to incorporate one or more non-naturally encoded amino acids site-specifically into the heavy and / or light chain amino acid sequence, conjugated to a drug or payload via a linker.

[0192] The antibodies or antibody fragments or variants of the present disclosure may be human, humanized, engineered, non-human, and / or chimeric antibodies or antibody fragments. The antibodies or antibody fragments or variants provided herein may comprise two or more amino acid sequences. The first amino acid sequence may comprise a first antibody chain, and the second amino acid sequence may comprise a second antibody chain. The first antibody chain may comprise a first amino acid sequence, and the second antibody chain may comprise a second amino acid sequence. An antibody chain may refer to an antibody heavy chain, an antibody light chain, or a combination of a region or all of an antibody heavy chain and a region or all of an antibody light chain. As a non-limiting example, the antibodies provided herein include a heavy chain or a fragment or variant thereof, and a light chain or a fragment or variant thereof. The two amino acid sequences of an antibody comprising two antibody chains may be connected, bonded, or linked by one or more disulfide bonds, chemical linkers, peptide linkers, or combinations thereof. Chemical linkers include linkers via unnatural amino acids. Chemical linkers include linkers mediated by one or more unnatural amino acids. Chemical linkers may include chemical conjugates. Peptide linkers include any amino acid sequence that connects two amino acid sequences. Peptide linkers may contain 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, or 100 or more amino acids. Peptide linkers may be any portion of an antibody, including antibody domains such as variable domains, CH1, CH2, CH3, and / or CL domains. In some embodiments, the heavy and light chains are connected, bonded, or linked, for example, via a peptide linker. In some cases, the heavy and light chains are connected, for example, by one or more disulfide bonds.

[0193] The antibodies, antibody fragments, and antibody variants of the present disclosure may interact with or engage antigens on effector cells. Effector cells may include, but are not limited to, immune cells, genetically modified cells with increased or decreased cytotoxic activity, cells involved in host defense mechanisms, anti-inflammatory cells, leukocytes, lymphocytes, macrophages, erythrocytes, platelets, neutrophils, monocytes, eosinophils, basophils, mast cells, NK cells, B cells, or T cells. In some embodiments, the immune cells may be T cells, such as cytotoxic T cells or natural killer T cells. The antibodies or antibody fragments may interact with receptors on T cells, such as, but not limited to, T cell receptors (TCRs). TCRs may include TCR alpha, TCR beta, TCR gamma, and / or TCR delta or TCR zeta. The antibodies or antibody fragments of the present disclosure may bind to receptors on lymphocytes, dendritic cells, B cells, macrophages, monocytes, neutrophils, and / or NK cells. The antibodies or antibody fragments of the present disclosure can bind to cell surface receptors. The antibodies or antibody fragments of the present disclosure can bind to antigen receptors such as, for example, the CD70 antigen receptor. The antibodies or antibody fragments of the present disclosure can be conjugated to T cell surface antigens.

[0194] Several cell surface antigens have high overexpression patterns in many tumors, making them excellent targets for the development of ADCs. Accordingly, the present disclosure provides novel anti-CD70 antibodies or corresponding antibody fragments, and antibody-drug conjugates thereof, for use as therapeutic agents. Disclosed herein are novel anti-CD70 antibodies, antibody fragments, or variants thereof, each having a non-naturally encoded amino acid that facilitates conjugation of the antibody to a drug (e.g., a drug, payload, or toxin molecule) or drug-linker compound.

[0195] The antibodies, antibody fragments, or variants provided in the present disclosure can be human, humanized, engineered, non-human, and / or chimeric antibodies or antibody fragments that bind to the extracellular domain of target antigens, which may be overexpressed in some cancers. Thus, novel antibodies, compositions, and antibody-drug conjugates for the treatment and / or diagnosis of antigen-expressing cancers, including, but not limited to, CD70-expressing cancers, would be beneficial.

[0196] Antibodies or antibody fragments or variants disclosed herein include, but are not limited to, analogs, isoforms, mimetics, fragments, or hybrids of CD70. Antibodies or antibody fragments or variants of CD70 of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc.

[0197] Antibodies comprising non-naturally occurring amino acids are also disclosed herein. In certain embodiments, antibodies or antibody fragments or variants include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. In some embodiments, an anti-CD70 antibody or antibody fragment or variant comprises one or more non-naturally occurring amino acids.

[0198] Non-limiting examples of antibodies or antibody fragments or variants of the present disclosure include the sequences listed in Table 1.

[0199] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-CD70 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-CD70 antibodies or antibody fragments or variants disclosed herein can be humanized. The anti-CD70 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, CD70 analogs, isoforms, mimetics, fragments, or hybrids. The anti-CD70 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. The anti-CD70 antibodies or antibody fragments or variants of the present disclosure can contain one or more polypeptide chains (e.g., one or more heavy and / or light chains) and can be characterized by the amino acid sequence of one or more polypeptide chains. The anti-CD70 antibody or antibody fragment or variant of the present disclosure comprises the amino acid sequence of SEQ ID NO:1-9 (Table 1). The antibody, fragment, or variant of the present disclosure can be an anti-CD70 antibody, fragment, or variant. In certain embodiments, the anti-CD70 antibody comprises heavy and light chain amino acid sequences selected from the group consisting of SEQ ID NO:1-9. In certain embodiments, the anti-CD70 antibody consists of heavy and light chain amino acid sequences selected from the group consisting of SEQ ID NO:1-9. In certain embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a light chain amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In certain embodiments, the anti-CD70 antibody comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence is SEQ ID NO:3 and the light chain amino acid sequence is SEQ ID NO:2.

[0200] The present disclosure provides, by way of non-limiting example, anti-CD70 ADCs, which are humanized monoclonal antibody-drug conjugates that function by promoting cell survival and proliferation of antigen-primed CD8 T cells, the formation of memory T cells, and the proliferation of B cells. CD70 receptors are found at high densities in cancer tissues, e.g., 34,000 to 189,000 copies per cell (Caki-1, 786-O, L-428, UMRC3, LP-1, DBTRG-05MG) (McDonagh, C. F., Engineered anti-CD70 antibody-drug conjugates with increased therapeutic index, Molecular Cancer Therapeutics, 7(9):2913-2923 (2008)). In non-cancerous and / or normal tissues, the receptor is present on 5% to 15% of activated T cells and 10% to 25% of activated B cells. CD70 expression has been found in approximately 40% of multiple myeloma isolates (Preclinical Characterization of SGN-70, a Humanized Antibody Directed against CD70, Cancer Therapy: Preclinical, 2008). For example, CD70 expression has been confirmed in a high percentage of Hodgkin's lymphoma Reed-Sternberg cells, non-Hodgkin's lymphoma, and renal cell carcinoma tumors. CD70 is a type II integral membrane protein of the TNF family. For purposes of the present invention, the anti-CD70 antibody can be any known CD70 antibody containing one non-naturally encoded amino acid. For illustrative purposes, anti-CD70 antibodies are listed in Table 1.

[0201] In one embodiment of the invention, the ADC comprises a heavy chain, wherein the heavy chain amino acid sequence is SEQ ID NO: 3 with one non-naturally encoded amino acid at position A114 (Kabat numbering). In some embodiments, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine (pAF). In another embodiment of the invention, the antibody comprises a light chain, wherein the light chain amino acid sequence is SEQ ID NO: 2.

[0202] In some other embodiments of the invention, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 90% identity with SEQ ID NO: 1. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence optionally comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 90% identity with SEQ ID NO: 2. In other embodiments, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 90% identity with SEQ ID NO: 3. In other embodiments, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 90% identity with SEQ ID NO: 4. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 90% identity with SEQ ID NO: 5. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 90% identity with SEQ ID NO: 6. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 90% identity with SEQ ID NO: 7. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 90% identity with SEQ ID NO: 8. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 90% identity with SEQ ID NO: 9.

[0203] In some other embodiments of the invention, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 95% identity with SEQ ID NO: 1. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence optionally comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 95% identity with SEQ ID NO: 2. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 95% identity with SEQ ID NO: 3. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 95% identity with SEQ ID NO: 4. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 95% identity with SEQ ID NO: 5. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 95% identity with SEQ ID NO: 6. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 95% identity with SEQ ID NO: 7. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 95% identity with SEQ ID NO: 8. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 95% identity with SEQ ID NO: 9.

[0204] In some other embodiments of the invention, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 96% identity with SEQ ID NO: 1. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence optionally comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 96% identity with SEQ ID NO: 2. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 96% identity with SEQ ID NO: 3. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 96% identity with SEQ ID NO: 4. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 96% identity with SEQ ID NO: 5. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 96% identity with SEQ ID NO: 6. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 96% identity with SEQ ID NO: 7. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 96% identity with SEQ ID NO: 8. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 96% identity with SEQ ID NO: 9.

[0205] In some other embodiments of the invention, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 97% identity with SEQ ID NO: 1. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence optionally comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 97% identity with SEQ ID NO: 2. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 97% identity with SEQ ID NO: 3. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 97% identity with SEQ ID NO: 4. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 97% identity with SEQ ID NO: 5. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 97% identity with SEQ ID NO: 6. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 97% identity with SEQ ID NO: 7. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 97% identity with SEQ ID NO: 8. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 97% identity with SEQ ID NO: 9.

[0206] In some other embodiments of the invention, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 98% identity with SEQ ID NO: 1. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence optionally comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 98% identity with SEQ ID NO: 2. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 98% identity with SEQ ID NO: 3. In other embodiments, an anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 98% identity with SEQ ID NO: 4. In other embodiments, an anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 98% identity with SEQ ID NO: 5. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 98% identity with SEQ ID NO: 6. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 98% identity with SEQ ID NO: 7. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 98% identity with SEQ ID NO: 8. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 98% identity with SEQ ID NO: 9.

[0207] In some other embodiments of the invention, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 99% identity with SEQ ID NO: 1. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence optionally comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 99% identity with SEQ ID NO: 2. In other embodiments, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 99% identity with SEQ ID NO: 3. In other embodiments, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares at least 99% identity with SEQ ID NO: 4. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 99% identity with SEQ ID NO: 5. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 99% identity with SEQ ID NO: 6. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 99% identity with SEQ ID NO: 7. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 99% identity with SEQ ID NO: 8. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence comprising one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares at least 99% identity with SEQ ID NO: 9.

[0208] In some other embodiments of the invention, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence, wherein the heavy chain amino acid sequence shares 100% identity with SEQ ID NO: 1. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence, optionally including one or more non-naturally encoded amino acids, wherein the one or more non-naturally encoded amino acids replace one or more amino acids in the light chain amino acid sequence that shares 100% identity with SEQ ID NO: 2. In other embodiments, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares 100% identity with SEQ ID NO: 3. In other embodiments, the anti-CD70 antibody comprises a heavy chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the heavy chain amino acid sequence shares 100% identity with SEQ ID NO: 4. In other embodiments, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares 100% identity with SEQ ID NO: 5. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares 100% identity with SEQ ID NO: 6. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares 100% identity with SEQ ID NO: 7. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares 100% identity with SEQ ID NO: 8. In another embodiment, the anti-CD70 antibody comprises a light chain having an amino acid sequence with one or more non-naturally encoded amino acids, wherein the light chain amino acid sequence shares 100% identity with SEQ ID NO: 9.

[0209] In some preferred embodiments of the invention, the antibody is an anti-CD70 antibody comprising (i) a light chain having an amino acid sequence that shares 100% identity with SEQ ID NO:2, and (ii) a heavy chain having an amino acid sequence that has one non-naturally encoded amino acid that shares 100% identity with SEQ ID NO:3, where the non-naturally encoded amino acid is para-acetyl-L-phenylalanine (pAF). In some other preferred embodiments of the invention, the anti-CD70 antibody comprises (i) two light chains, each having an amino acid sequence, where each light chain amino acid sequence has one non-naturally encoded amino acid that shares 100% identity with SEQ ID NO:2, and (ii) two heavy chains, each having an amino acid sequence, where each heavy chain amino acid sequence has one non-naturally encoded amino acid that shares 100% identity with SEQ ID NO:3, where each non-naturally encoded amino acid is para-acetyl-L-phenylalanine (pAF). Selection of non-naturally encoded amino acid sites is described herein, and as it relates specifically to anti-CD70 antibodies, sites were selected based on surface exposure / site accessibility within the antibody, and hydrophobic / neutral amino acid sites were selected to maintain charge on the antibody.

[0210] Table 1. Anti-CD70 heavy chain (HC) and light chain (LC) amino acid sequences with amber sites for incorporation of non-natural amino acids (nnAA). Also disclosed are all sequences in Table 1 in which X is replaced by any nnAA, all sequences in Table 1 in which any amino acid is replaced by any non-natural amino acid, all sequences in Table 1 in which X is pAF, all heavy chain sequences in Table 1 in which a non-naturally encoded amino acid has been site-specifically incorporated at position 114 according to Kabat numbering, as known to those of skill in the art, and all heavy chain sequences in Table 1 in which EEM has been replaced with DEL. WT: wild type, HC: heavy chain, LC: light chain, X represents nnAA.

[0211] [Table 1-1]

[0212] [Table 1-2]

[0213] [Table 1-3]

[0214] In some aspects, the present disclosure provides isolated anti-CD70 antibodies, or fragments thereof, comprising at least one amino acid sequence selected from the group consisting of the sequences listed in Table 1. In some embodiments, isolated anti-CD70 antibodies, or fragments thereof, are provided, consisting of at least one amino acid sequence selected from the group consisting of the sequences listed in Table 1.

[0215] In some embodiments, an isolated anti-CD70 antibody or fragment thereof is provided that shares 100% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0216] In some embodiments, an isolated anti-CD70 antibody or fragment thereof that shares 100% sequence identity with the amino acid sequence of SEQ ID NO:3 is provided.

[0217] In some embodiments, an isolated anti-CD70 antibody or fragment thereof is provided, having a light chain that shares 100% sequence identity with the amino acid sequence of SEQ ID NO:2 and a heavy chain that shares 100% sequence identity with the amino acid sequence of SEQ ID NO:3.

[0218] In some embodiments, an isolated anti-CD70 antibody or fragment thereof that shares 100% sequence identity with the amino acid sequence of SEQ ID NO:4 is provided.

[0219] In some embodiments, an isolated anti-CD70 antibody or fragment thereof sharing 100% identity with the amino acid sequence of SEQ ID NO:5 is provided.

[0220] In some embodiments, an isolated anti-CD70 antibody or fragment thereof sharing 100% identity with the amino acid sequence of SEQ ID NO:6 is provided.

[0221] In some embodiments, an isolated anti-CD70 antibody or fragment thereof sharing 100% sequence identity with the amino acid sequence of SEQ ID NO:7 is provided.

[0222] In some embodiments, an isolated anti-CD70 antibody or fragment thereof sharing 100% sequence identity with the amino acid sequence of SEQ ID NO:8 is provided.

[0223] In some embodiments, an isolated anti-CD70 antibody or fragment thereof sharing 100% sequence identity with the amino acid sequence of SEQ ID NO:9 is provided.

[0224] In some embodiments, a nucleic acid encoding any one of SEQ ID NO:1 through SEQ ID NO:9 is provided.

[0225] In some embodiments, a nucleic acid is provided that encodes any one of SEQ ID NO: 3 to SEQ ID NO: 9. In some embodiments, a nucleic acid is provided that encodes SEQ ID NO: 3.

[0226] In some general aspects, the disclosure provides a vector comprising a nucleic acid encoding any one of SEQ ID NO:1 through SEQ ID NO:9. In some embodiments, the disclosure provides a nucleic acid encoding any one of SEQ ID NO:3 through SEQ ID NO:9. In some embodiments, the disclosure provides a nucleic acid encoding SEQ ID NO:3.

[0227] Drug-Linker In some aspects, the present disclosure relates to linkers for intracellular delivery of drug conjugates. Many procedures and linker molecules are known for the attachment of various compounds to peptides. See, for example, European Patent Application No. 0188256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, 4,569,789, and 10,550,190, PCT Publication Nos. WO2012 / 166559(A1), WO2012 / 166560(A1), WO2013 / 185117(A1), WO2013 / 192360(A1), and WO2022 / 040596(A1), and U.S. Patent Application Publication No. 2017 / 0182181(A1). the contents of each of which are incorporated herein by reference in their entirety.

