Humanized Anti-prostate-specific membrane antigen (PSMA) antibody drug conjugates

Abstract

To provide novel anti-PSMA antibodies, variants, and antibody drug conjugate compositions, and to provide methods and compositions for using anti-PSMA antibody drug conjugates in inhibiting, preventing or treating PSMA related diseases or cancers.SOLUTION: The present invention provides an anti-prostate specific membrane antigen (anti-PSMA) antibody, comprising a specific heavy chain amino acid sequence and a specific light chain amino acid sequence.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 650,277, filed March 29, 2018, and entitled "Novel Anti-Prostate Specific Membrane Antigen (PSMA) Antibody Drug Conjugates," which is incorporated by reference in its entirety.

[0002] [Sequence Listing] This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on March 29, 2019 is named AMBX_0225_00PCT_ST25.txt and is 37,015 bytes in size.

[0003] [Technical field] The present disclosure relates to novel prostate-specific membrane antigen (anti-PSMA) antibodies and antibody-drug conjugates. More particularly, the present disclosure relates to methods and compositions for use with anti-PSMA antibody-drug conjugates in inhibiting, preventing or treating PSMA-related diseases or cancers.

[0004] [Background technology] Prostate cancer is the most commonly diagnosed non-skin-associated malignancy in men in developed countries. It is estimated that one in six men will be diagnosed with prostate cancer. Diagnosis of prostate cancer has improved following the use of serum-based markers such as prostate-specific antigen (PSA). Furthermore, prostate tumor-associated antigens provide targets for tumor imaging, diagnosis, and targeted therapy. Prostate-specific membrane antigen (PSMA), a prostate tumor-associated marker, is one such target. PSMA is significantly overexpressed in androgen-independent prostate cancer. Overexpression of PSMA is associated with high tumor grade, high risk of disease progression and recurrence (Perner et al, Human Pathology, 2007). High expression of PSMA is associated with negative clinical prognosis and significantly shorter survival. PSMA expression is observed both at the primary disease site or at metastatic sites such as bone and lymph nodes (Olson and Israel, Frontiers in Bioscience, 2014).

[0005] Although several targeted therapies have been pursued over the years, these radiotherapy-related treatments have made the most progress toward clinical development as they have demonstrated both efficacy and acceptable tolerability. Other clinical developments of modalities involving targeted delivery of non-radioactive cytotoxic agents have met with limited success.

[0006] Given the physical properties of PSMA and its expression patterns associated with prostate cancer progression, PSMA is an excellent target for the development of antibody-drug conjugates. Antibody-drug conjugates (ADCs) are a powerful class of therapeutic constructs that allow targeted delivery of cytotoxic agents to target cells, such as cancer cells. Due to their targeting function, these compounds exhibit a much higher therapeutic index compared to similar systemically delivered agents. ADCs are developed as complete antibodies or antibody fragments, such as scFvs. The antibody or fragment is linked to one or more copies of the drug via a linker that is stable under physiological conditions but can be cleaved once inside the target cell. To date, only two to three ADCs have been approved for treatment, including gemtuzumab ozogamicin (since withdrawn from the market) for AML, brentuximab vedotin for ALCL and Hodgkin lymphoma, and trastuzumab emtansine for HER2-positive metastatic breast cancer. (Verma et al, N Engl J Med 367:1783-91,2012; Bross et al, Clin Cancer Res 7:1490-96,2001; Francisco et al, Blood 102:1458-65,2003). A variety of drugs targeting many ADCs are in clinical trials. However, ADCs targeting PSMA face challenges due to lack of therapeutic index and toxicity.

[0007] Thus, there is a need for better or improved therapeutics that target the PSMA antigen and associated cancers. To overcome this deficiency in the art, the present disclosure herein provides novel anti-PSMA antibodies, variants, and antibody-drug conjugate compositions.

[0008] Summary of the Invention The present disclosure provides anti-PSMA antibodies, antibody variants, and antibody-drug conjugate compositions. In further embodiments, the present disclosure provides antibody-drug conjugates (ADCs) comprising such anti-PSMA antibodies.

[0009] The present disclosure provides anti-PSMA antibodies, antibody variants, and antibody compositions comprising SEQ ID NOs: 1-17. In some embodiments, the present disclosure provides antibody drug conjugates (ADCs) comprising SEQ ID NOs: 1-17.

[0010] The present disclosure provides anti-PSMA antibodies, antibody variants, and antibody compositions consisting of SEQ ID NOs: 1 to 17. In some embodiments, the present disclosure provides antibody-drug conjugates (ADCs) consisting of SEQ ID NOs: 1 to 17.

[0011] The present invention provides anti-prostate specific membrane antigen antibody (anti-PSMA) variants comprising a heavy chain and a light chain, wherein the heavy chain sequence is selected from the group of SEQ ID NO: 8, 10, 12, 14, or 16, and the light chain sequence is selected from the group of SEQ ID NO: 9, 11, 13, 15, or 17. In some embodiments, the present invention provides anti-prostate specific membrane antigen antibody (anti-PSMA) variants comprising a heavy chain sequence of SEQ ID NO: 8, 10, 12, 14, or 16. In some embodiments, the present invention provides anti-prostate specific membrane antigen antibody (anti-PSMA) variants comprising a light chain sequence of SEQ ID NO: 9, 11, 13, 15, or 17.

[0012] In other embodiments, the invention provides anti-prostate specific membrane antigen antibody (anti-PSMA) variants consisting of the heavy chain sequence of SEQ ID NO: 8, 10, 12, 14, or 16. In other embodiments, the invention provides anti-prostate specific membrane antigen antibody (anti-PSMA) variants consisting of the light chain sequence of SEQ ID NO: 9, 11, 13, 15, or 17.

[0013] In another embodiment, the anti-PSMA antibody variant comprises a heavy chain comprising a variable sequence region selected from the group of SEQ ID NO: 1 or 6. In yet another embodiment, the anti-PSMA antibody variant comprises a light chain sequence comprising a variable region selected from the group of SEQ ID NO: 2-5 or 7. In yet another embodiment, the anti-PSMA antibody heavy chain variant consists of the variable region sequence of SEQ ID NO: 1 or 6. In yet another embodiment, the anti-PSMA antibody light chain variant consists of a variable region selected from the group of SEQ ID NO: 2-5 or 7.

[0014] In further embodiments, the anti-PSMA antibody variant comprises a non-naturally encoded amino acid incorporated into the antibody heavy or light chain sequence. In another embodiment of the invention, the antibody comprises an anti-PSMA antibody variant having one or more non-naturally encoded amino acids substituted at one or more positions of the antibody heavy or light chain. In some embodiments, the heavy or light chain in the PSMA antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-naturally encoded amino acids. In other embodiments, the heavy or light chain sequence in the PSMA antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-naturally encoded amino acids at one or more positions in the antibody heavy or light chain. In one embodiment, the anti-PSMA antibody heavy chain sequence comprises a non-naturally encoded amino acid. In another embodiment of the invention, the anti-PSMA antibody variant comprises a heavy or light chain sequence as presented in Table 4.

[0015] In a further embodiment of the invention, the antibody is an anti-PSMA antibody variant comprising a light chain sequence from SEQ ID NO: 9, 11, 13, 15, or 17, where the non-naturally encoded amino acid is substituted at a position with high surface accessibility and / or at a site that is charge neutral to the antibody. In a further embodiment of the invention, the antibody is an anti-PSMA antibody variant comprising a heavy chain sequence from SEQ ID NO: 8, 10, 12, 14, or 16, where the non-naturally encoded amino acid is substituted at a position with high surface accessibility and / or at a site that is charge neutral to the antibody.

[0016] In one embodiment, the invention provides an anti-PSMA antibody drug conjugate (ADC or anti-PSMA ADC) in which the antibody is an anti-PSMA comprising light and heavy chain sequences. In a further embodiment of the invention, the anti-PSMA ADC comprises a light chain from SEQ ID NO: 9, 11, 13, 15, or 17, and a heavy chain from SEQ ID NO: 8, 10, 12, 14, or 16. In one embodiment of the invention, the anti-PSMA ADC comprises one or more non-naturally encoded amino acids substituted at one or more positions in the antibody heavy or light chain.

[0017] In another embodiment, the invention provides an anti-PSMA ADC comprising a heavy chain of SEQ ID NO: 8 and a light chain of SEQ ID NO: 9. In another embodiment, the invention provides an anti-PSMA ADC comprising SEQ ID NO: 12 and SEQ ID NO: 13. In another embodiment, the invention provides an anti-PSMA ADC comprising SEQ ID NO: 14 and SEQ ID NO: 15. In another embodiment, the invention provides an anti-PSMA ADC comprising SEQ ID NO: 16 and SEQ ID NO: 17.