[0228] Methods for selecting and designing linkers are well known in the art. Linkers can be designed ab initio, by way of example only, as part of a high-throughput screening process (where large numbers of polypeptides can be designed, synthesized, characterized, and / or tested) or based on the researcher's goals. Linkers can also be designed based on the structure of a known or partially characterized polypeptide. Principles for selecting which amino acids to replace and / or modify, as well as the selection of modifications to use, are described, for example, in WO 2013 / 185117. Linkers can be designed to meet the needs of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic efficacy of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacology, and / or pharmacodynamics of the polypeptide, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Additionally, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating antibodies, and any combination of the foregoing modifications.

[0229] Generally, the linker of the present disclosure can be a unit that can be combined with one or more additional units so that the combined linker unit can be bound to one or more drugs or payloads. Each linker unit can be composed of one or more moieties, each of which can appear one or more times. A non-limiting example of a linker unit can include a divalent -(CH2CH2-O)- moiety. The linker can further contain a reactive moiety, such as an aminooxy group. The reactive moiety can be attached, for example, to the distal end of the linker, and the drug can be attached, for example, to the proximal end of the linker. Thus, the linker can act as a spacer or bridge between the drug and the reactive moiety.

[0230] Drugs with linkers containing an aminooxy group can react with various electrophilic groups to form conjugates. Similar to hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of the aminooxy group allows it to react efficiently and selectively with various molecules containing carbonyl groups (including, but not limited to, ketones). See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117: 3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9): 727-736 (2001). Oximes generally result from the reaction of an aminooxy group with a carbonyl-containing group (e.g., a ketone, such as an acyl group).

[0231] Thus, in certain embodiments, described herein are drug-linkers that include an aminooxy group. Such drug-linkers may be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and may optionally be post-translationally modified.

[0232] In some embodiments, the linkers disclosed herein are connected to a drug and also to an antibody, antibody fragment, or variant thereof via a linking or adduct moiety, thus bridging the drug / payload and the antibody, antibody fragment, or variant thereof.

[0233] In some aspects, the present disclosure provides a drug-linker / payload, wherein the drug or payload is a cytotoxic drug or agent. In some aspects of the present disclosure, the cytotoxic agent has the following structure:

[0234] [ka] or a salt thereof.

[0235] Drug-linker compounds such as Compound 6 can be used or conjugated with any targeting ligand, such as an antibody or antibody fragment, selected based on its specificity for an antigen expressed on a target cell or at a target site of interest. The drug or payload linkers of the present invention can be used with antibodies or antibody fragments directed against various antigens, including, but not limited to, tumor-associated antigens, tumor-specific antigens, cancer antigens, or disease-specific antigens. In some embodiments, drug-linker compounds such as Compound 6 can be used with anti-CD70 antibodies, antibody fragments, or antibody-drug conjugates of the present invention. The synthesis of such drug-linkers is well known to those skilled in the art. See, for example, EP 14874745; Dubowchik et al., Bioconjugate Chem. 13:855-869, (2002); Doronina et al., Nature Biotechnology 21(7):778-784, 2003; WO 2012 / 166560; and WO 2013 / 185117. Each is incorporated herein by reference.

[0236] The present disclosure provides drug moieties having linkers that reduce the toxicity of the drug moiety in vivo while retaining pharmacological activity. In some embodiments, the toxicity of the linked drug is reduced or eliminated when administered to an animal or human compared to the free toxic group or a toxic group derivative containing a labile linkage while retaining pharmacological activity. In some embodiments, increased doses of the linked drug group can be administered to an animal or human with greater safety. In certain embodiments, non-natural amino acid polypeptides linked to the drug moiety provide in vitro and in vivo stability. In some embodiments, non-natural amino acid polypeptides linked to the drug moiety are effective and have reduced toxicity compared to the free drug moiety.

[0237] Antibody-drug conjugates The antibody-drug conjugates (ADCs) of the present disclosure offer novel therapeutic approaches by combining the selectivity of antibodies containing one or more unnatural amino acids with cytotoxic agents conjugated to the antibodies. Targeted delivery of cytotoxic drugs to tumor tissue significantly increases the therapeutic window of these agents. The ADCs of the present disclosure comprise antibodies conjugated to cytotoxic drugs via linkers. The stability of the linker between the antibody and the cytotoxic drug is essential for ADC integrity in circulation. Successful development of an ADC for a given target antigen depends on optimization of antibody selection, linker design and stability, drug potency, and the conjugation mode of the drug and linker to the antibody. pH- and redox-sensitive and protease-sensitive linker properties affect the circulatory stability and release of the drug moiety.

[0238] In some embodiments of the present disclosure, the antibody of the ADC comprises a full-length antibody or fragment thereof that binds to an antigen and is conjugated to a cytotoxic or immunosuppressive agent, wherein the antibody-drug conjugate exerts (a) a cytotoxic or cytostatic effect on antigen-expressing cells or antigen-targeted cells, or (b) a cytotoxic, cytostatic, or immunosuppressive / immunostimulatory effect on antigen-expressing immune cells, and wherein the conjugation occurs at a non-naturally encoded amino acid in the antibody. In some embodiments, the antigen of the antigen-expressing cells, antigen-targeted cells, or antigen-expressing immune cells is, but is not limited to, CD70.

[0239] In some embodiments, an antibody, variant, or composition of the present disclosure may be an antibody, variant, or composition that binds to an antigen receptor. In other embodiments, an antibody, variant, or composition may be an antibody, variant, or composition that binds to the extracellular surface of an antigen receptor. In some embodiments, an antibody, variant, or composition of the present disclosure may be an antibody, variant, or composition having a CDR grafted into a framework region of a variable region. In other embodiments, an antibody, variant, or composition of the present disclosure may be an antibody, variant, or composition having a non-naturally encoded amino acid. In some embodiments, an antibody, variant, or composition may be an antibody, variant, or composition described by two or more of the embodiments elsewhere in this disclosure. In some embodiments, an antibody, antibody variant, or antibody composition disclosed herein may be fully humanized. In other embodiments, an antibody, antibody variant, or antibody composition disclosed herein may be chimeric. In some embodiments, the antibody can be an antibody that is a full length antibody (variable + Fc region), a Fab, a bispecific, a Fab dimer, a Fab bispecific, a Fab trispecific, a bispecific T cell engager, a dual affinity retargeting antibody, an IgG1 / IgG3 bispecific antibody, a diabody, a bispecific diabody, an scFv-Fc, a minibody.

[0240] In one embodiment, an ADC comprises an antibody conjugated to a drug, wherein conjugation occurs via a non-naturally encoded amino acid in the antibody. In one embodiment, an ADC comprises an antibody conjugated to a drug, wherein conjugation occurs via a non-naturally encoded amino acid in the heavy chain of the antibody. In one embodiment, an ADC comprises an antibody conjugated to a drug, wherein conjugation occurs via a non-naturally encoded amino acid in the light chain of the antibody. In one embodiment, an ADC comprises a full-length antibody conjugated to a drug, wherein conjugation occurs via a non-naturally encoded amino acid in the heavy chain of the antibody. In one embodiment, an ADC comprises a full-length antibody conjugated to a drug, wherein conjugation occurs via a non-naturally encoded amino acid in the light chain of the antibody.

[0241] In some embodiments, the drug of the ADC is a cytotoxic drug or cytotoxic agent. In some aspects of the present disclosure, the cytotoxic drug is Compound 6. In some embodiments, the drug is produced as described in the Examples herein. In some embodiments, the ADC comprises an antibody, antibody fragment, or variant thereof, engineered to have one or more non-naturally encoded amino acids site-specifically incorporated into the heavy and / or light chain amino acid sequence, conjugated to the drug via a linker.

[0242] In some embodiments, the invention provides an anti-CD70 ADC, wherein the antibody is an anti-CD70 antibody comprising a light chain and a heavy chain, and the antibody is conjugated to a drug via a non-naturally encoded amino acid. In some more specific embodiments, the anti-CD70 ADC comprises (a) an anti-CD70 antibody comprising (i) two light chain amino acid sequences, each amino acid sequence sharing 100% identity with SEQ ID NO: 2, and (ii) two heavy chain amino acid sequences, each amino acid sequence having the non-naturally encoded amino acid para-acetyl-L-phenylalanine (pAF) at position 114 (Kabat numbering) and sharing 100% identity with SEQ ID NO: 3, and (b) a drug conjugated to the anti-CD70 antibody via a pAF. In some even more specific embodiments, a drug is conjugated to each pAF such that the ADC comprises two drug payloads. Thus, the drug-to-antibody ratio (DAR) is about 2. In some embodiments, the drug is Compound 6. In some embodiments, a drug is conjugated to each pAF via an oxime bond. It is understood that the stoichiometry of the drug to the antibody during the conjugation reaction may be less than 2, or the conjugation reaction between the drug and the antibody may be incomplete, resulting in a DAR of less than 2. Therefore, the DAR may be a non-integer value, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9.

[0243] In some aspects, the present disclosure provides: The following structure:

[0244] [ka] and Provided is an antibody drug conjugate (ADC) comprising a drug-linker group having an anti-CD70 antibody or fragment thereof comprising one or more heavy chains, wherein at least one member of the one or more heavy chains (a) comprises SEQ ID NO:3 or (b) has an amino acid sequence that shares at least 90% identity with SEQ ID NO:3; During the ceremony,

[0245] [ka] represents a single bond or a double bond, # denotes connection to an anti-CD70 antibody or fragment thereof.

[0246] In some embodiments, at least one member of the one or more heavy chains has an amino acid sequence that shares at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 3. In some embodiments, at least one member of the one or more heavy chains has the amino acid sequence of SEQ ID NO: 3. In some embodiments, at least one member of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 3. In some embodiments, each of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 3.

[0247] In some embodiments, the anti-CD70 antibody or fragment thereof comprises two heavy chains, each heavy chain having an amino acid sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3. In some embodiments, each heavy chain has the amino acid sequence of SEQ ID NO: 3. In some embodiments, each heavy chain is the amino acid sequence of SEQ ID NO: 3.

[0248] In some embodiments, the anti-CD70 antibody or fragment thereof further comprises one or more light chains, wherein at least one of the one or more light chains has an amino acid sequence that shares at least 90% identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, at least one of the one or more light chains has an amino acid sequence that shares at least 90% identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, at least one of the one or more light chains has the amino acid sequence of SEQ ID NO: 2.

[0249] In some embodiments, the anti-CD70 antibody or fragment thereof comprises two light chains. In some embodiments, each light chain has an amino acid sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the anti-CD70 antibody or fragment thereof comprises two light chains, each light chain having the amino acid sequence of SEQ ID NO: 2.

[0250] In some other embodiments, at least one of the one or more light chains has an amino acid sequence that contains a non-naturally encoded amino acid, and the amino acid sequence is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0251] In some embodiments, each

[0252] [ka] represents a double bond.

[0253] In some embodiments, the drug-linker group is one or more drug linker groups.

[0254] In some aspects, the present disclosure provides: The following structure:

[0255] [ka] and one or more drug-linker groups having Provided is an antibody drug conjugate (ADC) comprising an anti-CD70 antibody or fragment thereof comprising one or more heavy chains, wherein at least one of the one or more heavy chains has an amino acid sequence that contains a non-naturally encoded amino acid, the amino acid sequence being selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; During the ceremony, each

[0256] [ka] represents a single or double bond covalently attaching one of the one or more drug-linker groups to the anti-CD70 antibody or fragment thereof; Each # represents a site of attachment to an anti-CD70 antibody or fragment thereof.

[0257] In some embodiments, at least one of the one or more heavy chains has the amino acid sequence of SEQ ID NO: 3. In some embodiments, at least one of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 3. In some embodiments, each of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 3.

[0258] In some other embodiments, at least one of the one or more heavy chains has the amino acid sequence of SEQ ID NO: 4. In some other embodiments, at least one of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 4. In some other embodiments, each of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 4.

[0259] In some other embodiments, at least one of the one or more heavy chains has the amino acid sequence of SEQ ID NO: 5. In some other embodiments, at least one of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 5. In some other embodiments, each of the one or more heavy chains is the amino acid sequence of SEQ ID NO: 5.

[0260] In some embodiments, the anti-CD70 antibody or fragment thereof comprises two heavy chains, and each heavy chain comprises one non-naturally encoded amino acid. In some embodiments, each heavy chain has the amino acid sequence of SEQ ID NO:3.

[0261] In some other embodiments, the anti-CD70 antibody or fragment thereof comprises two heavy chains, and each heavy chain comprises one non-naturally encoded amino acid. In some embodiments, each heavy chain has the amino acid sequence of SEQ ID NO:4.

[0262] In some other embodiments, the anti-CD70 antibody or fragment thereof comprises two heavy chains, and each heavy chain comprises one non-naturally encoded amino acid. In some embodiments, each heavy chain has the amino acid sequence of SEQ ID NO:5.

[0263] In some embodiments, the anti-CD70 antibody or fragment thereof further comprises one or more light chains, wherein at least one of the one or more light chains has an amino acid sequence that shares at least 90% identity with SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, at least one of the one or more light chains has an amino acid sequence that shares at least 90% identity with the amino acid sequence of SEQ ID NO:2. In some embodiments, at least one of the one or more light chains has the amino acid sequence of SEQ ID NO:2. In some embodiments, at least one of the one or more light chains is the amino acid sequence of SEQ ID NO:2. In some embodiments, each of the one or more light chains is the amino acid sequence of SEQ ID NO:2.

[0264] In some embodiments, the anti-CD70 antibody or fragment thereof comprises two light chains, in some embodiments, each light chain having an amino acid sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the anti-CD70 antibody or fragment thereof comprises two light chains, each light chain having the amino acid sequence of SEQ ID NO: 2.

[0265] In some other embodiments, at least one of the one or more light chains has an amino acid sequence that contains a non-naturally encoded amino acid, and the amino acid sequence is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0266] In some embodiments, each

[0267] [ka] represents a double bond. In some embodiments, each double bond covalently attaches one of the one or more drug-linker groups to a non-naturally encoded amino acid of an anti-CD70 antibody or fragment thereof. In some embodiments, each non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

[0268] Thus, in some embodiments, the double bond is between the Drug-Linker group and the non-naturally encoded para-acetyl-L-phenylalanine incorporated into the antibody or fragment thereof. In some embodiments, the double bond is the product of a reaction between a terminal aminooxy group of the Drug-Linker compound and the acetyl group of the non-naturally encoded para-acetyl-L-phenylalanine incorporated into the anti-CD70 antibody or fragment thereof. Thus, in some embodiments, the Drug-Linker moiety is attached to the antibody or fragment thereof via an oxime group. In some embodiments, the Drug-Linker compound having a terminal aminooxy group has the following structure:

[0269] [ka] Compound 6 has the formula:

[0270] In some further embodiments, the ADC has formula (I):

[0271] [ka] ADC of During the ceremony, Ab is an anti-CD70 antibody or fragment thereof; each R is an unsubstituted C1-C8 alkyl; d is at least 1 and at most 10.

[0272] Thus, an ADC comprises at least one and up to ten drug-linker groups, each of the one or more drug-linker groups having the following structure:

[0273] [ka] and where each # represents a site of attachment to an anti-CD70 antibody or fragment thereof.

[0274] In some embodiments, d is 1, 2, 3, or 4. Thus, in some embodiments, the ADC comprises 1, 2, 3, or 4 drug-linker groups, respectively. In some embodiments, d is at least 1 and at most 2. Thus, in some embodiments, the ADC comprises at least 1 and at most 2 drug-linker groups.

[0275] In some embodiments, d is 2. Thus, the ADC comprises two drug-linker groups.

[0276] In some embodiments, each R is methyl. The R group may represent the methyl group of the acyl moiety of para-acetyl-L-phenylalanine incorporated into the anti-CD70 antibody.

[0277] In some embodiments, the anti-CD70 antibody or fragment thereof is humanized.

[0278] In some embodiments, the anti-CD70 antibody or fragment thereof is a humanized monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO: 3 and each light chain has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 3 comprises one non-naturally encoded amino acid, wherein the one non-naturally encoded amino acid is para-acetyl-L-phenylalanine. In some embodiments, d is 2.

[0279] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO: 4 and each light chain has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 4 comprises one non-naturally encoded amino acid, wherein the one non-naturally encoded amino acid is para-acetyl-L-phenylalanine. In some embodiments, d is 2.