[0018] In some embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that binds to the PSMA receptor. In other embodiments of the invention, the antibody, variant, or composition may be an antibody, variant, or composition that binds to the extracellular surface of the PSMA receptor. In another embodiment of the invention, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that binds to a PSMA dimer. In some embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that has a CDR from J591 introduced onto the framework region of the variable region. In other embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that has a non-naturally encoded amino acid. In some embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition described by one or more of the embodiments elsewhere in the disclosure of the invention herein. In some embodiments, the disclosed antibody, antibody variant, or antibody composition may be fully humanized. In other embodiments, the disclosed antibody, antibody variant, or antibody composition may be chimeric. In some embodiments of the invention, the antibody may be a full length antibody (variable + Fc region), Fab, bispecific, Fab-dimer, Fab-bispecific, Fab-trispecific, bispecific T cell engager, dual affinity retargeting antibody, IgG1 / IgG3 bispecific antibody, diabody, bispecific diabody, scFv-Fc, minibody.

[0019] In other embodiments, the anti-PSMA antibody, antibody variant, or antibody composition incorporates a non-naturally encoded amino acid that comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. In one embodiment of the invention, the non-naturally encoded amino acid comprises an aminooxy group.In yet other embodiments, the non-naturally encoded amino acid is para-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, p-acylphenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMephenylalanine, o-OMephenylalanine, m-OMephenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro substituted Leu, nitro substituted His, nitro substituted De, nitro substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 8-nitroTrp, 9-nitroTrp, 10-nitroTrp, 11-nitroTrp, 12-nitroTrp, 13-nitroTrp, 14-nitroTrp, 15-nitroTrp, 16-nitroTrp, 17-nitroTrp, 18-nitroTrp, 19-nitroTrp, 20-nitroTrp, 21-nitroTrp, 22-nitroTrp, 23-nitroTrp, 24-nitroTrp, 25-nitroTrp, 26-nitroTrp, 27-nitroTrp, 28-nitroTrp, 29-nitroTrp, 30-nitroTrp, 31-nitroTrp, 32-nitroTrp, 33-nitroTrp, 34-nitroTrp, 35-nitroTrp, -NitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-L-tyrosine, In another embodiment of the invention, the unnatural amino acid is para-acetylglucosamine.

[0020] In other embodiments of the invention, the anti-PSMA antibody variant is a monoclonal antibody, a humanized antibody, or a chimeric antibody. In one embodiment, the anti-PSMA humanized antibody variant comprises a heavy chain sequence selected from the group of SEQ ID NO: 8, 10, 12, or 14, and a light chain sequence selected from the group of SEQ ID NO: 9, 11, 13, or 15. In another embodiment, the anti-PSMA chimeric antibody variant comprises a heavy chain sequence of SEQ ID NO: 16 and a light chain sequence of SEQ ID NO: 17.

[0021] In another embodiment, the present invention provides a composition of an antibody drug conjugate comprising an anti-PSMA antibody variant covalently attached to a toxin, payload, or drug linker. In one embodiment, the toxin, payload, or drug linker comprises a cytotoxic agent. In one embodiment, the cytotoxic agent is a dolastatin, a dolastatin derivative, or an analog thereof. In another embodiment, the present invention provides a composition of an antibody drug conjugate comprising an anti-PSMA antibody variant covalently attached to a drug-linker, the conjugation occurring through a non-naturally encoded amino acid in the antibody. In another embodiment, the present invention provides a composition of an antibody drug conjugate comprising an anti-PSMA antibody variant of any of SEQ ID NOs: 1-17 covalently attached to one or more dolastatins, the conjugation occurring through a non-naturally encoded amino acid in the antibody. In another embodiment of the present invention, a composition of an antibody drug conjugate comprising an anti-PSMA antibody variant of any of SEQ ID NOs: 8-17 covalently attached to one or more dolastatins, the conjugation occurring through a non-naturally encoded amino acid in the antibody is provided. One embodiment of the present invention provides a composition of an antibody drug conjugate comprising an anti-PSMA antibody heavy chain sequence variant of SEQ ID NO: 8, 10, 12, 14, or 16 covalently linked to one or more dolastatins. The conjugate is formed via a non-naturally encoded amino acid in the antibody. Another embodiment of the present invention provides a composition of an antibody drug conjugate comprising an anti-PSMA antibody light chain variant of SEQ ID NO: 9, 11, 13, 15, or 17 covalently linked to one or more dolastatins. The conjugate is formed via a non-naturally encoded amino acid in the antibody. In another embodiment, the antibody is covalently linked to at least two dolastatins. In one embodiment, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dolastatins derivatives or analogs. In one embodiment, the composition comprises a dolastatin derivative or is a dolastatin analog. In another embodiment, the dolastatin derivative or analog is a monomethyl auristatin, and the monomethyl auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).In other embodiments, the one or more dolastatins are non-cleavable MMAF, non-cleavable MMAE, cleavable MMAF, cleavable MMAE, short cleavable MMAF, or short cleavable MMAE. In further embodiments of the invention, the antibody is linked to a linker, polymer, or biologically active molecule. In some embodiments, the linker is polyethylene glycol (PEG). In other embodiments, the linker is a branched or linear polyethylene glycol. In further embodiments of the invention, the anti-PSMA variants can be PEGylated. In still further embodiments of the invention, the anti-PSMA variants can be PEGylated on a non-naturally encoded amino acid. The anti-PSMA antibodies of the invention can be PEGylated with about 5 kDa PEG, about 10 kDa PEG, about 20 kDa PEG, about 30 kDa PEG, about 40 kDa or more PEG. The poly(ethylene glycol) molecule can have a molecular weight of about 0.1 kDa to about 100 kDa. The poly(ethylene glycol) molecule can have a molecular weight of 0.1 kDa to 50 kDa, 20 kDa to 40 kDa, and any integer value between 25 kDa and 35 kDa. The poly(ethylene glycol) molecule can have a molecular weight of about 30 kDa. The poly(ethylene glycol) molecule can be a linear molecule having a molecular weight of 0.1 kDa to 50 kDa, 20 kDa to 40 kDa, and any integer value between 25 kDa and 35 kDa. The poly(ethylene glycol) molecule can be a linear molecule having a molecular weight of 30 kDa. The poly(ethylene glycol) molecule can have an aminooxy group capable of reacting with an acetyl group on a synthetic amino acid. The poly(ethylene glycol) molecule can be, but is not limited to, a 30 kDa aminooxy-activated linear PEG capable of forming an oxime bond with the acetyl side chain of a non-naturally encoded amino acid (e.g., para-acetylphenylalanine).

[0022] In one embodiment, the invention provides an antibody drug conjugate comprising a humanized anti-PSMA antibody comprising a heavy chain sequence selected from SEQ ID NO: 8, 10, 12, 14, or 16, and a light chain sequence selected from SEQ ID NO: 9, 11, 13, 15, conjugated to at least one drug linker selected from Tables 1-3. The conjugation occurs via a non-naturally encoded amino acid incorporated into the heavy chain sequence. In some embodiments, the drug linker comprises a cytotoxic agent. The cytotoxic agent is a dolastatin, a dolastatin derivative, or an analog thereof. In one embodiment, the dolastatin, dolastatin derivative, or analog is a monomethylauristatin selected from monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE). In another embodiment, the monomethylauristatin is a cleavable MMAE or MMAF, a non-cleavable MMAE or MMAF, or a short cleavable MMAE or MMAF.

[0023] In one embodiment, the invention provides a method for reducing or inhibiting tumor growth or progression in a PSMA-expressing cancer or cancer cell, comprising contacting the PSMA-expressing cancer or cancer cell with an effective amount of an antibody-drug conjugate. The antibody-drug conjugate comprises a humanized anti-PSMA antibody comprising a heavy chain sequence selected from SEQ ID NO: 8, 10, 12, 14, or 16, and a light chain sequence selected from SEQ ID NO: 9, 11, 13, 15, conjugated to at least one drug linker selected from Tables 1-3. The conjugation occurs via a non-naturally encoded amino acid incorporated into the heavy chain sequence. In another embodiment, the invention provides a method further comprising contacting the PSMA-expressing cancer or cancer cell with an effective amount of a therapeutic agent. In another embodiment, the therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulant, an immunomodulatory agent, a corticosteroid, or a combination thereof. In one embodiment, the hormonal agent is enzalutamide.

[0024] In other embodiments, the anti-PSMA ADCs of the invention may be administered with one or more immunostimulants to induce or enhance an immune response. Such immunostimulants include, but are not limited to, IL-2, immunostimulatory oligonucleotides (e.g., CpG motifs), interferons, and tumor necrosis factor alpha. In other embodiments, the anti-PSMA ADCs of the invention may be administered with one or more immunomodulatory agents, including, but not limited to, cytokines, chemokines, adjuvants, or combinations thereof.

[0025] In a further embodiment, the present invention provides a method of inhibiting tumor growth or progression in a subject, comprising providing to the subject a therapeutically effective amount of a PSMA antibody drug conjugate of the present invention. Additionally, in a further embodiment, the present invention provides a method of treating a subject having prostate cancer, comprising providing to the subject a therapeutically effective amount of a composition of the present disclosure.

[0026] In one embodiment, the invention provides a method of treating a subject having a PSMA-expressing cancer cell or cancer: administering to the subject an effective amount of an antibody drug conjugate to treat the PSMA-expressing cancer cell or cancer cell. Wherein the antibody drug conjugate is conjugated to a drug linker via a non-naturally encoded amino acid incorporated into the antibody, and includes a PSMA antibody comprising a heavy and light chain sequence disclosed herein; and where the drug linker is selected from Tables 1-3. In another embodiment, a method of treating a subject having a PSMA-associated disease or cancer, comprising providing to the subject a therapeutically effective amount of an anti-PSMA antibody drug conjugate and a therapeutic agent. The therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulant, an immunomodulatory agent, a corticosteroid, or a combination thereof. The therapeutic agent can be provided before, after, or in conjunction with the anti-PSMA ADC of the invention.

[0027] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-PSMA antibody variant and at least one pharma- ceutically acceptable adjuvant, binder, buffer, carrier, diluent or excipient. A pharmaceutical composition comprising an antibody-drug conjugate of the present invention and at least one pharma- ceutically acceptable adjuvant, binder, buffer, carrier, diluent or excipient. The pharmaceutical composition further comprises a therapeutic agent. In one embodiment, the present invention provides a drug for the manufacture and treatment of cancer in a subject in need thereof.

[0028] In a further embodiment, the present invention provides a nucleic acid encoding any of SEQ ID NOs: 1 to 17. In one embodiment, the present invention provides a vector comprising the nucleic acid of SEQ ID NOs: 1 to 17.

[0029] It should be understood that the methods and compositions described herein are not limited to the particular methodologies, protocols, cell lines, constructs, and reagents described herein, and as such may vary. It should also be 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 scope of the appended claims.

[0030] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0031] 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.

[0032] All publications and patents cited herein are incorporated by reference in their entirety for the purposes of describing and disclosing, for example, the chemistry, chemical syntheses, compositions and other methodologies described in the publications that might be used in connection with the present invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application.

[0033] In an embodiment of the present disclosure, a novel anti-PSMA amino acid sequence is provided. The term "amino acid" refers to naturally occurring amino acids and non-natural or unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner 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 pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, by way of example only, an α-carbon that is bonded to a hydrogen, a carboxyl group, an amino group, and a functional group R. Such analogs may have modified R groups (by way of example only, norleucine) or may have modified peptide backbones while still retaining the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acids may be referred to herein by either their name, 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.

[0034] "Amino or carboxy terminal modification group" refers to any molecule that can be attached to a terminal amine group or terminal carboxy group, respectively. By way of example, such terminal amine group or terminal carboxy group may be at the end of a polymer molecule, including, but not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example only, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups can be used to modify the therapeutic properties of polymer molecules, including, but not limited to, increasing the serum half-life of peptides, polypeptides, or proteins.

[0035] In some embodiments, the present disclosure provides novel anti-PSMA antibodies and antibody variants. The term "antibody" herein refers to a protein consisting of one or more polypeptides substantially encoded by all or a portion 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 a myriad of immunoglobulin variable region genes. Antibodies herein are also meant to include full length antibodies and antibody fragments, and further include naturally occurring antibodies in any organism, antibody variants, modified antibodies, and antibody fragments. Antibodies herein are also meant to include intact antibodies, monoclonal or polyclonal antibodies. Antibodies herein also encompass multispecific and / or bispecific antibodies. Antibodies of the present invention include human antibodies. Human antibodies are usually made of two light chains and two heavy chains, each of which includes a variable region and a constant region. The light chain variable region comprises 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.

[0036] The term "antibody fragment" as used herein refers to any form of antibody other than the full-length form. Antibody fragments as used herein include antibodies that are small components present in a full-length antibody, and modified antibodies such as antibody variants. 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, etc. (Maynard & Georgiou, Annnu. Rev. Biomed. Eng. 2:339-76, 2000; Hudson, Curr. Opin. Biotechnol. 9:395-402, 1998). Another functional substructure is the single chain Fv (scFv), consisting of the variable regions of immunoglobulin heavy and light chains covalently linked by a peptide linker (Hu et al., Cancer Research, 56, 3055-3061, 1996). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide, yet can provide convenient building blocks for larger antigen-specific molecules. Unless otherwise specified, the specification and claims using the term "antibody" or "antibodies" specifically include "antibody fragment" and "antibody fragments."

[0037] In an embodiment of the present invention, a novel anti-PSMA antibody drug conjugate (ADC) is disclosed. The term "antibody-drug conjugate" or "ADC" as used herein refers to an antibody molecule or a fragment thereof that is 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. ADCs are a powerful class of therapeutic constructs that allow targeted delivery of cytotoxic agents to target cells, such as cancer cells. Due to the targeting function, these compounds exhibit a much higher therapeutic index compared to the same systemically delivered agent. ADCs are developed as intact antibodies or antibody fragments, such as scFvs. The antibody or fragment is linked to one or more copies of the drug via a linker. The linker is stable under physiological conditions, but may be cleaved once inside the target cell.

[0038] 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 fragments of an intact antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by 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 consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1 (iv) an Fd fragment consisting of the VL and VH domains of a single arm of an antibody; (v) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; H (vi) isolated complementary determining regions (CDRs), e.g., V domains with or without additional sequences (linker, framework regions, etc.); HCDR3; and (V) a combination of 2 to 6 isolated CDRs, with or without additional sequences (linker, framework regions, etc.). In addition, Fv fragments, V L , and V H Although the two domains of V are encoded by separate genes, they can be linked using recombinant methods with a synthetic linker that allows them to be produced as a single polypeptide chain. L and V H The domains pair to form monovalent molecules known as single chain Fvs (scFvs); see, e.g., Birdet al., Science 242:423-426,1988); and (Hustone et al., Proc. Natl. Acad. Sci. USA 85:5879-5883,1988). Such single chain antibodies are also intended to be encompassed by the above term "antigen-binding fragment" of an antibody. Furthermore, antigen-binding fragments include binding domain immunoglobulin fusion proteins. A binding domain immunoglobulin fusion protein includes (i) a binding domain polypeptide (such as 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 CH3 constant region fused to the CH2 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 with skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0039] A typical antigen-binding site contains a variable region formed by pairing a light immunoglobulin chain with a heavy immunoglobulin chain. The structure of antibody variable regions is highly consistent and also shows a highly similar structure. These variable regions typically contain three hypervariable regions called complementarity determining regions (CDRs) and relatively homologous framework regions (FRs) spaced apart. The overall binding activity of an antigen-binding fragment is often determined by the sequence of the CDRs. The FRs often play a role in the proper positioning and alignment of the CDRs in three dimensions for optimal antigen binding. Indeed, since the CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that exhibit the properties of a particular naturally occurring antibody by constructing expression vectors that contain the CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., Nature 332:323-327, 1998; Jones, P. et al., Nature 321:522-525, 1986; and Queen, C. et al., Proc. Natl. Acad. USA 86:10029-10033, 1989). Such framework sequences can be obtained from public DNA databases that contain germline antibody gene sequences. These germline sequences do not contain the fully assembled variable genes that are formed by V(D)J joining during B cell maturation. Thus, these germline sequences differ from mature antibody gene sequences. Germline gene sequences also differ from the sequences of high affinity secondary repertoire antibodies, which contain mutations throughout the variable genes, but typically cluster in 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 the 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. The partial sequences are used to determine the germline variable gene fragments and joining gene fragments that contributed to the recombinant antibody variable genes. The germline sequences are then used to fill in the missing portions of the variable regions. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add the missing sequences, the 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 complete 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. Of course, the whole or part of the framework regions of the antibodies described herein may be used in complex with the CDRs to optimize the affinity, specificity, or any other desired properties of the antibody.

[0040] In some embodiments, the present invention relates to polymers such as bifunctional polymers. "Bifunctional polymer", also referred to as "bifunctional linker", refers to a polymer that contains two functional groups that can specifically react with other moieties to form covalent or non-covalent bonds. Such moieties can include, but are not limited to, side groups on natural or non-natural amino acids, or peptides that contain such natural or non-natural amino acids. The other moieties that can be linked to the bifunctional linker or bifunctional polymer can be the same or different moieties. By way of example only, a bifunctional linker can have a functional group reactive with a group on a first peptide and another functional group reactive with a group on a second peptide, thereby forming a complex that includes the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules are known for attaching various compounds to peptides. For example, European Patent Application No. 0188256; U.S. Patent Nos. 4,659,839; 4,414,148; 4,699,784; 4,680,338; and 4,569,789 are incorporated herein by reference in their entirety. "Multifunctional polymer" also refers to a "multifunctional linker" and refers to a polymer that contains two or more functional groups that can react with other moieties. Such moieties may include, but are not limited to, side groups on natural or unnatural amino acids, or peptides that contain such natural or unnatural amino acids. Form covalent or non-covalent bonds (including, but not limited to, amino acid side groups). Bifunctional or multifunctional polymers may be of any desired length or molecular weight. And, bifunctional or multifunctional polymers may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and the molecule or molecules that bind to the compound.