[0280] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO: 5 and each light chain has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 5 comprises one non-naturally encoded amino acid, wherein the one non-naturally encoded amino acid is para-acetyl-L-phenylalanine. In some embodiments, d is 2.

[0281] In some embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains, two light chains, and two non-naturally encoded amino acids; each such heavy chain has the amino acid sequence of SEQ ID NO:3, wherein SEQ ID NO:3 contains one non-naturally encoded amino acid, and each non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position A114 (Kabat numbering) of SEQ ID NO:3; each said light chain having the amino acid sequence of SEQ ID NO:2; each R is methyl; the one or more Drug-Linker groups are two Drug-Linker groups, and d is 2, Each of the drug-linker groups is attached to one of the para-acetyl-L-phenylalanines at position A114, thereby attaching each drug-linker group to the anti-CD70 monoclonal antibody.

[0282] In some embodiments, the ADC is the ADC of FIG.

[0283] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains, two light chains, and two non-naturally encoded amino acids; each such heavy chain has the amino acid sequence of SEQ ID NO:4, wherein SEQ ID NO:4 contains one non-naturally encoded amino acid, and each non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position A136 (Kabat numbering) of SEQ ID NO:4; each said light chain having the amino acid sequence of SEQ ID NO:2; each R is methyl; the one or more Drug-Linker groups are two Drug-Linker groups; d is 2, Each of the drug-linker groups is attached to one of the para-acetyl-L-phenylalanines at position A136, thereby attaching each drug-linker group to the anti-CD70 monoclonal antibody.

[0284] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains, two light chains, and two non-naturally encoded amino acids; each such heavy chain has the amino acid sequence of SEQ ID NO:5, wherein SEQ ID NO:5 contains one non-naturally encoded amino acid, and each non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position L159 (Kabat numbering) of SEQ ID NO:5; each said light chain having the amino acid sequence of SEQ ID NO:2; each R is methyl; the one or more Drug-Linker groups are two Drug-Linker groups; d is 2, Each of the drug-linker groups is attached to one of the para-acetyl-L-phenylalanines at position L159, thereby attaching each drug-linker group to the anti-CD70 monoclonal antibody.

[0285] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains and two light chains; each such heavy chain has the amino acid sequence of SEQ ID NO:3, wherein SEQ ID NO:3 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid being para-acetyl-L-phenylalanine at position A114 (Kabat numbering) of SEQ ID NO:3; Each such light chain comprises: SEQ ID NO:6 (SEQ ID NO:6 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position V110 of SEQ ID NO:6); SEQ ID NO:7 (SEQ ID NO:7 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position A112 of SEQ ID NO:7), SEQ ID NO:8 (SEQ ID NO:8 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position S114 of SEQ ID NO:8), and having an amino acid sequence selected from the group consisting of: SEQ ID NO:9, which contains one non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position S121 of SEQ ID NO:9; each R is methyl; the one or more Drug-Linker groups are four Drug-Linker groups; d is 4, Each of the drug-linker groups is connected to one of the para-acetyl-L-phenylalanines, thereby connecting each drug-linker group to the anti-CD70 monoclonal antibody.

[0286] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains and two light chains; each such heavy chain has the amino acid sequence of SEQ ID NO:4, wherein SEQ ID NO:4 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid being para-acetyl-L-phenylalanine at position A136 (Kabat numbering) of SEQ ID NO:4; Each such light chain comprises: SEQ ID NO:6 (SEQ ID NO:6 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position V110 of SEQ ID NO:6); SEQ ID NO:7 (SEQ ID NO:7 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position A112 of SEQ ID NO:7), SEQ ID NO:8 (SEQ ID NO:8 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position S114 of SEQ ID NO:8), and having an amino acid sequence selected from the group consisting of: SEQ ID NO:9, which contains one non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position S121 of SEQ ID NO:9; each R is methyl; The one or more drug-linker groups are four drug-linker groups. d is 4, Each of the drug-linker groups is connected to one of the para-acetyl-L-phenylalanines, thereby connecting each drug-linker group to the anti-CD70 monoclonal antibody.

[0287] In some other embodiments, the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains and two light chains; each such heavy chain has the amino acid sequence of SEQ ID NO:5, wherein SEQ ID NO:5 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid being para-acetyl-L-phenylalanine at position L159 (Kabat numbering) of SEQ ID NO:5; Each such light chain comprises: SEQ ID NO:6 (SEQ ID NO:6 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position V110 of SEQ ID NO:6); SEQ ID NO:7 (SEQ ID NO:7 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position A112 of SEQ ID NO:7), SEQ ID NO:8 (SEQ ID NO:8 contains one non-naturally encoded amino acid, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position S114 of SEQ ID NO:8), and having an amino acid sequence selected from the group consisting of: SEQ ID NO:9, which contains one non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine at position S121 of SEQ ID NO:9; each R is methyl; the one or more Drug-Linker groups are four Drug-Linker groups; d is 4, Each of the drug-linker groups is connected to one of the para-acetyl-L-phenylalanines, thereby connecting each drug-linker group to the anti-CD70 monoclonal antibody.

[0288] It is understood that ADCs are typically produced as a population of ADCs, i.e., a composition containing a mixture of ADCs that are essentially identical except for drug loading. As disclosed herein, ADC compositions can be characterized by a drug-to-antibody ratio (DAR), which reports on the average number of drugs conjugated to antibodies in an ADC composition. Thus, in some aspects, the present disclosure provides ADC compositions comprising a mixture of ADCs, each ADC in the mixture being identical except for the number of drugs or drug-linkers conjugated to each antibody, which may vary.

[0289] In a non-limiting example, an ADC of the disclosure includes a first ADC, a second ADC, a third ADC, and a fourth ADC, wherein the first ADC, the second ADC, the third ADC, and the fourth ADC are identical except that the first ADC includes one drug or drug-linker, the second ADC includes two drugs or drug-linkers, the third ADC includes three drugs or drug-linkers, and the fourth ADC includes four drugs or drug-linkers.

[0290] In another non-limiting example, an ADC composition is provided, the ADC composition comprising: (a) an ADC of formula (I), wherein d is 1; (b) an ADC of formula (I), wherein the ADC is identical to formula (a), except that d is 2; (c) an ADC of formula (I), wherein the ADC is identical to formula (a), except that d is 3; (d) an ADC of formula (I), wherein the ADC is identical to formula (a), except that d is 4; or a combination of any two or more of the foregoing, wherein the composition is characterized by having a DAR of at least about 1 and at most about 4.

[0291] In some embodiments, the ADC compositions of the present disclosure are characterized by having a DAR of at least about 1 and at most about 8. In some embodiments, the ADC compositions are characterized by having a DAR of at least about 1 and at most about 4. In some embodiments, the ADC compositions are characterized by having a DAR of at least about 1 and at most about 2. In some other embodiments, the ADC compositions are characterized by having a DAR of about 2. In some other embodiments, the ADC compositions are characterized by having a DAR of about 3. In some other embodiments, the ADC compositions are characterized by having a DAR of about 4.

[0292] Methods and Techniques The present disclosure encompasses methodologies and techniques well known in the art. These include conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology that are within the skill of the art. The compounds of the present disclosure can be synthesized using several processes or schemes used in the art. See, for example, Dubowchik et al., Bioconjugate Chem. 13:855-869, 2002; Doronina et al., Nature Biotechnology 21(7):778-784, 2003; WO 2012 / 166560; and WO 2013 / 185117, each of which is incorporated herein by reference. Many methodologies and techniques for the synthesis of pharmaceutical, diagnostic, or therapeutic compounds are well known to those skilled in the art.

[0293] The present disclosure also embraces, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), cell biology, biochemistry and immunology, all of which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (Sambrook et al. Eds., 2001), Oligonucleotide Synthesis: Methods And Applications (Methods in Molecular Biology), Herdewijn, P., Ed., Humana Press, Totowa, NJ, Oligonucleotide Synthesis (Gait, MJ, Ed., 1984), Methods In Molecular Biology, Humana Press, Totowa, NJ, Cell Biology: A Laboratory Notebook, Academic Press, New York, NY (Cellis, JE, Ed., 1998), Animal Cell Culture (Freshney, RI, Ed., 1987), Introduction To Cell And Tissue Culture Plenum Press, New York, NY, (Mather, JP and Roberts, PE, Eds., 1998), Cell And Tissue Culture: Laboratory Procedures John Wiley and Sons, Hoboken, NJ, (Doyle, A. et al., Eds., 1993-8), Methods In Enzymology (Academic Press, Inc.) New York, NY, Weir's Handbook Of Experimental Immunology Wiley-Blackwell Publishers, New York, NY, (Herzenberg, LA et al. Eds.,1997)、Gene Transfer Vectors For Mammalian Cells Cold Spring Harbor Press,Cold Spring Harbor,NY,(Miller,J.M.et al.Eds.,1987)、Current Protocols In Molecular Biology,Greene Pub.Associates,New York,NY,(Ausubel,F.M.et al.,Eds.,1987)、PCR:The Polymerase Chain Reaction,Birkhauser,Boston,MA,(Mullis,K.et al.,Eds.,1994)、Current Protocols In Immunology,John Wiley and Sons,Hoboken,NJ,(Coligan,J.E.et al.,eds.,1991)、Short Protocols In Molecular Biology,Hoboken,NJ,(John Wiley and Sons,1999)、Immunobiology 7 Garland Science,London,UK,(Janeway,C.A.et al.,2007)、Antibodies.Stride Publications,Devoran,UK,(P.Finch,1997)、Antibodies:A Practical Approach Oxford University Press,USA,New York,NY,(D.Catty.,ed.,1989)、Monoclonal Antibodies:A Practical Approach Oxford University Press,USA,New York NY,(Shepherd,P.et al.Eds.,2000)、Using Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,(Harlow,E.et al.Eds.,1998)、The Antibodies Harwood Academic Publishers,London,UK,(Zanetti,M.This is fully explained in literature such as (Eds. et al. 1995).

[0294] Therapeutic Uses of ADCs The antibodies or ADCs of the present disclosure are useful for treating a wide range of diseases, disorders, conditions, or cancers. The present disclosure includes compositions for use in, and methods of treating, mammals at risk for, having, or / and having a disease or condition, such as cancer, that responds to CD70 overexpression, amplification, mutation, and / or targeted therapy. The compositions disclosed herein can be used to modulate an immune response. Modulating an immune response can include stimulating, activating, increasing, enhancing, or upregulating the immune response. Modulating an immune response can include suppressing, inhibiting, preventing, reducing, or downregulating the immune response. In some embodiments, the ADCs of the present invention can be used to reduce or inhibit tumor growth or progression in antigen-expressing cancers or cancer cells, comprising an effective amount of the ADC.

[0295] Administration of an ADC may result in a short-term effect, i.e., an immediate beneficial effect on some observed clinical parameter, which may occur within 12 or 24 hours from the time of administration, and / or a long-term effect, e.g., a beneficial slowing of tumor growth progression or a reduction in tumor size. The ADCs of the present disclosure may be administered by any means known to those of skill in the art, and may advantageously be administered via infusion, e.g., intraarterial, intraperitoneal, or intravenous injection and / or infusion, at a dosage sufficient to achieve the desired pharmacological effect. In some embodiments, the ADC, or a composition or formulation comprising the ADC, is administered orally, intradermally, intratumorally, intravenously, or subcutaneously. In some embodiments, the ADC, or a composition or formulation comprising the ADC, is administered intravenously.

[0296] Disclosed herein are methods of treating a subject for a disease or condition using an ADC or pharmaceutical composition of the present disclosure. The present invention provides methods of treating a disease or condition, such as a tumor or cancer, by administering to a patient (e.g., a human subject) in need thereof a therapeutically effective amount of an ADC of the present disclosure, or a pharmaceutical composition comprising an ADC of the present disclosure.

[0297] In the context of the present disclosure, the dose administered to a patient may be sufficient to induce a beneficial response in the subject over time. The ADC dosage may be given as a bolus injection and / or as an infusion for a clinically necessary period, e.g., a period ranging from several minutes to several hours, e.g., up to 24 hours. If necessary, ADC administration may be repeated once or several times. The ADC dosage may be an effective amount or dose.

[0298] In some embodiments, the effective amount of the ADC is a dose of at least about 0.05 mg / kg for a human subject. In some embodiments, the effective amount of the ADC is a dose of at least about 0.1 mg / kg for a human subject. In some embodiments, the effective amount of the ADC is a dose of at least about 0.15 mg / kg for a human subject. In some embodiments, the effective amount of the ADC is a dose of at least about 0.2 mg / kg for a human subject.

[0299] In some embodiments, an effective amount of an ADC is a dose in the range of about 0.05 mg / kg to about 10 mg / kg for a human subject.

[0300] In some embodiments, an effective amount of ADC is a dose in the range of about 0.05 mg / kg to 2 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of ADC is a dose in the range of about 0.05 mg / kg to 1.9 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of ADC is a dose in the range of about 0.05 mg / kg to 1.8 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of ADC is a dose in the range of about 0.05 mg / kg to 1.7 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of ADC is a dose in the range of about 0.05 mg / kg to 1.6 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of ADC is a dose in the range of about 0.05 mg / kg to 1.5 mg / kg for a human subject, or any value therebetween. In some other embodiments, an effective amount of an ADC is a dose within the range of about 0.1 mg / kg to 2 mg / kg, 0.1 mg / kg to 1.9 mg / kg, 0.1 mg / kg to 1.8 mg / kg, 0.1 mg / kg to 1.7 mg / kg, 0.1 mg / kg to 1.6 mg / kg, or 0.1 mg / kg to 1.5 mg / kg for a human subject. In some other embodiments, an effective amount of an ADC is a dose within the range of about 0.2 mg / kg to 2 mg / kg, 0.2 mg / kg to 1.9 mg / kg, 0.2 mg / kg to 1.8 mg / kg, 0.2 mg / kg to 1.7 mg / kg, 0.2 mg / kg to 1.6 mg / kg, or 0.2 mg / kg to 1.5 mg / kg for a human subject.

[0301] In some embodiments, an effective amount of an ADC is about 0.05 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, about 0.2 mg / kg, about 0.22 mg / kg, about 0.24 mg / kg, about 0.26 mg / kg, about 0.28 mg / kg, about 0.3 mg / kg, about 0.3 2mg / kg, approx. 0.34mg / kg, approx. 0.36mg / kg, approx. 0.38mg / kg, approx. 0.4mg / kg, approx. 0.42mg / kg, approx. 0.44mg / kg, approx. 0.46mg / kg, about 0.48mg / kg, about 0.5mg / kg, about 0.52mg / kg, about 0.54mg / kg, about 0.56mg / kg, about 0.58mg / kg, about 0.6mg / kg, about 0.62mg / kg, about 0.64mg / kg, about 0.66mg / kg, about 0.68mg / kg, about 0.7mg / kg, about 0.72mg / kg, about 0.74mg / kg, about 0.7 6mg / kg, approx. 0.78mg / kg, approx. 0.8mg / kg, approx. 0.82mg / kg, approx. 0.84mg / kg, approx. 0.86mg / kg, approx. 0.88mg / kg, approx. 0.9mg / k g, about 0.92 mg / kg, about 0.94 mg / kg, about 0.96 mg / kg, about 0.98 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg or about 2 mg / kg.

[0302] In some other embodiments, an effective amount of the ADC is a dose in the range of about 2 mg / kg to about 5 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of the ADC is a dose of about 2 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.6 mg / kg, about 3.8 mg / kg, about 4 mg / kg, about 4.2 mg / kg, about 4.4 mg / kg, about 4.6 mg / kg, about 4.8 mg / kg, or about 5 mg / kg for a human subject.

[0303] In some other embodiments, an effective amount of the ADC is a dose in the range of about 5 mg / kg to about 10 mg / kg for a human subject, or any value therebetween. In some embodiments, an effective amount of the ADC is a dose of about 5 mg / kg, about 5.2 mg / kg, about 5.4 mg / kg, about 5.6 mg / kg, about 5.8 mg / kg, about 6 mg / kg, about 6.2 mg / kg, about 6.4 mg / kg, about 6.6 mg / kg, about 6.8 mg / kg, about 7 mg / kg, about 7.2 mg / kg, about 7.4 mg / kg, about 7.6 mg / kg, about 7.8 mg / kg, about 8 mg / kg, about 8.2 mg / kg, about 8.4 mg / kg, about 8.6 mg / kg, about 8.8 mg / kg, about 9 mg / kg, about 9.2 mg / kg, about 9.4 mg / kg, about 9.6 mg / kg, about 9.8 mg / kg, or about 10 mg / kg for a human subject.