[0041] The term "bioavailability" as used herein refers to the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increasing bioavailability refers to increasing the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of example, increasing bioavailability can be shown as an increase in the concentration of a substance or its active portion in the blood compared to other substances or active portions.

[0042] The term "biologically active molecule", "biologically active moiety" or "biologically active agent" as used herein contemplates any substance capable of affecting any physical or biochemical property of a biological system, pathway, molecule, or interaction associated with an organism. Organisms include, but are not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, 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 health 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, bactericides, antiviral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, steroidal and non-steroidal agents, microbial toxins, and the like.

[0043] "Modulating biological activity" means increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, enhancing or decreasing the substrate selectivity of a polypeptide. Analysis of altered biological activity can be performed by comparing the biological activity of the non-natural polypeptide with the biological activity of the natural polypeptide.

[0044] In some embodiments, the present disclosure relates to amino acids biosynthetically incorporated into antibodies. The term "biosynthetically" as used herein refers to any method that utilizes a translation system (cellular or non-cellular). Any method that utilizes a translation system (cellular or non-cellular) includes the use of at least one of the following components: polynucleotides, codons, tRNA, and ribosomes. By way of example, non-natural amino acids may be "biosynthetically incorporated" into non-natural amino acid polypeptides using the methods and techniques described herein and also well known in the art, see, e.g., WO 2010 / 011735 and WO 2005 / 074650.

[0045] The term "conservatively modified variants" 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 natural and non-natural nucleic acids that code for identical or essentially identical natural and non-natural amino acid sequences, or natural and non-natural nucleic acids that do not code for essentially identical sequences. By way of example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described, without changing the encoded polypeptide. Such a mutation in the nucleic acid is a "silent mutation," which is one type of conservatively modified mutation. Thus, by way of example, any natural or non-natural nucleic acid sequence herein that codes for a natural or non-natural polypeptide also describes any possible silent mutation of the natural or non-natural nucleic acid. Those skilled in the art should recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent variation of a natural or non-natural nucleic acid that encodes a natural or non-natural polypeptide is present in each described sequence, even if not described. With respect to amino acid sequences, each individual substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence is a "conservatively modified variant." The individual substitution, deletion, or addition is a change, addition, or deletion of a single natural or non-natural amino acid, or a small percentage of natural and non-natural amino acids, in the encoded sequence. Here, the change results in the deletion of an amino acid, the addition of an amino acid, or the replacement of a natural or non-natural amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art.Conservative substitution tables providing functionally similar amino acids are known to those of skill 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., Clayton, Proteins: Structural and Molecular Properties (W. (See H Freeman & Co.; 2nd ed., 1993.) Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the compositions described herein.

[0046] The term "drug" as used herein refers to any substance used in the prevention, diagnosis, mitigation, treatment, or cure of a disease or condition, such as cancer, including prostate cancer.

[0047] The term "effective amount" as used herein refers to a sufficient amount of an agent, compound or composition administered to relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. By way of example, the agent, compound or composition administered includes, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, a modified non-amino acid polypeptide, or an antibody or variant thereof. Compositions including such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, modified non-natural amino acid polypeptides, or antibodies or variants thereof can be administered for prophylactic, enhancing, and / or therapeutic treatments. An appropriate "effective" amount in any individual case may be determined using techniques such as a dose escalation study.

[0048] The term "enhance" or "enhancing" refers to an increase or prolongation of a desired effect, either in potency or duration. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong, either in 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 the therapeutic agent in the treatment of a disease, disorder, or condition. When used in a patient, the amount 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 agent, and the judgment of the treating physician.

[0049] The term "humanized or chimeric antibody" refers to a molecule, typically prepared using recombinant techniques, that has an antigen-binding site derived from an immunoglobulin from a non-human species (e.g., mouse) and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains. All or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework residues / regions (FR) are those of a human immunoglobulin sequence. A humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized forms of rodent antibodies essentially contain the same CDR sequences of the parental rodent antibody, although certain amino acid substitutions may be included to increase affinity, to increase the stability of the humanized antibody, or for other reasons. However, since CDR loop exchange does not uniformly result in antibodies with the same binding properties as the original antibody, changes in framework residues (FR), which are residues involved in CDR loop support, may also be introduced into the humanized antibody to preserve antigen binding affinity. The antigen binding site may comprise either complete variable domains fused onto constant domains, or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domains. The antigen binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant regions as immunogens in human individuals, although the possibility of an immune response against the foreign variable regions remains (LoBuglio, AF et al., "Mouse / Human "Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response", Proc. Natl. Acad. Sci. (USA) 86:4220-4224, 1989). Another approach focuses on not only providing constant regions of human origin, but also modifying the variable regions to reshape them as close as possible to the human form. The variable regions of both heavy and light chains are known to contain three complementarity determining regions (CDRs). The CDRs change in response to the antigen in question and determine the binding ability. The CDRs are flanked by four framework regions (FRs) that are relatively conserved in a given species and presumably provide a scaffold for the CDRs. When a non-human antibody is prepared for a particular antigen, the variable region can be "humanized" by grafting the CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. The application of the above approach to various antibodies is described in Kettleborough, CA et al., “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation”, Protein Engineering 4:773-3783, 1991; Co., MSet al., “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA) 88:2869-2873, 1991; Carter, P. et al., “Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289, 1992; and Co. MSet al., “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen”, J. Immunol. 148:1149-1154, 1992. In certain embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six). The one or more CDRs are altered relative to the original antibody and are also referred to as one or more CDRs "derived from" the one or more CDRs from the original antibody.

[0050] 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 sequence units that are the same when compared and aligned for maximum correspondence over a comparison window, i.e., a specified 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 sequence 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 to a particular region. Such percentages describe the "percent identity" of two sequences. Sequence identity can exist over a region that is at least about 75-100 sequence units in length, over a region that is about 50 sequence units in length, or, if not specified, across 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 identical. On the other hand, two or more polypeptide sequences are "substantially identical" if the amino 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 to about 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 polypeptide sequence. Further, by way of example only, two or more polynucleotide sequences are identical if the nucleic acid residues are identical. On the other hand, 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. The identity can exist over a region that is at least about 75 to about 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.

[0051] The term "immunogenicity" as used herein refers to an 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 for the detection of 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), chemiluminescent 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 to the antibody response upon administration of the therapeutic natural amino acid polypeptide.

[0052] 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. Such solutions include, but are not limited to, aqueous solutions. An isolated component can be in a homogenous state, or the isolated component can be part of a pharmaceutical composition that includes additional pharma-ceutically acceptable carriers and / or excipients. Purity and homogeneity may be determined using analytical chemistry techniques. Analytical chemistry techniques include, but are not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. Additionally, a component of interest is described herein as substantially purified when 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, at least 99% or more pure. By way of example only, a nucleic acid or protein is "isolated" when it is free from at least some of the cellular components with which it is naturally associated, or when the nucleic acid or protein is concentrated to a level greater than its in vivo or in vitro production concentration. Also, by way of example, a gene is isolated when it is separated from open reading frames which flank it and which encode proteins other than the gene of interest.

[0053] The term "bond" as used herein refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent and non-covalent bonds. Meanwhile, such chemical moieties may include, but are not limited to, esters, carbonates, imine phosphate esters, hydrazones, acetals, orthoesters, peptide bonds, and oligonucleotide bonds. A hydrolytically stable bond means that the bond is substantially stable in water for extended periods of time, possibly indefinitely, under physiological conditions, and does not react with water at useful pH values. A hydrolytically unstable or degradable bond means that the bond is degradable in water or in an aqueous solution. An aqueous solution includes, for example, blood. An enzymatically unstable or degradable bond means that the bond can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable bonds in the polymer backbone or in the linker group between the polymer backbone and the terminal functional group of one or more polymer molecules. Such degradable bonds include, but are not limited to, ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with the alcohol group on biologically active agent.Such ester groups are generally hydrolyzed under physiological conditions to release biologically active agents.Other hydrolytically degradable bonds include carbonate bonds; imine bonds resulting from the reaction of amines and aldehydes; phosphate ester bonds formed by the reaction of alcohols with phosphate groups; hydrazone bonds, which are the reaction products of hydrazides and aldehydes; acetal bonds, which are the reaction products of aldehydes and alcohols; orthoester bonds, which are the reaction products of formates and alcohols; peptide bonds formed by amine groups (including but not limited to those at the end of polymers such as PEG) and the carboxyl group of peptides; and oligonucleotide bonds formed by phosphoramidite groups (including but not limited to those at the end of polymers) and the 5' hydroxyl group of oligonucleotides.

[0054] The term "metabolite", as used herein, refers to a derivative of a compound (by way of example only, 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 (by way of example only, 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 "pharmacologically active metabolite" or "active metabolite" refers to a derivative of a biologically active compound (by way of example only, 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 such a compound (by way of example only, 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.

[0055] The term "metabolized" as used herein refers to the sum of the processes by which a particular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information regarding metabolism may be obtained from The Pharmacological Basis of Therapeutics, 9th 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 may 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.

[0056] The term "modified" as used herein refers to the presence of an alteration to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. Such an alteration or modification can be obtained by post-synthetic modification of the natural amino acid, the non-natural amino acid, the natural amino acid polypeptide, or the non-natural amino acid polypeptide, or can be obtained by co-translation, or can be obtained by post-translational modification of the natural amino acid, the non-natural amino acid, the natural amino acid polypeptide, or the non-natural amino acid polypeptide.

[0057] "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 "unnatural amino acid" are "non-naturally encoded amino acid", "non-natural 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 naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that are not 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 naturally and can be obtained synthetically, or that can be obtained by modification of unnatural amino acids.

[0058] The term "nucleic acid" as used herein refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof in either single-stranded 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 as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs, including but not limited to PNA (peptide nucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.); (iii) conservatively modified variants thereof (including but not limited to degenerate codon substitutions), as well as complementary sequences and sequences explicitly shown. By way of example, degenerate codon substitutions may 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).

[0059] The term "pharmaceutical acceptable" as used herein refers to a material, including but not limited to a salt, binder, adjuvant, excipient, carrier, or diluent. The material does not eliminate the biological activity or properties of the compound, and is relatively non-toxic. That is, the material may be administered individually without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is included.

[0060] In some embodiments, the present invention relates to polymers. The term "polymer" as used herein refers to a molecule composed of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides, or polyalkylene glycols. The polymers of the present invention can be linear or branched polymeric polyether polyols, including, but 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). By way of example only, such polymers have an average molecular weight of about 0.1 kDa to about 100 kDa. Such polymers include, but are not limited to, about 100 Da to about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including about 100,000 Da, about 95,000 Da, about 90,000, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da. , about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, about 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments, the molecular weight of the polymer is about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 1,000 Da to about 40,000 Da.In some embodiments, the molecular weight of the polymer is from about 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 10,000 Da to about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched PEG may be from about 1,000 Da to about 100,000 Da, and may be about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,0 The molecular weight of the branched PEG includes, but is not limited to, about 1,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched PEG is about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the branched PEG is about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is about 5,000 Da to about 40,000 Da. In some embodiments, the branched PEG has a molecular weight of about 5,000 Da to about 20,000 Da. In other embodiments, the branched PEG has a molecular weight of about 2,000 to about 50,000 Da. The term "PEGylation" or "PEGylated" is meant to refer to the covalent attachment of a particular synthetic amino acid to a polyethylene glycol (PEG) molecule. The method can include contacting an isolated α-PSMA ADC polypeptide that includes a synthetic amino acid with a water-soluble polymer that includes a moiety that reacts with the synthetic amino acid.

[0061] 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 to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are unnatural amino acids. Moreover, such "polypeptides", "peptides" and "proteins" include amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0062] The term "post-translational modification" 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 cell-free translation systems), post-translational in vivo modifications, and post-translational in vitro modifications.

[0063] The term "prodrug" or "pharmaceutical acceptable prodrug" as used herein refers to an agent that is converted to the parent drug in vivo or in vitro. Such agents do not inhibit the biological activity or properties of the drug and are relatively non-toxic. That is, the substance can be administered individually without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained. Prodrugs are generally drug precursors that are converted to an active or more active species through some process, such as conversion by metabolic pathways, after administration to a subject and subsequent absorption. Some prodrugs have chemical groups present on the prodrug that reduce the activity of the drug and / or confer solubility or other properties to the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is generated. The prodrug is converted to the active drug in the body by enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemical properties (e.g., better solubility), improved delivery properties (e.g., specific targeting to certain 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 may be designed to regulate the amount of a drug or biologically active molecule that reaches the desired site of action through manipulation of the drug's properties (e.g., physicochemical, biopharmaceutical, or pharmacokinetic properties). A non-limiting example of a prodrug is a non-natural amino acid polypeptide. Non-natural amino acid polypeptides are administered as esters ("prodrugs") to facilitate transport across cell membranes where water solubility is detrimental to mobility, and are then metabolically hydrolyzed to the active entity, a carboxylic acid, once inside the cell, where water solubility is beneficial.Prodrugs may be designed as reversible drug derivatives for use as modifiers to enhance drug transport to site-specific tissues.

[0064] The term "prophylactically effective amount," as used herein, refers to an amount of a composition comprising at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide that is applied prophylactically to a patient to 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, or the like. It is considered well within the skill of one in the art to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, dose escalation clinical trials.

[0065] The term "recombinant host cell", also referred to as "host cell", refers to a cell that contains an exogenous polynucleotide. Here, the method used to insert the exogenous polynucleotide into the cell includes, but is not limited to, direct uptake, transduction, f-mating, or other methods known in the art for making recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrated vectors (including, but not limited to, plasmids) or may be integrated into the host genome.

[0066] The term "subject," as used herein, refers to an animal that is the object of treatment, observation, or experiment. By way of example only, and without limitation, a subject may be a mammal, including, but not limited to, a human.

[0067] The term "substantially purified" as used herein refers to a component of interest that is substantially or essentially free from other components that typically coexist 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 impurity components. Thus, a "substantially purified" component of interest has a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. By way of example only, a natural or non-natural amino acid polypeptide may be purified from a native or host cell in the case of a recombinantly produced natural or non-natural amino acid polypeptide. By way of example, a preparation of natural or non-natural amino acid polypeptides may be "substantially purified" if it 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 contaminants. 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 no more than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% by 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 may 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 may 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. Purity levels are determined by appropriate methods, including, but not limited to, SDS / PAGE measurement, RP-HPLC, SEC, and capillary electrophoresis.

[0068] The term "therapeutically effective amount," as used herein, refers to an amount of a composition containing at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide that is administered to a patient already suffering from a disease, condition, or disorder 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 will depend on conditions including, but not limited to, the severity and course of the disease, disorder, or condition, previous treatments, 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, dose escalation clinical trials.

[0069] The terms "toxic" or "toxic moiety" or "toxic group" or "cytotoxic" or "cytotoxic payload" or "payload", as used herein, refer to a compound that can cause harm, injury, or death. Toxic moieties include auristatins, DNA minor groove binders, DNA minor groove alkylators, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, vinca alkaloids, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, , combretastatin, calicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide, etc.), baccatin and its derivatives, antitubulin agents, cryptophycin, combretastatin bretin, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colchimide, estramustine, cemadotin, discadermolide, maytansine , erythemoglobin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, itarabin, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, pentostatin. , 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (anthracyclines, gentamicin, cephalothin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin,Rocithromycin, furazolidone, amoxicillin, ampyridine, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral drugs (abacavir, acyclovir, ampligen, cidofovir, delavirdine, etc.), , didanosine, efavirenz, entecavir, phosphonet, ganciclovir, ibasitabine, 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, phenoxyzombis Cyclopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrroloindolediones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide de), doloxafine, allocolchicine, halichondrin B, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysterin, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur. "Taxanes" include paclitaxel in addition to any active taxane derivatives or prodrugs.

[0070] The terms "treat", "treating" or "treatment" as used herein include alleviating, preventing, reducing or ameliorating symptoms of a disease or condition, preventing additional symptoms, improving or preventing the underlying metabolic causes of symptoms, inhibiting the onset of a disease or condition (e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, reducing 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 treatments. The terms "treat", "treating" or "treatment" can refer to the reduction, reduction or amelioration of one or more symptoms associated with prostate cancer.

[0071] The term "water-soluble polymer" as used herein refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C 10These include, but are not limited to, alkoxy or aryloxy derivatives thereof (as described in U.S. Pat. No. 5,252,714, incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α-β-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. By way of example only, coupling of such water-soluble polymers to natural amino acid polypeptides or non-natural polypeptides can result in changes, including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life compared to the unmodified form, increased bioavailability, modulated biological activity, increased circulation time, modulated immunogenicity, modulated physical association properties (including, for example, but not limited to, aggregation and multimerization), altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. Additionally, such water-soluble polymers may or may not have biological activity of their own.

[0072] 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 the 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, serum half-life is measured by taking blood samples at various time points after administration of a biologically active molecule or 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. By way of example, an adjusted serum half-life may be an increase in serum half-life. An increase in serum half-life may allow for 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 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.

[0073] 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 the 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. An increased therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. By way of example, an increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased another parameter or mechanism of action of the unmodified molecule, or increased or decreased degradation of the molecule by enzymes (e.g., by way of example only, proteases). Methods for assessing therapeutic half-life are known in the art and may be used to assess the therapeutic half-life of the antibodies and antibody drug conjugates of the present invention.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS 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. The accompanying drawings, in which:

[0075] Figure 1 shows pharmacokinetic studies in mice with anti-PSMA antibody-drug conjugates (ADCs) using non-cleavable MMAF, short cleavable MMAF, cleavable MMAF and short cleavable MMAE. F1 represents detection of antibody-drug conjugates in serum by antibody. F3 represents detection of antibody-drug conjugates in serum by drug-linker.