[0304] The average amount of ADC can vary, and can be based on the recommendations and prescriptions of qualified physicians.The exact amount of ADC is a matter of preference depending on factors such as the exact type of condition being treated, the condition of the patient being treated, and other components in the composition.The present disclosure also provides for the administration of a therapeutically effective amount of another active agent.The amount to be administered can be easily determined by those skilled in the art based on the treatment with ADC.

[0305] The tumor or cancer treated by the ADCs of the invention can be a solid tumor or a hematological tumor or cancer. In some embodiments, the tumor or cancer is a hematological cancer such as lymphoma, multiple myeloma, or leukemia. In some embodiments, the tumor or cancer is kidney cancer, brain cancer, breast cancer, Burkitt's lymphoma, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, testicular cancer, prostate cancer, colorectal cancer, esophageal cancer, bladder cancer, lung cancer, non-small cell lung cancer (NSCLC), urothelial carcinoma, cholangiocarcinoma, colorectal carcinoma, pancreatic cancer, renal cell carcinoma, nasopharyngeal carcinoma, mantle cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, or acute myeloid leukemia, or a cancer or disease or condition related to any of these cancers. In some embodiments, the tumor or cancer is renal cell carcinoma, brain cancer, multiple myeloma, mantle cell lymphoma, or lung cancer. In some embodiments, the tumor or cancer is renal cell carcinoma (RCC). In some embodiments, the tumor or cancer is clear cell renal cell carcinoma (ccRCC). In some other embodiments, the tumor or cancer is brain cancer. In some embodiments, the tumor or cancer is multiple myeloma. In some embodiments, the tumor or cancer is mantle cell lymphoma. In some embodiments, the tumor or cancer is lung cancer.

[0306] In some embodiments, the tumor or cancer is renal cell carcinoma (RCC). In some embodiments, the renal cell carcinoma is metastatic renal cell carcinoma. In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma. In some embodiments, the tumor or cancer is MDR-positive renal cell carcinoma. In some embodiments, the tumor or cancer is MDR-positive clear cell renal cell carcinoma. In some embodiments, the patient being treated is resistant or refractory to previous standard therapies. In some embodiments, the patient is an adult.

[0307] In some embodiments, the tumor or cancer being treated is a CD70-expressing cancer. Thus, in some embodiments, the ADCs of the present disclosure can be used to treat cancers in which cells express high CD70 surface counts. In some embodiments, the tumor or cancer being treated is a CD70-expressing cell with at least about 10,000 CD70 / cell, or at least about 15,000 CD70 / cell. Cancer can be treated by recruiting cytotoxic T cells to antigen receptor-expressing tumor cells. In some embodiments, the present disclosure provides a method of treating any cancer, disease, or condition associated with high expression of an antigen receptor by administering to a patient a therapeutically effective amount of an antibody or ADC of the present disclosure. In some embodiments, the antibody or antibody fragment of the ADC binds to the tumor-associated CD70 antigen.

[0308] In some embodiments, the patient being treated has a CD70-expressing cancer and / or cancer metastasis from the same or a different cancer.

[0309] In some embodiments, the treatment method improves or optimizes cancer cell killing, hi some embodiments, the method delays tumor or cancer progression or recurrence.

[0310] In some aspects, the antibodies or ADCs of the present disclosure may be used in conjunction with additional therapies or treatments, including, but not limited to, surgery, radiation, cryosurgery, hyperthermia, hormone therapy, chemotherapy, vaccines, and other immunotherapies.

[0311] Thus, in some embodiments, the present disclosure provides methods of treating a disease or condition, such as a tumor or cancer, comprising administering to a subject an effective amount of an ADC of the present disclosure and an additional therapeutic agent and / or radiation therapy (radiotherapy). In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, an immunomodulatory agent, or an immunotherapeutic agent, or a combination thereof. In some other embodiments, the additional therapeutic agent is a checkpoint inhibitor, a CD70 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, a hypomethylating agent, or a platinum-based therapeutic agent, or a combination thereof. In some other embodiments, the therapeutic agent is a CD70-targeted therapeutic agent.

[0312] In some aspects, a subject is treated with an anti-CD70 antibody or ADC of the present disclosure and an additional agent, wherein the additional agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors (or simply "checkpoint inhibitors" herein) can function as tumor suppressors by modulating interactions between immune cells and tumor cells (see, e.g., Alsaab, HO et al., Frontiers in Pharmacology, Vol. 8, Article 561 (2017); https: / / doi.org / 10.3389 / fphar.2017.00561). In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor that targets or inhibits cytotoxic T-cell lymphocyte-associated protein 4 (CTLA-4). In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some other embodiments, the checkpoint inhibitor is a PD-1 inhibitor that targets or inhibits programmed death receptor 1 (PD-1). In some other embodiments, the checkpoint inhibitor is a PD-L1 inhibitor that targets or inhibits PD-1 ligand 1 (PD-L1). In some embodiments, the checkpoint inhibitor targets or inhibits PD-1 and / or PD-L1, and such checkpoint inhibitors may be referred to herein as "PD-1 / PD-L1 inhibitors." Non-limiting examples of PD-1 / PD-L1 inhibitors include AMP-224, atezolizumab, avelumab, BMS-936558, BMS-936559, CT-001, durvalumab, MEDI0680, nivolumab, PDR001, pembrolizumab, pidilizumab, and REGN2810. Accordingly, in some embodiments, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC of the disclosure and an additional therapeutic agent, wherein the additional therapeutic agent is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor or a CTLA-4 inhibitor.In some embodiments, the checkpoint inhibitor is selected from the group consisting of AMP-224, atezolizumab, avelumab, BMS-936558, BMS-936559, CT-001, durvalumab, ipilimumab, MEDI0680, nivolumab, PDR001, pembrolizumab, pidilizumab, and REGN2810, and combinations thereof.

[0313] As disclosed herein, treatment with several therapeutic agents can upregulate CD70, and the present disclosure provides for the use of such therapeutic agents in combination with an anti-CD70 antibody or ADC of the present disclosure.

[0314] In some aspects, a subject is treated with an anti-CD70 antibody or ADC of the present disclosure and an additional agent, wherein the additional agent is a hypomethylating agent. Hypomethylating agents, also known as demethylating agents, are chemotherapeutic agents that have been shown to upregulate CD70 in conditions including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (see, e.g., Stomper, J. et al., Leukemia 35:1873-1889 (2021)). Non-limiting examples of hypomethylating agents include azacitidine (5-azacytidine), cytidine, decitabine (5-aza-2'-deoxycytidine), and guadecitabine. Accordingly, in some embodiments, a method of treating a disease or condition in a subject is provided, the method comprising administering to the subject an anti-CD70 ADC of the present disclosure and an additional therapeutic agent, wherein the additional therapeutic agent is a hypomethylating agent. In some embodiments, the hypomethylating agent is selected from the group consisting of azacitidine, cytidine, decitabine, and guadecitabine. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is AML. In some other embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the RCC is clear cell RCC. In some other embodiments, the disease or condition is MDS.

[0315] In some aspects, a subject is treated with an anti-CD70 antibody or ADC of the present disclosure and an additional agent, wherein the additional agent is a platinum-based chemotherapeutic agent. Non-limiting examples of platinum-based chemotherapeutic agents include cisplatin, oxaliplatin, and carboplatin. Cisplatin has been reported to upregulate CD70 in cancers, including non-small cell lung cancer (NSCLC) (see, e.g., Flieswasster, T. et al., J Exp Clin Cancer Res. 41:12 (2022)). Accordingly, in some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC of the present disclosure and an additional therapeutic agent, wherein the additional therapeutic agent is a platinum-based therapeutic agent. In some embodiments, the platinum-based therapeutic agent is cisplatin, oxaliplatin, or carboplatin. In some embodiments, the platinum-based therapeutic agent is cisplatin. In some embodiments, the cancer is NSCLC. In some other embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the RCC is clear cell RCC.

[0316] In some embodiments, a subject is treated with an anti-CD70 antibody or ADC of the present disclosure and an additional agent, wherein the additional agent is a tyrosine kinase inhibitor (TKI). Non-limiting examples of TKIs include axitinib, cabozantinib, dasatinib, everolimus, erlotinib, gefitinib, imatinib, lapatinib, lenvatinib, pazopanib, and sunitinib. TKIs have been reported to upregulate CD70 in chronic myelogenous leukemia (CML) (see Riether, C. et al., https: / / doi.org / 10.7892 / boris.77241). TKIs are also used to treat patients with metastatic renal cell carcinoma (RCC) (see, e.g., Stitt, TM et al., Journal of Hematology Oncology Pharmacy, 12(3):138-144(2022)). Cancers characterized by epidermal growth factor receptor (EGFR) mutations can acquire resistance to EGFR TKIs. Epithelial-mesenchymal transition (EMT) is associated with acquired EGFR-TKI resistance in NSCLC, and CD70 is highly upregulated in EMT-associated resistance. Anti-CD70 ADCs have shown potent activity against EGFR TKI-resistant cells, suggesting that CD70 is a suitable therapeutic target for EGFR-mutated tumors with acquired EGFR TKI resistance (see, e.g., Nilsson, MB et al., Cancer Cell, 41(2):340-355(2023)). Accordingly, in some embodiments, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC of the disclosure and an additional therapeutic agent, where the additional therapeutic agent is a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is selected from the group consisting of axitinib, cabozantinib, dasatinib, everolimus, erlotinib, gefitinib, imatinib, lapatinib, lenvatinib, pazopanib, and sunitinib, and combinations thereof.In some embodiments, the cancer is an EGFR-mutated tumor with acquired EGFR TKI resistance. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is NSCLC. In some other embodiments, the cancer is CML. In some other embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the RCC is clear cell RCC.

[0317] In some aspects, a subject is treated with an anti-CD70 antibody or ADC of the present disclosure and an additional agent, wherein the additional agent is radiation. Radiation therapy (radiotherapy) can also upregulate CD70 expression, for example, in glioma, leukemia, and lymphoma (Flieswasster, T. et al., J Exp Clin Cancer Res. 41:12 (2022)). Accordingly, in some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC of the present disclosure in combination with radiation therapy. In some embodiments, the cancer is glioma, leukemia, or lymphoma. In some other embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the RCC is clear cell RCC.

[0318] It will be understood that an antibody, compound, ADC, or composition of the disclosure can be used in the manufacture of a medicament for treating a disease or condition, including cancer.

[0319] Pharmaceutical compositions containing the antibodies or ADCs of the present invention can be formulated in potencies effective for administration by various means to human patients experiencing disorders that can be affected by antibody agonists or antagonists, such as, but not limited to, antiproliferative, anti-inflammatory, or antiviral agents, used alone or as part of a condition or disease. The average amount of the antibody or ADC can vary and should be based on the recommendations and prescriptions of a qualified physician. The exact amount of antibody or ADC is a matter of preference, subject to factors such as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The present disclosure also provides for the administration of a therapeutically effective amount of another active agent, such as, but not limited to, an anti-cancer chemotherapeutic or immunotherapeutic agent. The amount to be administered can be readily determined by one of skill in the art based on treatment with the antibodies or ADCs of the present invention.

[0320] CD70-related disorders As disclosed herein, the anti-CD70 antibodies and ADCs described herein are useful for treating or preventing immunological disorders characterized by CD70-expressing cancers or CD70 expression due to inappropriate activation of immune cells (e.g., lymphocytes or dendritic cells). Such expression of CD70 can result from, for example, increased CD70 protein levels on the cell surface and / or changes in the antigenicity of the expressed CD70. Treatment or prevention of immunological disorders according to the methods described herein is achieved by administering to a subject in need of such treatment or prevention an effective amount of an anti-CD70 antibody or derivative, whereby the antibody or derivative (i) binds to activated immune cells that express CD70 and are associated with the disease state, and (ii) exerts a cytotoxic, cytostatic, or immunomodulatory effect on the activated immune cells. In some embodiments, the cytotoxic, cytostatic, or immunomodulatory effect is exerted without conjugation to a cytotoxic, cytostatic, or immunomodulatory agent. In some embodiments, the cytotoxic, cytostatic, or immunomodulatory properties are exerted by conjugation to a cytotoxic, cytostatic, or immunomodulatory agent.

[0321] The anti-CD70 antibodies and ADCs described herein are also useful for treating or preventing CD70-expressing cancers. Treatment or prevention of CD70-expressing cancers according to the methods described herein is achieved by administering to a subject in need of such treatment or prevention an effective amount of an anti-CD70 antibody or derivative or ADC, whereby the antibody or derivative or ADC (i) binds to CD70-expressing cancer cells and (ii) exerts a cytotoxic or cytostatic effect, depleting or inhibiting the proliferation of the CD70-expressing cancer cells. In some embodiments, the cytotoxic, cytostatic, or immunomodulatory effect is exerted without conjugation to a cytotoxic, cytostatic, or immunomodulatory agent. In some embodiments, the cytotoxic, cytostatic, or immunomodulatory effect is exerted by conjugation to a cytotoxic, cytostatic, or immunomodulatory agent. In some embodiments, the cytotoxic, cytostatic, or immunomodulatory effect is exerted by an anti-CD70 ADC comprising an anti-CD70 antibody conjugated to the drug-linker AS269, i.e., an anti-CD70-AS269 ADC of the disclosure.

[0322] CD70-expressing cancers that can be treated or prevented by the methods described herein include, for example, different subtypes of non-Hodgkin's lymphoma (indolent NHL, follicular NHL, small lymphocytic lymphoma, lymphoplasmacytic NHL, or marginal zone NHL); Hodgkin's disease (Hodgkin's lymphoma, e.g., Reed-Sternberg cell); cancers of the B-cell lineage, e.g., diffuse large B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, Mannheim's lymphoma, and the like. CD70-expressing cancers include, for example, Toll cell lymphoma, B-cell lymphocytic leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia); Epstein-Barr virus-positive B-cell lymphoma; renal cell carcinoma (e.g., clear cell renal cell carcinoma, papillary renal cell carcinoma); nasopharyngeal carcinoma; thymic carcinoma; brain cancer; glioma; glioblastoma; neuroblastoma; astrocytoma; meningioma; Waldenstrom's macroglobulinemia; multiple myeloma; lung cancer; pancreatic cancer; and colon, stomach, and rectal carcinoma. The cancer may be, for example, newly diagnosed, previously treated, or refractory or relapsed. In some embodiments, the CD70-expressing cancer has at least about 15,000, at least about 10,000, or at least about 5,000 CD70 molecules / cell. In some embodiments, the cancer or tumor may be a multidrug-resistant CD70-expressing cancer or tumor. In some embodiments, the cancer is multidrug resistance (MDR)-positive renal cell carcinoma. In some more particular embodiments, the cancer is MDR-positive clear cell renal cell carcinoma.

[0323] In some other embodiments, the disease or condition being treated is myelodysplastic syndrome, hi some embodiments, the subject has previously been treated with a hypomethylating agent or is currently undergoing treatment with a hypomethylating agent.

[0324] Pharmaceutical Composition In other aspects, pharmaceutical compositions or formulations containing the antibodies, antibody fragments, variants, or ADCs of the present disclosure are provided. Such pharmaceutical compositions can use a variety of pharmaceutically acceptable excipients, stabilizers, buffers, and other components for administration to animals. See, for example, Remington, The Science and Practice of Pharmacy, 19th ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1995. Identifying a composition or formulation suitable for stability, subject administration, and activity will vary for each compound, as several components (e.g., purification components, stabilization components) must be considered. Suitable salts for inclusion in the composition or formulation include, but are not limited to, sodium chloride, potassium chloride, or calcium chloride. Buffers and / or stabilizers, such as sodium acetate, can be used. Suitable buffers may include phosphate-citrate buffer, phosphate buffer, citrate buffer, histidine buffer, L-histidine, L-arginine hydrochloride, bicarbonate buffer, succinate buffer, citrate buffer, and TRIS buffer, either alone or in combination. Surfactants may also be used, including polysorbates (e.g., polysorbate 80), sodium dodecyl sulfate (SDS), and lecithin, either alone or in combination.

[0325] In some embodiments, the pharmaceutical composition may be a formulation comprising an ADC of the disclosure and one or more pharmaceutically acceptable excipients, stabilizers, or buffers.

[0326] In some embodiments, the formulation may contain an ADC and a buffer, cryoprotectant, or surfactant, or any combination thereof.