[0076] FIG. 2 shows a pharmacokinetic study performed in rats with an anti-PSMA ADC that exhibits non-cleavable MMAF at various concentrations.

[0077] FIG. 3 shows a pharmacokinetic comparison of unconjugated anti-PSMA antibodies (naked antibodies) compared to ADCs using MMAF, which are non-cleavable.

[0078] Figures 4A-4B show single dose responses in the C4-2 xenograft prostate model in terms of tumor growth (Figure 4A) and body weight change (Figure 4B) with the anti-PSMA ADC noncleavable MMAF at various doses.

[0079] Figures 5A-5B show single dose responses in the C4-2 xenograft prostate model with respect to tumor growth (Figure 5A) and body weight change (Figure 5B) by the non-cleavable anti-PSMA ADC MMAF at various doses.

[0080] 6A-6B show pharmacokinetic studies in tumor and non-tumor bearing mice (FIG. 6A) and cynomolgus monkeys (FIG. 6B) following a single dose of anti-PSMA ADC with non-cleavable MMAF.

[0081] Figures 7A-7B show repeat dosing responses in the C4-2 xenograft prostate model in terms of tumor growth (Figure 7A) and body weight change (Figure 7B) at various doses of MMAF, a non-cleavable anti-PSMA ADC, and MMAF + enzalutamide, a non-cleavable anti-PSMA ADC.

[0082] Figures 8A-8B show repeat dosing responses in a PDX prostate model in terms of tumor growth (Figure 8A) and body weight change (Figure 8B) at various doses of MMAF, a non-cleavable anti-PSMA ADC, and MMAF + enzalutamide, a non-cleavable anti-PSMA ADC.

[0083] Before describing the invention in detail, it should be understood that the invention is not limited to specific methodologies, or compositions, or biological systems, which can, of course, vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "cell" includes a combination of two or more cells, and the like.

[0084] Although preferred embodiments of the present invention have been disclosed and described herein, it will be apparent to those skilled in the art that these embodiments are merely exemplary. Those skilled in the art may make numerous changes, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be employed to implement the present invention. The following claims define the scope of the present invention, and it is intended to cover methods and configurations within the scope of these claims and their equivalents.

[0085] Unless otherwise defined herein 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.

[0086] Antibody-based therapeutics have emerged as a key component of treatment for a growing number of human malignancies in areas such as oncology, inflammatory and infectious diseases. In most cases, the basis for therapeutic function is the high degree of specificity and affinity that an antibody-based drug has for its target antigen. Arming monoclonal antibodies with drugs, toxins, or radionuclides is yet another strategy by which monoclonal antibodies can induce therapeutic effects. By combining the precise targeting specificity of antibodies with the tumoricidal power of toxic effector molecules, immunoconjugates allow for highly sensitive discrimination between targets and normal tissues, thereby producing fewer side effects than most conventional chemotherapy drugs. The toxins utilized can be specifically, stably, and irreversibly bound to specific sites in the antibody. This specific binding process allows for precise control of the location of the toxin on the antibody, and the number of toxins attached to each antibody. Both of these features are important for controlling the biophysical properties and toxicity associated with ADCs. (See, e.g., Jackson et al., 2014; Tian et al., 2014).

[0087] The anti-PSMA antibody drug conjugates provided in the present disclosure comprise humanized or chimeric monoclonal antibodies and variants that bind to the extracellular domain of prostate specific membrane antigen.Prostate specific membrane antigen is a type II membrane protein that is highly expressed in, for example, prostatic intraepithelial neoplasia (PIN), primary prostate cancer, and metastatic prostate cancer, and the anti-PSMA antibody disclosed herein can be any known PSMA antibody that has at least one non-naturally or unnaturally encoded amino acid.

[0088] The present invention provides anti-PSMA antibodies and variants thereof with non-naturally encoded amino acids that facilitate antibody conjugation to drugs (e.g., drugs, toxin molecules). In one embodiment, the ADC comprises an anti-PSMA antibody conjugated to a drug, where conjugation occurs through a non-naturally encoded amino acid in the antibody. In one embodiment, the ADC comprises an anti-PSMA antibody conjugated to a drug, where conjugation occurs through a non-naturally encoded amino acid in the heavy chain of the antibody. In one embodiment, the ADC comprises an anti-PSMA antibody conjugated to a drug, where conjugation occurs through a non-naturally encoded amino acid in the light chain of the antibody. In one embodiment, the ADC comprises a full-length antibody conjugated to a drug, where conjugation occurs through a non-naturally encoded amino acid in the antibody. In one embodiment, the ADC comprises a full-length antibody conjugated to a drug, where conjugation occurs through a non-naturally encoded amino acid in the heavy chain of the antibody. In one embodiment, the ADC comprises a full-length antibody conjugated to a drug, where conjugation occurs through a non-naturally encoded amino acid in the light chain of the antibody.

[0089] In some embodiments, the drug of the ADC is a cytotoxic drug or agent. In some aspects of the invention, the cytotoxic drug is selected from the group consisting of auristatins, DNA minor groove binders, DNA minor groove alkylators, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, and vinca alkaloids. In some aspects of the invention, the cytotoxic drug is, but is not limited to, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatostatin, calicheamicin, maytansine, DM-1, or netropsin.

[0090] In some aspects of the present invention, the cytotoxic drug is an antitubulin agent.In some embodiments, the antitubulin agent is an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophycin, a maytansinoid, a combretastatin, or a dolastatin.In other aspects of the present invention, the antitubulin agent is, but is not limited to, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, colchimide, estramustine, cemadotin, discodenolide, maytansine, DM-1, or eleutherobin.

[0091] In other embodiments of the invention, the cytotoxic drug of the ADC is ganciclovir, etanercept, cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a 5-epoxygenase inhibitor, or a leukotriene receptor antagonist.

[0092] In some embodiments of the invention, the antibody of the ADC comprises a full-length antibody or a fragment thereof. The antibody (a) binds PSMA and (b) is conjugated to a cytotoxic or immunosuppressant agent, where the antibody-drug conjugate exerts (a) a cytotoxic or cytostatic effect on a PSMA-expressing cancer cell line, or (b) a cytotoxic, cytostatic, or immunosuppressive effect on a PSMA-expressing immune cell, where the conjugation occurs at a non-naturally encoded amino acid in the antibody.

[0093] In some embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that binds to the PSMA receptor. In other embodiments of the invention, the antibody, variant, or composition may be an antibody, variant, or composition that binds to the extracellular surface of the PSMA receptor. In another embodiment of the invention, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that binds to a PSMA dimer. In some embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that has a CDR from J591 grafted onto the framework region of the variable region. In other embodiments, the disclosed antibody, variant, or composition may be an antibody, variant, or composition that has a non-naturally encoded amino acid. In some embodiments, the antibody, variant, or composition may be an antibody, variant, or composition described in the disclosure of the invention by one or more embodiments anywhere herein. In some embodiments, the disclosed antibody, antibody variant, or antibody composition may be fully humanized. In other embodiments, the disclosed antibody, antibody variant, or antibody composition may be chimeric. In some embodiments of the invention, the antibody may 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, a scFv-Fc, a minibody.

[0094] Methods, compositions, and techniques for making and using dolastatin linker derivatives or analogs that include at least one of carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione are well known to those skilled in the art. (See, for example, International Publication No. 2013 / 185117, the entirety of which is incorporated herein by reference.) Methods, compositions, and techniques for making and using dolastatin linker derivatives or analogs that include at least one unnatural amino acid or modified unnatural amino acid with oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.) are also well known to those skilled in the art, and are described, for example, in International Publication No. 2013 / 185117, the entirety of which is incorporated herein by reference. Such dolastatin linker derivatives containing unnatural amino acids may contain additional functional groups. Functional groups include, but are not limited to, polymers; water-soluble polymers; derivatives of polyethylene glycol; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; and any combination thereof. Please note that the various aforementioned functional groups are not meant to imply that a member of one functional group cannot be classified as a member of another functional group. In fact, there is overlap depending on the specific situation. As a mere example, water-soluble polymers overlap in scope with derivatives of polyethylene glycol, but the overlap is not complete, and therefore both functional groups are cited above.

[0095] In some embodiments herein, toxic group linker derivatives are provided that include carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketoamine, ketoalkyne, alkyne, cycloalkyne, or enedione. In some embodiments, the toxic group derivatives include any of the linkers disclosed herein. Methods, compositions, and techniques for making and using toxic group derivatives or analogs that include at least one unnatural amino acid or modified unnatural amino acid with an oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.) are described in WO 2013 / 185117, which is incorporated herein by reference in its entirety. In some embodiments, such toxic derivatives that include unnatural amino acids may include additional functional groups. Functional groups include, but are not limited to, polymers; water-soluble polymers; derivatives of polyethylene glycol; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; and any combination thereof. In certain embodiments, the toxic group is a tubulin inhibitor. In certain specific embodiments, the toxic group is a dolastatin or auristatin. In other specific embodiments, the toxic group is a dolastatin or auristatin derivative. It should be noted that the various aforementioned functional groups are not meant to imply that elements of one functional group cannot be classified as elements of another functional group. In fact, there is overlap depending on the specific situation. As a mere example, water-soluble polymers overlap in scope with derivatives of polyethylene glycol, but the overlap is not complete, and therefore both functional groups are cited above.