[0327] In some embodiments, the formulation may contain the ADC at a concentration in the range of about 5 mg / mL to about 25 mg / mL. In some embodiments, the formulation may contain the ADC at a concentration of about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, or about 25 mg / mL. In some embodiments, the formulation may contain the ADC at a concentration in the range of about 5 mg / mL to about 15 mg / mL. In some embodiments, the formulation contains the ADC at a concentration of about 5 mg / mL. In some embodiments, the formulation contains the ADC at a concentration of about 10 mg / mL. In some embodiments, the formulation contains the ADC at a concentration of about 15 mg / mL.

[0328] In some embodiments, the formulation may contain a buffer. In some embodiments, the buffer is an acetate buffer, a succinate buffer, a histidine buffer, or a phosphate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the formulation may have a histidine buffer concentration in the range of about 10 mM to about 50 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the formulation may have a histidine buffer concentration in the range of about 5 mM to about 25 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, or about 25 mM. In some embodiments, the formulation may have a histidine buffer concentration in the range of about 15 mM to about 25 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 15 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 20 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 25 mM. In some embodiments, the histidine is L-histidine. In some embodiments, the histidine buffer comprises L-histidine and L-histidine hydrochloride. Various combinations of L-histidine and L-histidine hydrochloride concentrations can be used by one skilled in the art to achieve a target pH for the histidine buffer.

[0329] In some embodiments, the formulation is characterized as having a pH value. In some embodiments, the formulation may have a pH in the range of about 5 to about 7.4. In some embodiments, the formulation may have a pH of up to about 7, up to about 6.5, or up to about 6. In some embodiments, the formulation may have a pH in the range of about 5.4 to about 6.4. In some embodiments, the formulation may have a pH in the range of about 5.2 to about 6.2. In some embodiments, the formulation may have a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the formulation is less than 6. In some embodiments, the pH of the formulation is about 5.5. In some embodiments, the pH of the formulation is about 5.6. In some embodiments, the pH of the formulation is about 5.7. In some embodiments, the pH of the formulation is about 5.8. In some embodiments, the pH of the formulation is about 5.9.

[0330] In some embodiments, the formulation contains a cryoprotectant. In some embodiments, the cryoprotectant is polyvinylpyrrolidone, glycerol, trehalose, fructose, sucrose, glucose, or mannose, or a combination thereof. In some embodiments, the formulation has a cryoprotectant concentration in the range of about 1% (w / v) to about 20% (w / v). In some embodiments, the formulation has a cryoprotectant concentration of up to about 15% (w / v). In some other embodiments, the formulation has a cryoprotectant concentration in the range of about 5% (w / v) to about 15% (w / v). In some embodiments, the formulation has a cryoprotectant concentration of up to about 10% (w / v). In some embodiments, the formulation has a cryoprotectant concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w / v). In some embodiments, the cryoprotectant is sucrose. In some embodiments, the formulation has a sucrose concentration in the range of about 1% (w / v) to about 20% (w / v). In some embodiments, the formulation has a sucrose concentration of up to about 15% (w / v). In some other embodiments, the formulation has a sucrose concentration in the range of about 5% (w / v) to about 15% (w / v). In some embodiments, the formulation has a sucrose concentration of up to about 10% (w / v). In some embodiments, the formulation has a sucrose concentration in the range of about 5% (w / v) to about 8% (w / v). In some embodiments, the formulation has a sucrose concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w / v). In some embodiments, the formulation contains sucrose at a concentration of about 5% (w / v). In some embodiments, the formulation contains sucrose at a concentration of about 6% (w / v). In some embodiments, the formulation contains sucrose at a concentration of about 7% (w / v). In some embodiments, the formulation contains sucrose at a concentration of about 8% (w / v). In some embodiments, the formulation contains sucrose at a concentration of about 9% (w / v). In some embodiments, the formulation contains sucrose at a concentration of about 10% (w / v).

[0331] In some embodiments, the formulation may contain a surfactant. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 20. In some other embodiments, the surfactant is polysorbate 80. In some embodiments, the formulation has a surfactant concentration of up to about 1% (w / v). In some embodiments, the formulation has a surfactant concentration of up to about 0.1% (w / v). In some embodiments, the formulation has a surfactant concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the formulation has a surfactant concentration of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.10% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of up to about 1% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of up to about 0.1% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.10% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration in the range of about 0.01% (w / v) to about 0.05% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.01% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.02% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.03% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.04% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.05% (w / v).

[0332] In some embodiments, a formulation is provided comprising an ADC of the present disclosure, a histidine buffer, sucrose, and polysorbate 80. In some embodiments, the formulation comprises an ADC (i.e., an anti-CD70-AS269 ADC) at a concentration in the range of about 5 mg / mL to about 25 mg / mL, a histidine buffer at a concentration in the range of about 10 mM to about 50 mM, sucrose at a concentration in the range of about 1% (w / v) to about 20% (w / v), and polysorbate 80 at a concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the formulation has a pH in the range of about 5.4 to about 6.4. In some embodiments, the formulation has a pH in the range of about 5.2 to about 6.2. In some embodiments, the pH of the formulation is about 6 or less. In some embodiments, the pH of the formulation is less than 6. In some further embodiments, the formulation comprises an anti-CD70-AS269 ADC at a concentration in the range of about 5 mg / mL to about 15 mg / mL, a histidine buffer at a concentration in the range of about 10 mM to about 25 mM, sucrose at a concentration in the range of about 5% (w / v) to about 15% (w / v), and polysorbate 80 at a concentration in the range of about 0.01% (w / v) to about 0.05% (w / v), and the formulation has a pH in the range of about 5.4 to about 6. In some embodiments, the pH of the formulation is about 6 or less. In some embodiments, the pH of the formulation is less than 6. In some embodiments, the formulation contains an ADC at a concentration of about 10 mg / mL, a histidine buffer at a concentration of about 20 mM, sucrose at a concentration of about 8% (w / v), and polysorbate 80 at a concentration of about 0.02% (w / v), and the formulation has a pH in the range of about 5.4 to about 6. In some embodiments, the pH of the formulation is about 6 or less. In some embodiments, the pH of the formulation is less than 6. In some embodiments, the pH of the formulation is about 5.5. In some embodiments, the pH of the formulation is about 5.6. In some embodiments, the pH of the formulation is about 5.7. In some embodiments, the pH of the formulation is about 5.8. In some embodiments, the pH of the formulation is about 5.9.

[0333] In some embodiments, the formulation is a liquid formulation. In some embodiments, the liquid formulations of the present disclosure can be stored at room temperature. In some other embodiments, the liquid formulations can be stored frozen.

[0334] Formulations of the present disclosure can be lyophilized to produce lyophilized pharmaceutical preparations containing the ADCs or antibodies or fragments thereof of the present disclosure. The lyophilized pharmaceutical preparations can be reconstituted with a suitable diluent, for example, prior to administration. In some embodiments, the diluent is water, such as water-for-injection (WFI). Individual vials containing the lyophilized pharmaceutical preparations can contain, for example, about 5 mg of ADC, about 10 mg of ADC, about 15 mg of ADC, about 20 mg of ADC, about 25 mg of ADC, about 30 mg of ADC, about 35 mg of ADC, about 40 mg of ADC, about 45 mg of ADC, about 50 mg of ADC, about 55 mg of ADC, or about 60 mg of ADC, or more. In some embodiments, individual vials containing the lyophilized pharmaceutical preparations can contain about 60 mg of ADC (e.g., anti-CD70-AS269 ADC) per vial. After reconstitution of the lyophilized drug product with a suitable diluent, such as water (e.g., WFI), the reconstituted formulation may have an ADC concentration of about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, or more. The pH can be adjusted to provide acceptable stability and administration by a trained physician.

[0335] In some embodiments, the lyophilized pharmaceutical product, when reconstituted with a diluent, provides a reconstituted solution containing the ADC at a concentration in the range of about 5 mg / mL to about 25 mg / mL. In some embodiments, the reconstituted solution further comprises an L-histidine buffer at a concentration in the range of about 10 mM to about 50 mM, sucrose at a concentration in the range of about 1% (w / v) to about 20% (w / v), and polysorbate 80 at a concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the reconstituted solution has a pH in the range of about 5.4 to about 6.4. In some embodiments, the reconstituted solution has a pH in the range of about 5.2 to about 6.2. In some embodiments, the pH of the reconstituted solution is about 6 or less. In some embodiments, the pH of the reconstituted solution is less than 6. In some embodiments, the diluent is water.

[0336] In some further embodiments, the lyophilized pharmaceutical product, when reconstituted with a diluent, provides a reconstituted solution comprising an ADC (e.g., an anti-CD70-AS269 ADC) at a concentration in the range of about 5 mg / mL to about 15 mg / mL, an L-histidine buffer at a concentration in the range of about 10 mM to about 25 mM, sucrose at a concentration in the range of about 5% (w / v) to about 8% (w / v), and polysorbate 80 at a concentration in the range of about 0.01% (w / v) to about 0.05% (w / v). In some embodiments, the reconstituted solution has a pH in the range of about 5.5 to about 6.2. In some embodiments, the pH of the reconstituted solution is about 6 or less. In some embodiments, the pH of the reconstituted solution is less than 6. In some embodiments, the pH of the reconstituted solution is about 5.5. In some embodiments, the pH of the reconstituted solution is about 5.6. In some embodiments, the pH of the reconstituted solution is about 5.7. In some embodiments, the pH of the reconstituted solution is about 5.8. In some embodiments, the pH of the reconstituted solution is about 5.9. In some embodiments, the formulation comprises an ADC at a concentration in the range of about 5 mg / mL to about 15 mg / mL, an L-histidine buffer at a concentration in the range of about 10 mM to about 15 mM, sucrose at a concentration in the range of about 5% (w / v) to about 8% (w / v), and polysorbate 80 at a concentration in the range of about 0.01% (w / v) to about 0.05% (w / v), and the formulation has a pH in the range of about 5.5 to about 6.0. In some embodiments, the pH of the reconstituted solution is about 6 or less. In some embodiments, the pH of the reconstituted solution is less than 6. In some embodiments, the formulation contains an ADC at a concentration of about 10 mg / mL, a histidine buffer at a concentration of about 20 mM, sucrose at a concentration of about 8% (w / v), and polysorbate 80 at a concentration of about 0.02% (w / v), and the formulation has a pH in the range of about 5.5 to about 5.9. In some embodiments, the pH of the formulation is about 5.9 or less. In some embodiments, the pH of the formulation is less than 5.9. In some embodiments, the pH of the formulation is about 5.6. In some embodiments, the pH of the formulation is about 5.7. In some embodiments, the pH of the formulation is about 5.8.

[0337] The composition can be stored in, but is not limited to, a vial or cartridge, a pen delivery device, a syringe, an intravenous tube, or an intravenous bag. In other embodiments, the pharmaceutical composition of the present invention can be administered as a single dose, or as one or more subsequent doses minutes, days, or weeks after the initial dose. Further administrations may be contemplated when needed to treat, alleviate, or prevent a cancer, condition, disorder, or disease. [Example]

[0338] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims.

[0339] Example 1: General Experimental Procedure All commercially available anhydrous solvents are used without further purification and stored under nitrogen atmosphere. Chromatographic purification was carried out on a CombiFlash Rf from Teledyne ISCO using the conditions detailed in the experimental procedures.

[0340] Preparative HPLC was performed on a Shimadzu system using a Gemini-NX C18, 5 μm 100 × 30 mm, 150 × 30 mm, or 250 × 50 mm column, depending on the scale. Mass spectra (MS) were recorded on a Shimadzu LCMS-2020 system, and data were processed using Shimadzu Lab Solutions software. An Agilent 1260 Infinity Binary LC coupled to a 6230 Accurate-Mass TOFMS system was used for HR-ESI-TOF analysis. NMR spectral data were collected on a 500 MHz Bruker NMR spectrometer. Chemical shifts (δ) are reported in ppm and referenced to the deuterium solvent signal. Coupling constants (J) are reported in hertz (Hz). Spin multiplicities are described as s (singlet), br (broad), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), or m (multiplet).

[0341] Abbreviations used in the examples herein: AHZ: acetohydrazide, DIAD: diisopropyl azodicarboxylate, DMF: dimethylformamide, HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, MeOH: methanol, TFA: trifluoroacetic acid.

[0342] Chemical names of compounds were derived from chemical structures using ChemDraw version 20.1.1 (CambridgeSoft).

[0343] Example 2: Synthesis of Compounds 1-4

[0344] [ka]

[0345] 2-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethoxy)isoindoline-1,3-dione (Compound 1-3): Tetraethylene glycol 1-1 (10 g, 51.5 mmol), N-hydroxyphthalimide 1-2 (8.4 g, 51.15 mmol), and triphenylphosphine (17.6 g, 67 mmol) were dissolved in 300 mL of tetrahydrofuran, followed by the addition of DIAD (12.8 mL, 61.78 mmol) at 0 °C. The resulting solution was stirred overnight at room temperature and then concentrated to dryness. The residue was purified by flash column chromatography to give 5.47 g (31%) of compound 1-3.

[0346] 2-(2-(2-(2-((1,3-Dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)acetaldehyde (Compound 1-4): To a solution of compound 1-3 (200 mg, 0.59 mmol) in 15 mL of dichloromethane was added Dess-Martin periodinane (300 mg, 0.71 mmol). The reaction mixture was stirred at ambient temperature overnight. The reaction was quenched with a solution of sodium bisulfite in 15 mL of saturated sodium bicarbonate. The mixture was separated. The organic layer was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography to give 150 mg (75%) of compound 1-4.

[0347] Example 3: Synthesis of Compound 6

[0348] [ka]

[0349] (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (compound 6-1, Boc-Val-Dil-Dap-OH). Compound 6-1 is available from commercial suppliers, including MedChemExpress (Monmouth Junction, NJ), catalog number HY-130961.

[0350] Methyl ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate (Compound 6-2): To a solution of compound 6-1 (500 mg, 0.875 mmol) in 3 mL of DMF was added 283 mg of L-phenylalanine methyl ester hydrochloride (commercially available from Sigma-Aldrich, catalog number P1 7202), 433 mg of HATU, and 581 μL of N-methylmorpholine. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mL × 1, 50 mL × 2). The organic layers were combined, washed with brine, dried over sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to give 560 mg (76%) of compound 6-2.

[0351] Methyl ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-amino-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate (Compound 6-3): Compound 6-2 was dissolved in 15 mL of 4 N HCl / dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give 511 mg of compound 6-3.

[0352] Methyl ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate (Compound 6-4): To a solution of compound 6-3 (368 mg, 0.55 mmol) in 3 mL of DMF was added 255 mg of Boc-N-methyl-L-valine (commercially available from Sigma-Aldrich, catalog number 15538), 314 mg of HATU, and 303 μL of N-methylmorpholine. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mL x 1, 50 mL x 2). The organic layers were combined, washed with brine, dried over sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to give compound 6-4 (370 mg, 79%).

[0353] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (Compound 6-5): To a solution of compound 6-4 (170 mg) in 10 mL of MeOH was added 5 equivalents of 1N LiOH. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was acidified with 1N HCl, extracted with ethyl acetate, washed with brine, dried over sodium sulfate, and concentrated in vacuo to give 150 mg (90%) of compound 6-5.

[0354] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (compound 6-6): Compound 6-5 was dissolved in 4N HCl / dioxane. The reaction mixture was stirred at room temperature for 2 hours, concentrated in vacuo, and purified by HPLC to give 150 mg of compound 6-6.

[0355] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-14-((1,3-dioxoisoindolin-2-yl)oxy)-2-isopropyl-3-methyl-6,9,12-trioxa-3-azatetradecanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (Compound 6-7): To a solution of compound 6-6 (50 mg, 0.062 mmol) in 1 mL of DMF was added compound 1-4 (63 mg, 0.186 mmol) and 70 μL of acetic acid, followed by the addition of 8 mg of sodium cyanoborohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give 60 mg (80%) of compounds 6-7.

[0356] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-14-(aminooxy)-2-isopropyl-3-methyl-6,9,12-trioxa-3-azatetradecanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (Compound 6): Compound 6-7 (60 mg, 0.05 mmol) was dissolved in 1 mL of DMF. Hydrazine (32 μL) was added. The resulting solution was stirred at ambient temperature for 1 hour. The reaction was quenched with 1 N hydrochloric acid solution. The reaction mixture was purified by HPLC to give 33 mg (55%) of Compound 6.