[0096] Certain embodiments of the invention describe preparations of certain toxic moieties with linkers that reduce the toxicity of the moiety in vivo while the toxic moiety retains its pharmacological activity. In some embodiments, the toxicity of the attached toxic group 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 its pharmacological activity. In some embodiments, the dose of the attached toxic group (e.g., dolastatin linker derivatives, non-natural amino acid linked dolastatin derivatives) can be increased to more safely administer to an animal or human. In certain embodiments, the non-natural amino acid polypeptide attached to the toxic moiety (e.g., dolastatin derivatives) provides in vitro and in vivo stability. In some embodiments, the non-natural amino acid polypeptide attached to the toxic moiety (e.g., tubulin inhibitors, dolastatin-10 derivatives) is more effective and less toxic than the free toxic moiety (e.g., tubulin inhibitors, dolastatin-10).

[0097] [Methodology and Technology] 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 technology and pharmacology, within the scope of the art. The compounds of the present disclosure can be synthesized using several processes or schemes employed 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; WO 2013 / 185117. Many methodologies and techniques for the synthesis of pharmaceutical, diagnostic or therapeutic compounds are well known to those skilled in the art.

[0098] The present invention encompasses, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), cell biology, biochemistry and immunology, all of which are within the skill of one of ordinary skill in the art. Such techniques are fully described in the following publications: 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, L. A. 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. et al. Eds. 1995) ​In embodiments of the present disclosure, dolastatin linker derivatives or analogs can be utilized that contain at least one unnatural amino acid or modified unnatural amino acid with a carbonyl, dicarbonyl, oxime, or hydroxylamine group. Methods for selecting and designing dolastatin linker derivatives to be modified using methods, compositions, and techniques are well known in the art, see, for example, WO 2013 / 185117, which is incorporated herein by reference in its entirety. Dolastatin linker derivatives may be designed de novo, including, by way of example only, as part of a high-throughput screening process (where a large number of polypeptides may be designed, synthesized, characterized, and / or tested) or based on the researcher's goals. Novel dolastatin linker derivatives may 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, and the selection of which modifications to employ, are described, for example, in WO 2013 / 185117. Dolastatin linker derivatives may 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, modulating the pharmacokinetics, pharmacology and / or pharmacodynamics of the polypeptide (e.g., by way of example only, increasing water solubility, increasing bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time). Further, such modifications include, by way of example only, providing additional functional groups to the polypeptide, incorporating antibodies, and any combination of the foregoing modifications. Such dolastatin linker derivatives can be modified to include oxime, carbonyl, dicarbonyl, or hydroxylamine groups. Dolastatin linker derivatives may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more of a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected form thereof.The dolastatin linker derivatives may be the same or different, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites in the derivative, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different reactive groups.

[0099] For example, dolastatin derivatives with linkers containing hydroxylamine (also called aminooxy) groups allow for reaction with a variety of electrophilic groups to form conjugates, including but not limited to PEG or other water-soluble polymers. Like hydrates, hydrates, and hemicarbazides, the enhanced nucleophilicity of the aminooxy group allows for efficient and selective reaction with a variety of molecules containing carbonyl- or dicarbonyl-groups, including but not limited to ketones, aldehydes, or other functional groups with similar chemical reactivity. (See, e.g., 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). However, while the result of reaction with a hydrazine group is the corresponding hydrazone, an oxime generally results from the reaction of a carbonyl- or dicarbonyl-containing group with an aminooxy group (such as, by way of example only, a ketone, aldehyde, or other functional group with similar chemical reactivity). In some embodiments, dolastatin derivatives with linkers containing azides, alkynes, or cycloalkynes allow for the attachment of molecules through cycloaddition reactions (e.g., the 1,3-dipropionyl cycloaddition reaction described in U.S. Pat. No. 7,807,619, the azide-alkyne-Huesgen cycloaddition reaction, etc., herein incorporated by reference, depending on the extent of the reaction).

[0100] Thus, certain embodiments described herein are dolastatin derivatives having linkers containing hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, keto-alkyne, and ene-dione hydroxylamine groups, hydroxylamine-like groups (having a reactivity similar to that of a hydroxylamine group and structurally similar to that of a hydroxylamine group), masked hydroxylamine groups (which can be easily converted to a hydroxylamine group), or protected hydroxylamine groups (having a reactivity similar to that of a hydroxylamine group upon deprotection). In some embodiments, the dolastatin derivatives having linkers contain azides, alkynes, or cycloalkynes. Examples of such dolastatin linker derivatives are included elsewhere herein, as well as in International Publication Nos. WO 2013 / 185117 and WO 2005 / 074650, each of which is incorporated herein by reference in its entirety.

[0101] [Unnatural Amino Acids] The selection of the non-naturally encoded amino acid site was based on surface exposure / site accessibility within the antibody, with hydrophobic or neutral amino acid sites selected to maintain the charge on the antibody. Methods for introducing non-natural amino acids inserted into sites in proteins are described, for example, in WO 2010 / 011735 and WO 2005 / 074650. The present invention employs such methodologies and techniques. The unnatural 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, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine); (2) the introduced unnatural amino acid is substantially chemically inert toward the 20 common genetically encoded amino acids; (3) the unnatural amino acid is capable of being stably incorporated into a polypeptide, preferably with stability comparable to that of natural amino acids or under typical physiological conditions, and more preferably, such attachment can occur through in vivo systems; (4) the unnatural amino acid has an oxime functional group or a functional group that can be converted to an oxime group by reaction with a reagent under the following conditions: The conditions are preferably those that do not destroy the biological properties of the polypeptide that includes the unnatural amino acid, or those that allow the transformation to occur under aqueous conditions at a pH between about 4 and about 8, or those that the reactive site of the unnatural amino acid is an electrophilic site. Any number of unnatural amino acids can be introduced into the polypeptide. The unnatural amino acid can also contain a protected or masked oxime, or a protected or masked group that can be transformed into an oxime group after deprotection or unmasking.The unnatural amino acid may also further comprise a protected or masked carbonyl group or a protected or masked dicarbonyl group that can be transformed into a carbonyl group or a dicarbonyl group after deprotection or unmasking, thereby reacting with a hydroxylamine or an oxime to form an oxime group. Oxime-based unnatural amino acids may be synthesized by methods well known in the art, including (a) reacting a hydroxylamine-containing unnatural amino acid with a carbonyl- or dicarbonyl-containing reagent; (b) reacting a carbonyl- or dicarbonyl-containing unnatural amino acid with a hydroxylamine-containing reagent; or (c) reacting an oxime-containing unnatural amino acid with a particular carbonyl- or dicarbonyl-containing reagent (see, e.g., WO 2013 / 185117 and WO 2005 / 074650).

[0102] Unnatural amino acids that may be used in the methods and compositions described herein include, but are not limited to, amino acids containing amino acids with novel functional groups, amino acids that interact covalently or noncovalently with other molecules, glycosylated amino acids such as sugar-substituted serines, other carbohydrate-modified amino acids, keto-containing amino acids, aldehyde-containing amino acids, polyethylene glycol or other polyether-containing amino acids, heavy atom-substituted amino acids, chemically cleavable and / or photodegradable amino acids, amino acids with elongated side chains compared to natural amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids with one or more toxic moieties. Amino acids with elongated side chains compared to natural amino acids include, but are not limited to, those with polyethers or long chain hydrocarbons (more than about 5 carbons or more than about 10 carbons).

[0103] In some embodiments, the unnatural amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-bond between the amino acid and the sugar is replaced by a covalent bond not commonly found in nature, including, but not limited to, an alkene, an oxime, a thioether, an amide, and the like. Examples of such amino acids also include sugars not commonly found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, and the like.

[0104] Chemical moieties incorporated into polypeptides through the incorporation of unnatural amino acids into such polypeptides provide various advantages and manipulations of the polypeptide. For example, the unique reactivity of carbonyl or dicarbonyl functional groups (including keto or aldehyde functional groups) allows for selective modification of proteins with any number of hydrazine- or hydroxylamine-containing reagents in vivo and in vitro. Heavy atom unnatural amino acids can be useful, for example, for phasing X-ray structural data. Site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in choosing the location of the heavy atoms. Photoreactive unnatural amino acids (including, but not limited to, amino acids with benzophenone and aryl azide (including, but not limited to, phenyl azide side chains) side chains, for example, allow for efficient photocrosslinking of polypeptides in vivo and in vitro. Examples of photoreactive unnatural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Polypeptides with photoreactive unnatural amino acids can then be crosslinked at will by excitation of the photoreactive group, which provides temporal control. In a non-limiting example, the methyl group of an unnatural amino acid can be substituted with a labeled isotope, using, but not limited to, the methyl group as a probe of, but not limited to, local structure and dynamics using, but not limited to, nuclear magnetic resonance and vibrational spectroscopy.