[0357] Example 4 Methods using genome engineering techniques to generate cell lines for facilitating the production of unnatural amino acid-containing proteins (essentially described in WO 2018 / 223108, the entire contents of which are incorporated herein by reference in their entirety) can be applied to generate anti-CD70 antibodies containing a non-naturally encoded amino acid of the present disclosure.

[0358] Molecular cloning for expression of anti-CD70 antibodies. CHO cell codon-optimized antibody heavy and light chain cDNA sequences were obtained from a commercial DNA synthesis service (Integrated DNA Technologies (IDT), San Diego, CA). The synthesized DNA fragments were digested with Hind III and EcoR I (both from New England BioLabs (NEB), Ipswich, MA) and purified using a PCR purification kit (Qiagen, Valencia, CA). The digested antibody gene fragments were then ligated into an expression vector using a quick ligation kit (NEB) to obtain constructs for expression of wild-type antibody heavy and light chains. The resulting plasmids were propagated in E. coli and verified by a DNA sequencing service (Eton Biosciences, San Diego, CA).

[0359] Generation of Amber Codon-Containing Mutants. Based on the crystal structure of immunoglobulin G1 (IgG1) mAb, heavy chain amino acid A114 (Kabat numbering) was selected for genetic incorporation of the unnatural amino acid para-acetyl-L-phenylalanine (pAF). Alternative Fc mutations, including heavy chain amino acids A136 and L159, were selected for optimization of pharmacokinetic stability, antibody-dependent cellular phagocytosis (ADCP), and / or antibody-dependent cellular cytotoxicity. Light chain amino acid mutation sites were selected to serve as alternatives to heavy chain mutations and / or as additional conjugation sites to provide ADCs with higher drug-to-antibody ratios. The genetic codons at the selected sites were then mutated to an amber codon (TAG) via site-directed mutagenesis to generate expression plasmids for the antibody variants. Site-directed mutagenesis experiments were performed using the Q5 Site-Directed Mutagenesis Kit from New England Biolabs (NEB). Expression plasmids for the mutants were propagated in E. coli and verified by a DNA sequencing service (Eton Biosciences).

[0360] Table 1 provides a list of amino acid sequences containing amber mutation sites (Kabat numbering) in the heavy or light chain of anti-CD70 antibodies. SEQ ID NO:1 and SEQ ID NO:2 show the wild-type heavy and light chain amino acid sequences, respectively. Anti-CD70 mAb light chains with non-naturally encoded amino acids include the light chain sequences of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. Anti-CD70 mAb heavy chains with non-naturally encoded amino acids include the heavy chain sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0361] Protocol for the production of anti-CD70 antibodies containing pAF at position 114 of the heavy chain (Kabat numbering) Transient Expression. Platform cell lines were maintained in EX-Cell 302 (Sigma) supplemented with 3 mM L-glutamine (Gibco) and 3 mM GlutaMAX (Gibco). Cells were passaged every 3–4 days and seeded at a density of 400,000 cells per mL. One day before transfection, cells were seeded at 600,000 cells per mL. On day 0, cells were transfected with antibody expression plasmids encoding the light and heavy chains using the MaxCyte electroporation platform according to the manufacturer's instructions. After transfection, cells were rested in an empty 125 mL shake flask and incubated at 37°C in a static incubator for 30 minutes. The transfected cells were then transfected into 3 × 10 cells in a shake flask. 6 Transfected cells were seeded at a density of 100 cells / mL in basal expression medium (50% Dunamis: 50% ExCell 302 supplemented with 3 mM L-glutamine and 3 mM GlutaMAX). Transfected cells were incubated at 37°C and 5% CO2 on an orbital shaker set at 140 rpm. On day 1, pAF (final concentration in culture medium: 1 mM), Cell Boost 5 (GE Healthcare, final concentration in culture medium: 7 g / L), Long R3 IGF-1 (Sigma, final concentration in culture medium: 120 μg / L), and GlutaMAX (final concentration in culture medium: 2 mM) were added to the culture medium. The incubator temperature was shifted from 37°C to 32°C. Additional Cell Boost 5 (final concentration: 7 g / L) and GlutaMAX (final concentration: 2 mM) were added on day 3, and the supernatant was collected on day 5. Glucose levels in the culture medium were monitored using a glucose meter, and additional glucose was added to the culture when glucose levels fell below 2 g / L. Viable cell count and viability were measured using a Vi-Cell instrument. Antibody production was measured using an Octet protein G sensor.

[0362] Stable bulk pool generation. Expression plasmids were linearized using PvuI (NEB) digestion for 4 hours. After linearization, DNA was purified using phenol:chloroform:isoamyl alcohol extraction and dissolved in endotoxin-free water at a concentration of 2.5 μg / pL. Platform cell lines were maintained in EX-CELL 302 supplemented with 3 mM L-glutamine and 3 mM GlutaMAX. Cells were passaged every 3–4 days, reaching 0.3 × 10 6 One day before transfection, cells were seeded at a density of 0.6 × 10 cells / mL. 6 On day 0, 15 × 10 cells / mL were seeded. 6 Cells were transfected with 25 μg of linearized antibody expression plasmid using the MaxCyte Electroporation (OC-100) platform according to the manufacturer's instructions. After transfection, cells were rested in an empty 125 mL shaker flask and incubated at 37°C in a static incubator for 30 minutes. Then, 30 mL of recovery medium (50% Ex-302: 50% CD-CHO supplemented with 3 mM L-glutamine and 3 mM GlutaMAX) was added to the flask and shaken overnight. On day 1, transfected cells were counted, spun down, washed, and resuspended in selection medium (50% EXCELL 302: 50% CD-CHO with 50 μM MSX) for stable bulk pool generation. Viable cell count and viability were monitored, and the medium was changed every 3–4 days until the viability of the stable bulk pool returned to 90%. Once selection was complete, frozen cell stocks were made and the resulting stable bulk pool was used to generate material for fed-batch expression.

[0363] Fed-batch expression. On day 0, a stable bulk pool of pre-produced antibodies was cultured in shake flasks at 0.5 x 10 6Transfected cells were seeded at a density of 100 cells / mL in basal expression medium (50% Dunamis: 50% ExCell 302 supplemented with 50 μM MSX). The transfected cells were incubated at 37°C and 5% CO2 on an orbital shaker set at 150 rpm. On day 3, pAF (CAS#122555-04-8, final concentration in culture medium: 0.5 mM), Cell Boost 4 (GE Healthcare, final concentration in culture medium: 10 g / L), and Cell Boost 7b (GE Healthcare, final concentration in culture medium: 0.52 g / L) were added to the culture. On day 5, Long R3 IGF-1 (final concentration in culture medium: 120 μg / L) was added to the culture. The glucose level in the culture medium was monitored using a glucose meter, and additional glucose was added to the culture when the glucose level fell below 2 g / L. The number of viable cells and viability were measured using a Vi-Cell instrument. Supernatants were collected for purification on day 7. Antibody production was measured by Octet using a Protein G sensor.

[0364] Purification of nnAA-containing antibodies from the EuCODE expression system.

[0365] Clarified cell culture medium containing target antibodies containing non-naturally encoded amino acids was loaded onto a Protein A ProSep Ultra column (EMD Millipore) equilibrated with 20 mM sodium phosphate, 100 mM sodium chloride, pH 7.5. After loading, the column was washed with Buffer A (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.5) followed by Wash Buffer B (5 mM succinic acid, pH 5.8) to remove host cell contaminants. The target antibodies were eluted from the column using Elution Buffer C (50 mM glycine, 10 mM succinic acid, pH 3.2). The target antibodies were pooled and the pH was adjusted to pH 5.0 with 2.0 M Tris base. The target antibodies were further purified by loading the conditioned Protein A pool onto a Capto SP Impres column (GE Healthcare) equilibrated with 30 mM sodium acetate, pH 5.0. The target antibody was eluted from the column with a linear gradient of Buffer B (30 mM sodium acetate, 0.5 M sodium chloride, pH 5.0) to 100%, and fractions containing monomeric antibody were pooled, 0.22 μM filtered, and stored at ≦65°C until further use.

[0366] Example 5: Anti-CD70-AS269 ADC synthesis AS269 (Figure 1) was conjugated to a humanized anti-CD70 monoclonal antibody (mAb), subclass immunoglobulin G1 (IgG1), with specificity for human CD70 and containing the non-naturally encoded amino acid para-acetyl-L-phenylalanine at position 114 (Kabat numbering) of each heavy chain sequence, in the presence of acetohydrazide (AHZ) under controlled reaction conditions. Briefly, stock solutions of AS269 and AHZ were prepared in water for injection (WFI) and added to the anti-CD70 mAb solution at molar ratios of 8:1 (AS269:mAb) and 300:1 (AHZ:mAb). After addition, the pH was adjusted to 3.8-4.3 with 0.5 M citrate buffer and diluted to 30 ± 2.0 g / L. The conjugation reaction proceeded for 24-28 h at 22 ± 2 °C with continuous gentle mixing. Reversed-phase (RP) chromatography was used to monitor the reaction progress and ensure that a drug-to-antibody ratio (DAR) of 1.8–2.0 was met before further processing. RP-HPLC analysis was performed on an Agilent 1200 Series HPLC system using an Agilent Stablebond SB-C8, 5 μm, 4.6 × 150 mm column. Mobile phase A consisted of 0.1% TFA in water, and mobile phase B consisted of 0.1% TFA in acetonitrile. The flow rate was 1 mL / min, the column temperature was 75 °C, and detection was recorded at A280 nm. Elution of the mAb and ADC occurred during a 30–60% gradient increase in mobile phase B over 20 min. Representative HPLC chromatograms (Figure 2) show the unconjugated mAb before conjugation and the anti-CD70-AS269 ADC (DAR = 1 and DAR = 2) after 24 h.

[0367] After conjugation, the reaction mixture containing the anti-CD70-AS269 ADC was diafiltered into histidine formulation buffer via tangential flow filtration (TFF) using a 30 kDa molecular weight cutoff membrane (10 diafiltration volumes). The diafiltered ADC was formulated using a solution of histidine buffer, pH 5.7, sucrose, and polysorbate 80 (PS80) to provide a final formulation containing 10.0 mg / mL of ADC (active agent), histidine buffer, sucrose, and PS80, pH 5.7. The formulated ADC was filtered through a 0.22 micron filter, filled into polycarbonate (PC) bottles, and stored frozen at -70 ± 10°C. The frozen solution was thawed, aliquoted into glass vials, and subsequently lyophilized to a white lyophilized cake. The lyophilizate was reconstituted with WFI prior to administration.

[0368] The scheme below shows an exemplary ADC engineered by conjugating AS269 (compound 6) with an anti-CD70 mAb containing the non-naturally encoded amino acid pAF.

[0369] [ka]

[0370] Example 6: Anti-CD70-AS269 ADC Ex Vivo Mouse Plasma Stability The anti-CD70-AS269 ADC was incubated in mouse plasma at 37°C for 7 days. After T=0, 1, 3, 5, and 7 days of incubation, samples were processed to capture the antibody, extracted, and analyzed by LC-MS for intact AS269 drug-linker on the antibody heavy chain. The percent of intact drug-linker was determined relative to the T=0 time point. The anti-CD70-AS269 ADC demonstrated stability with 100% intact drug-linker after 7 days of incubation in mouse plasma (Figure 3).

[0371] Example 7: Anti-CD70-AS269 ADC Pharmacokinetics in CD-1 Mice A single dose of 1 mg / kg anti-CD70-AS269 ADC or 1 mg / kg unconjugated mAb was administered to CD-1 mice (n=5 / group). Small amounts of blood were collected from all animals before administration and at 2, 6, 24, 48, 96, 168, 240, 336, 504, and 672 hours after administration. Blood samples were immediately diluted in casein blocker buffer in tubes and frozen at ≤60°C. Samples were analyzed by a total antibody (TA) pharmacokinetic method, which detects the antibody scaffold (unconjugated and conjugated species). Anti-CD70-AS269 ADC samples were also measured by an intact ADC pharmacokinetic method, which detects only ADC species with a drug-to-antibody ratio of 2 (DAR=2). Any loss of drug-linker from the anti-CD70-AS269 ADC results in a separation of the ADC TA and intact ADC concentration-time curves. The ADC TA and intact ADC curves were in good agreement, demonstrating that anti-CD70-AS269 was stable in the mouse circulation through 672 hours and had a long ADC terminal half-life of 397 hours (Figure 4). Furthermore, the anti-CD70-AS269 ADC TA and intact ADC curves were similar to the unconjugated mAb TA curve, suggesting that anti-CD70-AS269 ADC clearance was similar to that of the unconjugated mAb and was not affected by AS269 drug-linker conjugation.

[0372] Example 8: 786-O S3 renal carcinoma xenograft model in mice Mice. Six- to seven-week-old nu / nu female mice were received from Charles River Laboratories and housed five per cage in a barriered, pathogen-free, restricted-access room. Animals received gamma-irradiated diet (Newco Item #15061) and autoclaved tap water ad libitum. Mice were individually identified by tail tattoo and allowed a minimum of three days to acclimate before the start of study activities.

[0373] Cells. 786-O renal carcinoma cells were purchased from ATCC (catalog number CRL-1932) and serially passaged three times in nude mice for faster growth. 786-O S3 cells were cultured in vitro for a minimum of two weeks in RPMI + 10% FBS + P / S before implantation. Freshly harvested 786-O S3 cells were suspended in PBS and mixed 1:1 with Matrigel. Mice were anesthetized with isoflurane anesthesia (2-3%, 2 L / min oxygen), and 5e6 cells / mouse in 0.2 mL of cell suspension were implanted subcutaneously into the right flank.

[0374] In-life measurements and study design. On day 14, when tumors reached an average size of 300 mm, mice were sorted into groups of 10 mice each. The vehicle group received formulation buffer, and treatments in all groups were administered IV at 10 mL / kg. Tumor size was measured by calipers consisting of the length (across the longest line of the tumor) and width (perpendicular to the length measurement). Tumor volume was calculated as L x W x W x 0.5. Data shown are the mean tumor volume ± SEM over time for each cohort (Figure 5).

[0375] Example 9: 786-O S3 renal carcinoma xenograft model in mice - Anti-CD70-AS269 ADC versus sunitinib 786-O S3 cells were implanted subcutaneously into nu / nu female mice, and tumor growth was monitored as described in Example 8. Mice (n=10 / group) were administered three different dose levels of the anti-CD70-AS269 ADC IV once weekly (dotted lines) for a total of five doses, and 30 mg / kg sunitinib PO daily for 35 days. Data shown are the mean tumor volume ± SEM over time for each cohort (Figure 6).

[0376] Example 10: Caki-1 renal carcinoma xenograft model in mice Mice: Taconic Laboratories NCRNU-F CrTac: 4-week-old NCR-Foxn1 <nu>Female mice were housed five per cage in a barriered, pathogen-free, restricted-access room. They were fed gamma-irradiated diet (Newco Item #15061) and provided with autoclaved tap water ad libitum. Mice were individually identified by ear tags.

[0377] Caki-1 renal cell carcinoma cells were purchased from ATCC and cultured in McCoys + 10% FBS + P / S for a minimum of 2 weeks before implantation. Cells were implanted at passage 5, with 5e6 cells per mouse inoculated into the right hind flank.

[0378] In-life measurements and study design. At day 9 after implantation, the average tumor size was approximately 115 mm. 3 When tumor volume reached 100 mg / kg, mice were sorted into four groups of 10 mice each. Test articles, as indicated in Figure 7, were administered weekly (dotted lines), and tumor volumes were measured as described in Example 8. Data shown are mean tumor volumes ± SEM over time for each cohort (Figure 7).

[0379] Example 11: Caki-1 renal carcinoma xenograft model in mice - individual tumor volumes Individual Caki-1 tumor volumes on day 41 are shown in FIG. 8 from the repeat-dose Caki-1 xenograft study described in Example 10, with the mean values ​​for each treatment group represented by the solid lines.

[0380] Example 12: Viability of MDR-positive 786-O cells treated with anti-CD70 ADCs 786-O cells were seeded into 96-well white plates at 1,000 cells / 80 μL / well and incubated overnight at 37°C in a 5% CO2 incubator. The following day, 5x concentrated anti-CD70-AS269 or anti-CD70-MMAE solutions were prepared in normal medium or medium containing 5x concentrated elacridar or verapamil. The final concentrations of elacridar and verapamil in the assay were 1 μM and 4 μM, respectively. Serially diluted ADCs (20 μL) were added to the wells, and the plates were incubated for 96 hours at 37°C in a 5% CO2 incubator. At the end of the incubation, CellTiter-Glo 2.0 (Promega, Madison, WI) was added to the plates equilibrated to room temperature, and luminescence was measured using a SpectraMax M5E luminometer. Cell viability was calculated as a percentage of the ADC-untreated control. IC 50 was determined by nonlinear four-parameter dose-response curve fitting using GraphPad Prism (GraphPad Software, San Diego, CA). The anti-CD70-AS269 ADC showed strong activity against MDR-positive 786-O cells, whereas the control anti-CD70-MMAE ADC showed weak activity that was improved by the addition of the MDR inhibitor elacridar (Figure 9). [Monomethyl auristatin E (MMAE) is a known substrate of MDR pumps. AS269 was not a substrate of MDR / P-gp or BCRP (data not shown).]

[0381] Example 13: Efficacy of anti-CD70-AS269 in the U266 multiple myeloma disseminated model Male NOG mice (n = 9 / group) were intravenously injected with U266 cells, and serum IgE lambda levels were measured as an indicator of tumor burden. After IgE lambda levels reached approximately 4500 ng / mL, animals were divided into groups and administered 0.5 mg / kg or 1.5 mg / kg of anti-CD70-AS269 or 1.5 mg / kg of unconjugated mAb. Animals were weighed twice weekly and monitored daily for survival. Mice were also bled once every two weeks by tail snipping; 50–100 μL of blood was collected in serum separator tubes and then measured by IgE lambda ELISA. A single administration of 1.5 mg / kg of anti-CD70-AS269 significantly prolonged survival compared to vehicle (Figure 11), delayed tumor burden, and reduced weight loss (data not shown).

[0382] Example 14: Co-culture CD27 reporter assay 786-O cells were seeded at 40,000 cells / well in a 96-well plate and incubated overnight in an incubator set at 37°C and 5% CO2. The next day, a vial of Jurkat / CD27 / NFkB-luciferase (Jurkat NFkB-luc) reporter cells was thawed, and the cells were resuspended in assay buffer. Jurkat NFkB-luc reporter cells were added to the seeded 786-O cells along with serially diluted anti-CD70-AS269 ADC or parental anti-CD70 mAb, negative control anti-HER2-AS269 ADC or anti-HER2 mAb, or positive control blocking anti-CD27 mAb. The 96-well co-culture plate was incubated at 37°C and 5% CO2, and luminescence signals were detected using a luminometer. Anti-CD70 mAb, anti-CD70-AS269 ADC, and positive control blocking anti-CD27 mAb all inhibited CD70-mediated CD27 signaling in Jurkat NFkB-luc reporter cells, whereas negative control anti-HER2 mAb and anti-HER2-AS269 ADC did not inhibit signaling (Figure 12).

[0383] Example 15: In vitro activity and CD70 expression across multiple cell types In vitro activity. All cell lines, except U266 and NCI-H929, were seeded into 96-well cell culture plates at optimized cell densities (ranging from 1,000 to 8,000 cells / well) for 5 days of incubation and incubated overnight in an incubator set at 37°C and 5% CO2. U266 and NCI-H929 cells were seeded at optimized cell densities for 4 days of incubation and were not incubated overnight in an incubator. The anti-CD70-AS269 ADC was serially diluted in the appropriate cell line-specific culture medium and added directly to the seeded cells in duplicate or triplicate. The total volume in the assay wells was 100 μL / well. The assay plate was incubated in an incubator set at 37°C and 5% CO2. After 4 days of incubation, plates were equilibrated to room temperature and cell viability was measured by adding Promega CellTiter-Glo or CellTiter-Glo 2.0 reagent, incubating on a plate shaker for 2 minutes to induce cell lysis, incubating at room temperature for 10 minutes to stabilize the signal, and reading on a luminometer. Cell viability was calculated as a percentage of untreated control cells and was compared with that of the Ati-CD70-AS269 IC. 50 Values ​​were determined using a four-parameter curve fit of the dose-response curve.

[0384] CD70 expression levels. CD70 cell surface counts in various cell lines were quantified using QiFiKit (Dako, catalog number K0078). Cell lines were harvested and stained with mouse anti-human CD70 Ki-24 antibody (BD Biosciences catalog number 555833) for 1 hour at 4°C. After washing, cells and QiFiKit control beads were stained with anti-mouse IgG-FITC secondary antibody (1:50 dilution) for 45 minutes at 4°C, washed twice, and analyzed on a flow cytometer in the FITC channel. CD70 counts per cell were calculated using a reference curve generated with QiFiKit control beads.

[0385] Anti-CD70-AS269 generally showed higher activity in tumor cell lines expressing higher CD70 / cell and lower activity in tumor cell lines expressing lower CD70 / cell, demonstrating the specificity of the anti-CD70-AS269 ADC (Table 2).

[0386] [Table 2]

[0387] Example 16: Anti-CD70-AS269 ADC affinity to CD70 from various species A surface plasmon resonance assay was developed in which anti-human IgG (Fc) was diluted to 25 μg / mL in immobilization buffer (10 nM sodium acetate, pH 5.0) and injected over an activated CM5 sensor chip at a flow rate of 10 μL / min for 360 seconds. Anti-CD70-AS269 ADC was then diluted to 5 or 20 μg / mL in running buffer (1× HEPES + 0.005% Tween-20, pH 7.4) and injected into the sample channel. Analyte human CD70, cynomolgus monkey CD70, rat CD70, or mouse CD70 protein was serially diluted in running buffer and injected at a flow rate of 30 μL / min for a 120-second association phase and a 300-second dissociation phase. After each cycle of interaction analysis, the sensor chip surface was regenerated with 3 M magnesium chloride for 30 seconds at a flow rate of 20 μL / min. Affinity analysis was performed using Biacore Insight Evaluation software, using a reference channel for background subtraction and a 1:1 binding method for curve fitting. Anti-CD70-AS269 bound to human and cynomolgus CD70 within 3-fold but showed no cross-reactivity to rodent CD70 (Figure 13).

[0388] Example 17: Anti-CD70-AS269 ADC toxicokinetics and pharmacokinetics The anti-CD70-AS269 ADC was administered intravenously at different dose levels to male and female cynomolgus monkeys (n=5 / sex / dose level). The ADC was administered every 3 weeks for a total of 3 doses (1.5 mg / kg, 4.5 mg / kg, and 9 mg / kg), followed by a 5-week recovery period. Serum samples were collected from the animals at various time points and measured in total antibody (TA) and anti-CD70-AS269 ADC ligand binding assays using a Meso Scale Discovery (MSD) readout. Comparison of the anti-CD70-AS269 ADC toxicokinetic (TK) concentration-time curve at the highest nonseverely toxic dose (HNSTD) concentration (4.5 mg / kg) in monkeys with the anti-CD70-AS269 intact ADC pharmacokinetic concentration-time curve at a pharmacologically active dose in mice (see Example 7) shows a clear therapeutic index (Figure 14).

[0389] Example 18: Phase I dose-escalation and dose-expansion study of ARX305, an anti-CD70-AS269 ADC, in subjects with relapsed or refractory clear cell renal cell carcinoma. This first-in-human (FIH), multicenter, open-label, dose-escalation and dose-expansion study will evaluate the safety, pharmacokinetics (PK), and antitumor activity of ARX305 in subjects with clear cell renal cell carcinoma (ccRCC) whose tumors are resistant or refractory to previous standard therapies. Subjects must have been previously treated with kinase inhibitors, antiangiogenic agents, mTOR inhibitors, and / or immune checkpoint inhibitors (either monotherapy or in combination).

[0390] This study includes a dose-escalation and dose-expansion portion in patients with relapsed or refractory ccRCC. ARX305 is administered intravenously in a Q3W cycle, with cycles continuing as long as the subject is eligible to continue treatment. Subjects receive an initial dose of 0.24 mg / kg of ARX305 on day 1 of the first 3-week cycle, followed by dose escalation in the Phase 1 study. Examples of escalating doses include about 0.05 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, about 0.2 mg / kg, about 0.22 mg / kg, about 0.24 mg / kg, about 0.26 mg / kg, about 0.28 mg / kg, about 0.3 mg / kg, about 0.32 mg / kg, and about 0.4 mg / kg for human subjects. 0.34mg / kg, about 0.36mg / kg, about 0.38mg / kg, about 0.4mg / kg, about 0.42mg / kg, about 0.44mg / kg, about 0.46mg / kg, about 0. 48mg / kg, approx. 0.5mg / kg, approx. 0.52mg / kg, approx. 0.54mg / kg, approx. 0.56mg / kg, approx. 0.58mg / kg, approx. 0.6mg / kg, approx. 0.62mg / kg, about 0.64mg / kg, about 0.66mg / kg, about 0.68mg / kg, about 0.7mg / kg, about 0.72mg / kg, about 0.74mg / kg, about 0.76mg / k g, about 0.78mg / kg, about 0.8mg / kg, about 0.82mg / kg, about 0.84mg / kg, about 0.86mg / kg, about 0.88mg / kg, about 0.9mg / kg, about 0 The doses may include about 0.92 mg / kg, about 0.94 mg / kg, about 0.96 mg / kg, about 0.98 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, or about 2 mg / kg. More specific doses may include about 0.24 mg / kg, 0.48 mg / kg, 0.8 mg / kg, 1.1 mg / kg, 1.3 mg / kg, and 1.5 mg / kg. Safety, efficacy, and objective response rate (ORR) will be evaluated using RECIST 1.1 criteria. ARX305 will be administered as monotherapy; no other anticancer agents will be administered in this study.

[0391] Example 19: ARX305 lyophilized powder for injection ARX305 formulation development included chemical and physical characterization of the ARX305 antibody-drug conjugate drug substance (e.g., amino acid sequence, charge, isoelectric point (pI), molecular weight, drug-to-antibody ratio (DAR), size and charge variants), solubility, excipient screening (e.g., excipient identity and concentration, buffer pH, identity and concentration), and stability studies, as well as consideration of the ability to minimize manufacturing processing steps between the bulk drug substance and the formulated drug product. The lead clinical formulation was selected based on the results of the aforementioned evaluations, including solubility (protein recovery), chemical and physical stability criteria, using high performance liquid chromatography (HPLC) analysis and capillary electrophoresis.

[0392] The excipients were selected according to their stabilizing effect on the pharmaceutical product. L-histidine and L-histidine monohydrochloride monohydrate, at a concentration of 20 mM, maintain the pH of the liquid state. Sucrose at a concentration of 8% (w / v) stabilizes the ARX305 active substance against aggregate formation in the liquid state and functions as a cryoprotectant. Polysorbate 80 at a concentration of 0.02% (w / v) was selected to stabilize the ARX305 active substance against stirring in the liquid state.

[0393] The ARX305 active drug substance is sterile filtered at a target protein concentration of 10 mg / mL in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80 (pH 5.7), filled into 20 mL glass vials, and lyophilized in a lyophilizer to produce the drug product.

[0394] The clinical formulation for intravenous administration, ARX305 Injection, lyophilized powder (hereafter referred to as ARX305 Drug Product), contains the ARX305 antibody-drug conjugate (drug substance, 60 mg / vial) formulated at 10 mg / mL in a solution containing 20 mM histidine buffer (L-histidine, L-histidine hydrochloride), 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80. The final solution pH before lyophilization (and after reconstitution of the lyophilized product) is 5.7 (see Table 3). The labeled fill volume is 6.0 mL per vial. Based on an extractable volume determination study, the overfill volume is set at 0.46 mL per vial.

[0395] [Table 3] 1 Amounts are based on a labeled volume of 6.0 mL / vial. Adequate amount = sufficient amount.

[0396] The excipients comply with the requirements of ...

Claims

1. An antibody-drug conjugate (ADC), comprising: The following structure: 【Chemistry 1】 and one or more drug-linker groups having an anti-CD70 antibody or fragment thereof comprising one or more heavy chains, wherein at least one of the one or more heavy chains comprises an amino acid sequence that contains a first non-naturally encoded amino acid, the amino acid sequence comprising SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; During the ceremony, each 【Chemistry 2】 represents a single or double bond covalently attaching one of the one or more drug-linker groups to the anti-CD70 antibody or fragment thereof, and each # represents a site of attachment to the anti-CD70 antibody or fragment thereof.

2. 2. The ADC of claim 1, wherein at least one of the one or more heavy chains comprises the amino acid sequence of SEQ ID NO:

3.

3. 3. The ADC of claim 1 or 2, wherein the anti-CD70 antibody or fragment thereof further comprises one or more light chains, and at least one of the one or more light chains comprises an amino acid sequence that shares at least 90% identity with SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

4. 4. The ADC of claim 3, wherein at least one of the one or more light chains comprises the amino acid sequence of SEQ ID NO:

2.

5. 4. The ADC of claim 1, 2, or 3, wherein at least one of the one or more light chains comprises an amino acid sequence containing a second non-naturally encoded amino acid, wherein the amino acid sequence comprises SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

6. 6. The ADC of any one of claims 1-5, wherein the anti-CD70 antibody or fragment thereof comprises two heavy chains, one heavy chain comprising the first non-naturally encoded amino acid and the other heavy chain comprising a third non-naturally encoded amino acid.

7. 7. The ADC of claim 6, wherein each of the one heavy chain and the other heavy chain comprises the amino acid sequence of SEQ ID NO:

3.

8. each 【Transformation 3】 The ADC of any one of claims 1 to 7, wherein represents a double bond.

9. each 【Chemistry 4】 covalently attaches one of the one or more drug-linker groups to a non-naturally encoded amino acid of one of the anti-CD70 antibody or fragment thereof.

10. 10. The ADC of any one of claims 1-9, wherein the first non-naturally encoded amino acid, the second non-naturally encoded amino acid, and the third non-naturally encoded amino acid are each para-acetyl-L-phenylalanine.

11. The ADC has formula (I): 【Transformation 5】 wherein: Ab is the anti-CD70 antibody or a fragment thereof; Each R is independently an unsubstituted C １ ~C ８ is alkyl, The ADC according to any one of claims 1 to 10, wherein d is an integer from 1 to 10.

12. 12. The ADC of claim 11, wherein d is 1, 2, 3, or 4.

13. 13. The ADC of claim 11 or 12, wherein each R is methyl.

14. 14. The ADC of claim 11, 12, or 13, wherein the anti-CD70 antibody or fragment thereof comprises two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:

3.

15. The ADC of any one of claims 11 to 14, wherein the anti-CD70 antibody or fragment thereof comprises two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:

2.

16. The ADC of any one of claims 11 to 15, wherein the anti-CD70 antibody or fragment thereof is humanized.

17. 17. The ADC of any one of claims 11 to 16, wherein the anti-CD70 antibody or fragment thereof is a humanized monoclonal antibody comprising two heavy chains and two light chains, each heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and each light chain comprising the amino acid sequence of SEQ ID NO:

2.

18. 18. The ADC of claim 17, wherein the amino acid sequence of SEQ ID NO: 3 comprises one non-naturally encoded amino acid, and said one non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

19. The ADC of any one of claims 11 to 18, wherein d is 2.

20. the anti-CD70 antibody or fragment thereof is a humanized anti-CD70 monoclonal antibody comprising two heavy chains, two light chains, and two non-naturally encoded amino acids; the amino acid sequence of each of the two heavy chains is SEQ ID NO:3, and SEQ ID NO:3 contains one non-naturally encoded amino acid, and the one non-naturally encoded amino acid in SEQ ID NO:3 is para-acetyl-L-phenylalanine at position A114 (Kabat numbering) of SEQ ID NO:3; the amino acid sequence of each of the light chains is SEQ ID NO:2, and each R is methyl; the one or more drug-linker groups are two drug-linker groups; d is 2, 12. The ADC of claim 11, wherein each of the two drug-linker groups is attached to a respective para-acetyl-L-phenylalanine at position A114 of SEQ ID NO:3, thereby attaching each of the two drug-linker groups to the humanized anti-CD70 monoclonal antibody.

21. A composition comprising the ADC of any one of claims 1 to 20.

22. 22. The composition of claim 21, further comprising an additional therapeutic agent.

23. 23. The composition of claim 22, wherein the additional therapeutic agent is an immunotherapeutic agent, a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, or an immunomodulatory agent, or a combination thereof.

24. 24. The composition of claim 22 or 23, wherein the additional therapeutic agent is a checkpoint inhibitor, a CD70 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, a hypomethylating agent, or a platinum-based therapeutic agent, or a combination thereof.

25. The composition of any one of claims 21 to 24, wherein the composition comprising the ADC is a pharmaceutical composition comprising an effective amount of the ADC, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

26. 100. A method of killing a cell, comprising contacting the cell with an ADC according to any one of claims 1 to 20, a composition according to any one of claims 21 to 25 comprising an effective amount of said ADC, a reconstituted solution according to any one of claims 91 to 94, or a formulation according to any one of claims 83 to 90, 95, and 96 comprising an effective amount of said ADC.

27. 27. The method of claim 26, wherein the cell is a tumor cell or a cancer cell.

28. 28. The method of claim 27, wherein the tumor cells or cancer cells are renal cell carcinoma (RCC) tumor or cancer cells.

29. 100. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the ADC of any one of claims 1 to 20, a composition of any one of claims 21 to 25 comprising an effective amount of said ADC, a reconstituted solution of any one of claims 91 to 94, or a formulation of any one of claims 83 to 90, 95, and 96 comprising an effective amount of said ADC.

30. 30. The method of claim 29, wherein the disease or condition is a tumor or cancer.

31. 31. The method of claim 30, wherein the tumor or cancer is a solid tumor.

32. 31. The method of claim 30, wherein the tumor or the cancer is a blood cancer.

33. 33. The method of claim 32, wherein the hematological cancer is lymphoma, multiple myeloma, or leukemia.

34. 31. The method of claim 30, wherein the tumor or cancer is kidney cancer, brain cancer, breast cancer, Burkitt's lymphoma, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, testicular cancer, prostate cancer, colorectal cancer, esophageal cancer, bladder cancer, lung cancer, non-small cell lung cancer, urothelial carcinoma, bile duct cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, nasopharyngeal carcinoma, mantle cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, or acute myeloid leukemia.

35. 31. The method of claim 30, wherein the tumor or cancer is renal cell carcinoma, brain cancer, multiple myeloma, mantle cell lymphoma, or lung cancer.

36. 31. The method of claim 30, wherein the tumor or cancer is renal cell carcinoma.

37. 37. The method of claim 36, wherein the renal cell carcinoma is clear cell renal cell carcinoma.

38. The method of any one of claims 30 to 37, wherein the tumor or cancer is a multidrug resistant tumor or cancer.

39. 39. The method of any one of claims 29 to 38, further comprising treating the subject with radiation therapy.

40. 40. The method of any one of claims 29 to 39, further comprising treating the subject with an effective amount of an additional therapeutic agent.

41. 41. The method of claim 40, wherein the additional therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, an immunomodulatory agent, or an immunotherapeutic agent, or a combination thereof.

42. 42. The method of claim 40 or 41, wherein the additional therapeutic agent is a checkpoint inhibitor, a CD70 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, a hypomethylating agent, or a platinum-based therapeutic agent, or a combination thereof.

43. 43. The method of claim 42, wherein the additional agent is a checkpoint inhibitor, and the checkpoint inhibitor is a PD-1 inhibitor.

44. 44. The method of claim 43, wherein the PD-1 inhibitor is AMP-224, atezolizumab, avelumab, BMS-936558, BMS-936559, CT-001, durvalumab, MEDI0680, nivolumab, PDR001, pembrolizumab, pidilizumab, or REGN2810.

45. 45. The method of claim 44, wherein the PD-1 inhibitor is pembrolizumab.

46. 46. ​​The method of any one of claims 30 to 45, wherein said method improves or optimizes cancer cell killing or delays the progression or recurrence of said tumor or said cancer.

47. The method of any one of claims 30 to 46, wherein the tumor or cancer is a CD70-expressing cancer.

48. 48. The method of claim 47, wherein the CD70-expressing cancer has at least about 5,000 CD70 molecules / cell.

49. 48. The method of claim 47, wherein the CD70-expressing cancer has at least about 10,000 CD70 molecules / cell.

50. 48. The method of claim 47, wherein the CD70-expressing cancer has at least about 15,000 CD70 molecules / cell.

51. 51. The method of any one of claims 30 to 50, wherein the subject, the cancer, or the tumor is resistant or refractory to conventional standard therapies.

52. 52. The method of any one of claims 30 to 51, wherein the subject has cancer metastasis from the same cancer or a different cancer.

53. 30. The method of claim 29, wherein the disease or condition is a myelodysplastic syndrome.

54. 54. The method of claim 53, wherein the subject is undergoing or has previously been treated with a hypomethylating agent.

55. 55. The method of any one of claims 29 to 54, wherein the subject is a human subject.

56. 56. The method of claim 55, wherein the human subject is an adult.

57. 57. The method of claim 55 or 56, wherein the effective amount of the ADC is a dose in the range of about 0.05 mg / kg to about 10 mg / kg of the human subject, or any value therebetween.

58. 58. The method of claim 57, wherein the effective amount of the ADC is a dose in the range of about 0.05 mg / kg to about 2 mg / kg of the human subject, or any value therebetween.

59. The effective amount of the ADC is about 0.05 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, about 0.2 mg / kg, about 0.22 mg / kg, about 0.24 mg / kg, about 0.26 mg / kg, about 0.28 mg / kg, about 0.3 mg / kg, about 0.32 mg / kg, about 0.40 mg / kg, about 0.42 mg / kg, about 0.46 mg / kg, about 0.48 mg / kg, about 0.49 mg / kg, about 0.50 mg / kg, about 0.52 mg / kg, about 0.54 mg / kg, about 0.56 mg / kg, about 0.58 mg / kg, about 0.59 mg / kg, about 0.60 mg / kg, about 0.61 mg / kg, about 0.62 mg / kg, about 0.63 mg / kg, about 0.64 mg / kg, about 0.65 mg / kg, about 0.66 mg / kg, about 0.67 mg / kg, about 0.68 mg / kg, about 0.69 mg / kg, about 0.70 mg / kg, about 0.71 mg / kg, about 0.72 mg / kg, about 0.73 mg / kg, about 0.74 mg / kg, about 0.75 mg / kg, about 0.76 mg / kg, about 0.77 mg / kg, about 0.78 mg / kg, about 0.79 mg / kg, about 0.80 mg / kg, about 0.81 mg / kg, about 0.82 mg / kg, about 0.83 mg / kg, about 0.84 mg / kg, about 0.85 mg / kg, about 0.86 .. 34mg / kg, about 0.36mg / kg, about 0.38mg / kg, about 0.4mg / kg, about 0.42mg / kg, about 0.44mg / kg, about 0.46mg / kg, about 0.48m g / kg, about 0.5 mg / kg, about 0.52 mg / kg, about 0.54 mg / kg, about 0.56 mg / kg, about 0.58 mg / kg, about 0.6 mg / kg, about 0.62 mg / kg, About 0.64 mg / kg, about 0.66 mg / kg, about 0.68 mg / kg, about 0.7 mg / kg, about 0.72 mg / kg, about 0.74 mg / kg, about 0.76 mg / kg, about 0.7 8mg / kg, about 0.8mg / kg, about 0.82mg / kg, about 0.84mg / kg, about 0.86mg / kg, about 0.88mg / kg, about 0.9mg / kg, about 0.92mg / kg 59. The method of claim 58, wherein the dose is about 0.94 mg / kg, about 0.96 mg / kg, about 0.98 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg or about 2 mg / kg.

60. 58. The method of claim 57, wherein the effective amount of the ADC is a dose in the range of about 2 mg / kg to about 5 mg / kg of the human subject, or any value therebetween.

61. 61. The method of claim 60, wherein the effective amount of the ADC is a dose of about 2 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.6 mg / kg, about 3.8 mg / kg, about 4 mg / kg, about 4.2 mg / kg, about 4.4 mg / kg, about 4.6 mg / kg, about 4.8 mg / kg, or about 5 mg / kg of the human subject.

62. 58. The method of claim 57, wherein the effective amount of the ADC is a dose in the range of about 5 mg / kg to about 10 mg / kg of the human subject, or any value therebetween.

63. 63. The method of claim 62, wherein the effective amount of the ADC is at a dose of about 5 mg / kg, about 5.2 mg / kg, about 5.4 mg / kg, about 5.6 mg / kg, about 5.8 mg / kg, about 6 mg / kg, about 6.2 mg / kg, about 6.4 mg / kg, about 6.6 mg / kg, about 6.8 mg / kg, about 7 mg / kg, about 7.2 mg / kg, about 7.4 mg / kg, about 7.6 mg / kg, about 7.8 mg / kg, about 8 mg / kg, about 8.2 mg / kg, about 8.4 mg / kg, about 8.6 mg / kg, about 8.8 mg / kg, about 9 mg / kg, about 9.2 mg / kg, about 9.4 mg / kg, about 9.6 mg / kg, about 9.8 mg / kg, or about 10 mg / kg of the human subject.

64. 64. The method of any one of claims 29 to 63, wherein the administration is oral, intradermal, intratumoral, intravenous, or subcutaneous.

65. 21. A method of treating renal cell carcinoma in a human subject in need thereof, comprising administering to the human subject an effective amount of the ADC of any one of claims 1-20, wherein the ADC is in a formulation comprising about 10 mg / mL of the ADC, about 20 mM histidine buffer, about 8% (w / v) sucrose, and about 0.02% (w / v) polysorbate 80, wherein the formulation has a pH of about 5.

7.

66. [0033] An effective amount of the ADC is about 0.05 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, about 0.2 mg / kg, about 0.22 mg / kg, about 0.24 mg / kg, about 0.26 mg / kg, about 0.28 mg / kg, about 0.3 mg / kg, about 0.32 mg / kg, about 0.34 mg / kg, about 0.36 mg / kg, about 0.38 mg / kg, about 0.4 mg / kg, about 0.42 mg / kg, about 0.44 mg / kg, about 0.46 mg / kg, about 0.48 mg / kg, about 0.5mg / kg, about 0.52mg / kg, about 0.54mg / kg, about 0.56mg / kg, about 0.58mg / kg, about 0.6mg / kg g, about 0.62 mg / kg, about 0.64 mg / kg, about 0.66 mg / kg, about 0.68 mg / kg, about 0.7 mg / kg, about 0.72 m g / kg, about 0.74 mg / kg, about 0.76 mg / kg, about 0.78 mg / kg, about 0.8 mg / kg, about 0.82 mg / kg, about 0. 84mg / kg, about 0.86mg / kg, about 0.88mg / kg, about 0.9mg / kg, about 0.92mg / kg, about 0.94mg / kg, About 0.96 mg / kg, about 0.98 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4mg / kg, about 1.5mg / kg, about 1.6mg / kg, about 1.7mg / kg, about 1.8mg / kg, about 1.9mg / kg, about 2m g / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3 mg / kg, about 3.2 mg / kg , about 3.4 mg / kg, about 3.6 mg / kg, about 3.8 mg / kg, about 4 mg / kg, about 4.2 mg / kg, about 4.4 mg / kg, about 4. 6 mg / kg, about 4.8 mg / kg, about 5 mg / kg, about 5.2 mg / kg, about 5.4 mg / kg, about 5.6 mg / kg, about 5.8 mg / kg, about 6 mg / kg, about 6.2 mg / kg, about 6.4 mg / kg, about 6.6 mg / kg, about 6.8 mg / kg, about 7 mg / kg, about 7.2 mg / kg, about 7.4 mg / kg, about 7.6 mg / kg, about 7.8 mg / kg, about 8 mg / kg, about 8.2 mg / kg, about 8.4 mg / kg, about 8.6 mg / kg, about 8.8 mg / kg, about 9 mg / kg, about 9.2 mg / kg, about 9.4 mg / kg, about 9.6 mg / kg,66. The method of claim 65, wherein the dose is about 9.8 mg / kg or about 10 mg / kg.

67. 67. The method of claim 65 or 66, wherein said administering an effective amount of said ADC is on a once every three weeks dosing schedule.

68. (i) the renal cell carcinoma is clear cell renal cell carcinoma, or (ii) the renal cell carcinoma is metastatic renal cell carcinoma, and / or (iii) 68. The method of claim 65, 66, or 67, wherein the human subject, the clear cell renal cell carcinoma, or the metastatic renal cell carcinoma is resistant or refractory to previous standard therapy.

69. An isolated anti-CD70 antibody or fragment thereof, comprising an amino acid sequence selected from the group consisting of the sequences listed in Table 1.

70. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

1.

71. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

2.

72. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

3.

73. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

4.

74. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

5.

75. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 6, 7, 8, or 9.

76. 70. The isolated anti-CD70 antibody or fragment thereof of claim 69, comprising a heavy chain and a light chain, wherein the heavy chain amino acid sequence is SEQ ID NO: 3 and the light chain amino acid sequence is SEQ ID NO:

2.

77. A nucleic acid encoding any one of SEQ ID NOs: 1 to 9.

78. A nucleic acid encoding any one of SEQ ID NOs: 3 to 9.

79. A nucleic acid encoding SEQ ID NO:

3.

80. A vector comprising a nucleic acid encoding any one of SEQ ID NOs: 1 to 9.

81. A vector comprising a nucleic acid encoding any one of SEQ ID NOs: 3 to 9.

82. 21. Use of any ADC of any one of claims 1 to 20, or an antibody or fragment thereof comprising an amino acid sequence listed in Table 1, in the manufacture of a medicament for the treatment of a disease or condition in a subject.

83. A formulation comprising the ADC of any one of claims 1 to 20.

84. 84. The formulation of claim 83, further comprising a buffer.

85. 85. The formulation of claim 84, wherein the buffer is a histidine buffer.

86. 86. The formulation of claim 83, 84, or 85, wherein the formulation further comprises a cryoprotectant.

87. 87. The formulation of claim 86, wherein the cryoprotectant is sucrose.

88. 88. The formulation of any one of claims 83 to 87, wherein the formulation further comprises a surfactant.

89. 89. The formulation of claim 88, wherein the surfactant is polysorbate 80.

90. The formulation of any one of claims 83 to 89, wherein the formulation is a lyophilized pharmaceutical product.

91. 91. A reconstituted solution comprising the formulation of claim 90 and a diluent, wherein the reconstituted solution comprises the ADC at a concentration in the range of about 5 mg / mL to about 25 mg / mL.

92. 92. The reconstituted solution of claim 91, wherein the histidine buffer is an L-histidine buffer at a concentration in the range of about 10 mM to about 50 mM, the sucrose is at a concentration in the range of about 1% (w / v) to about 20% (w / v), the polysorbate 80 is at a concentration in the range of about 0.01% (w / v) to about 0.1% (w / v), and the reconstituted solution has a pH in the range of about 5.2 to about 6.

2.

93. 93. The reconstituted solution of claim 91 or 92, wherein the pH of the reconstituted solution is less than 6.

94. 94. The reconstituted solution of claim 91, 92, or 93, wherein the diluent is water.

95. 21. A formulation comprising about 5 mg / mL to about 15 mg / mL of the ADC of any one of claims 1 to 20, about 15 mM to about 25 mM histidine buffer, about 5% (w / v) to about 15% (w / v) sucrose, and about 0.01% (w / v) to about 0.05% (w / v) polysorbate 80, wherein the formulation has a pH of about 5.4 to about 6.

0.

96. 96. The formulation of claim 95, comprising about 10 mg / mL of the ADC, about 20 mM histidine buffer, about 8% (w / v) sucrose, and about 0.02% (w / v) polysorbate 80, wherein the pH of the formulation is about 5.

7.

97. 69. The method of any one of claims 29 to 68, wherein administration of an effective amount of the ADC is performed in a dosing schedule.

98. 98. The method of claim 97, wherein the administration schedule is once every 1, 2, 3, 4, 5, or 6 weeks.

99. 98. The method of claim 97, wherein the administration schedule is once every two weeks.

100. 98. The method of claim 97, wherein the administration schedule is once every three weeks.

101. 98. The method of claim 97, wherein the administration schedule is once every four weeks.

102. 98. The method of claim 97, wherein the administration schedule is more than once within a three week cycle.

103. 103. The method of any one of claims 29-68 and 97-102, wherein the administration is at least once every four weeks for at least about eight weeks.

104. 104. The method of any one of claims 29-68 and 97-103, wherein the administration is once every three weeks for at least about eight weeks.

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