[0105] [Non-natural amino acid-linked dolastatin derivatives] Other embodiments of the invention described herein are methods, schemes and techniques for incorporating at least one dolastatin linker derivative into an unnatural amino acid. The invention described herein includes methods for producing, purifying, characterizing and using dolastatin linker derivatives containing at least one such unnatural amino acid. The embodiments also include compositions and methods for producing, purifying, characterizing and using oligonucleotides (including DNA and RNA) that can be used to at least partially produce dolastatin linker derivatives containing at least one unnatural amino acid. The embodiments also include compositions and methods for producing, purifying, characterizing and using cells that can express oligonucleotides that can be used to at least partially produce dolastatin linker derivatives containing at least one unnatural amino acid.

[0106] Thus, provided and described herein are dolastatin linker derivatives that include at least one unnatural amino acid or modified unnatural amino acid with a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group. In certain embodiments, the dolastatin linker derivatives that include at least one unnatural amino acid or modified unnatural amino acid with a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group include at least one post-translational modification at some positions on the polypeptide. In some embodiments, the co-translational or post-translational modification occurs via cellular machinery (e.g., glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid-linked modification, etc.), and in many cases, such cellular machinery-based co-translational or post-translational modification occurs at a naturally occurring amino acid site on the polypeptide, but in certain embodiments, the cellular machinery-based co-translational or post-translational modification occurs at a non-natural amino acid site on the polypeptide.

[0107] In other embodiments, the post-translational modification does not utilize cellular machinery, but instead, the functionality is provided by attaching a molecule (a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof) that contains a second reactive group to at least one unnatural amino acid having a first reactive group (including, but not limited to, an unnatural amino acid containing a ketone, aldehyde, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine functional group) using chemical methodologies described herein, or others appropriate for the particular reactive group. In certain embodiments, the co-translational or post-translational modification is performed in vivo in a eukaryotic or non-eukaryotic cell. In certain embodiments, the post-translational modification is performed in vitro without the use of cellular machinery. Also included in this aspect are methods for producing, purifying, characterizing, and using such dolastatin linker derivatives that contain at least one such co-translationally or post-translationally modified unnatural amino acid.

[0108] Also included within the scope of the methods, compositions, schemes and techniques described herein are reagents that can react with a dolastatin linker derivative (including a carbonyl or dicarbonyl group, an oxime group, an alkyne, a cycloalkyne, an azide, a hydroxylamine group, or masked or protected forms thereof) that is part of a polypeptide to cause any of the above-mentioned post-translational modifications. In certain embodiments, the resulting post-translationally modified dolastatin linker derivative contains at least one oxime group; the resulting modified oxime-containing dolastatin linker derivative can undergo subsequent modification reactions. Also included in this aspect are methods for producing, purifying, characterizing and using such reagents that allow any such post-translational modifications of such dolastatin linker derivatives.

[0109] In certain embodiments, the polypeptide or non-natural amino acid linked dolastatin derivative comprises at least one co-translational or post-translational modification made in vivo by one host cell, where the post-translational modification is not normally made by another type of host cell. In certain embodiments, the polypeptide comprises at least one co-translational or post-translational modification made in vivo by a eukaryotic cell, where the co-translational or post-translational modification is not normally made by a non-eukaryotic cell. Examples of such co-translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linked modification, and the like. In one embodiment, the co-translational or post-translational modification comprises the attachment of an oligosaccharide to asparagine via a GlcNAc-asparagine linkage. The oligosaccharide includes, but is not limited to, (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, and the like. In another embodiment, the co-translational or post-translational modification comprises the attachment of an oligosaccharide (including but not limited to Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, GalNAc-threonine, GalNAc-threonine, or GlcNAc-threonine linkage. In certain embodiments, the protein or polypeptide may comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, etc. Also included in this aspect are methods for producing, purifying, characterizing, and using such polypeptides that comprise at least one such co-translational or post-translational modification. In other embodiments, the glycosylated non-natural amino acid polypeptide is produced in an unglycosylated form.Such unglycosylated forms of glycosylated non-natural amino acids can be produced by chemical or enzymatic removal of oligosaccharide groups from isolated or substantially purified or unpurified glycosylated non-natural amino acid polypeptides; production of the non-natural amino acid in a host that does not glycosylate such non-natural amino acid polypeptides (such hosts include prokaryotes or eukaryotes that have been engineered or mutated so as not to glycosylate such polypeptides); and introduction of a glycosylation inhibitor into a cell culture medium in which such non-natural amino acid polypeptides are produced by eukaryotes that normally glycosylate such polypeptides; or a combination of any of these methods. Such unglycosylated forms of normally glycosylated non-natural amino acid polypeptides (by normally glycosylated, we mean a polypeptide that would be glycosylated if produced under conditions in which the naturally occurring polypeptide is glycosylated) are also described herein. Of course, such unglycosylated forms of normally glycosylated non-natural amino acid polypeptides (or indeed any polypeptides described herein) can be in unpurified, substantially purified, or isolated form.

[0110] [Oxime-containing linked dolastatin derivatives] The unnatural amino acid dolastatin-linked derivatives containing an oxime group allow for reaction with a variety of reagents that contain specific reactive carbonyl or dicarbonyl groups, including but not limited to those containing ketones, aldehydes, or other groups with similar reactivity, to form new unnatural amino acids with new oxime groups. Such oxime exchange reactions allow for further functionalization of the dolastatin-linked derivatives. Furthermore, the original dolastatin-linked derivatives containing an oxime group may themselves be useful, so long as the oxime bond is stable under the conditions required for incorporation of the amino acid into a polypeptide (e.g., in vivo, in vitro, and by the chemical synthesis methods described in WO 2013 / 185117 and WO 2005 / 074650).

[0111] Thus, certain embodiments described herein are unnatural amino acid dolastatin-linked derivatives that contain a side chain that contains an oxime group, an oxime-like group (which has similar reactivity to and is structurally similar to an oxime group), a masked oxime group (which can be readily converted to an oxime group), or a protected oxime group (which has similar reactivity to an oxime group upon deprotection).

[0112] The methods and compositions described herein include the incorporation of one or more non-natural amino acids into the dolastatin conjugate derivatives, which are known in the art, for example, as described in WO 2013 / 185117 and WO 2005 / 074650, each of which is incorporated herein by reference in its entirety. One or more non-natural amino acids may be incorporated into one or more specific positions that do not destroy the activity of the dolastatin conjugate derivatives. This may be achieved by "conservative" substitutions (including, but not limited to, substitution of hydrophobic amino acids with non-natural or natural hydrophobic amino acids, substitution of bulky amino acids with non-natural or natural bulky amino acids, substitution of hydrophilic amino acids with non-natural or natural hydrophilic amino acids), and / or insertion of non-natural amino acids at positions not required for activity.

[0113] A variety of biochemical and structural approaches can be used to select desired sites for substitution with unnatural amino acids within the dolastatin conjugated derivative. In some embodiments, the unnatural amino acid is attached to the C-terminus of the dolastatin derivative. In other embodiments, the unnatural amino acid is attached to the N-terminus of the dolastatin derivative. Any position of the dolastatin conjugated derivative is suitable for selection for incorporation of an unnatural amino acid, and selection can be based on rational design or any selection for any purpose or particularly undesired purpose. Selection of a desired site can be based on the production of a unnatural amino acid polypeptide (either further modified or left unmodified) that has any desired property or activity, including but not limited to, a receptor binding modulator, a receptor activity modulator, a modulator of binding to a binding partner, a modulator of the activity of a binding partner, a modulator of the conformation of a binding partner, formation of dimers or multimers, no change in activity and properties compared to the native molecule, or specific manipulation of the physical or chemical properties of the polypeptide, such as solubility, aggregation, or stability. Alternatively, sites identified as important for biological activity may be good candidates for substitution with unnatural amino acids, depending on the desired activity sought for the polypeptide. Another alternative is to simply generate a series of substitutions at each position on the polypeptide with unnatural amino acids and observe the effect on the activity of the polypeptide. Any technique, technique or method for selecting sites for substitution with unnatural amino acids for any polypeptide is suitable for use in the methods, techniques, and compositions described herein.

[0114] Additionally, the structure and activity of naturally occurring and deletion-containing polypeptide variants can be tested to determine regions of the protein that are likely to tolerate substitution with unnatural amino acids. Once other residues that are likely to be intolerant of substitution with unnatural amino acids have been eliminated, the impact of the proposed substitutions at each of the remaining positions can be tested using methods including, but not limited to, methods involving the three-dimensional structure of the relevant polypeptide, as well as any associated ligands or any binding proteins. X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Data Bank (PDB, www.rcsb.org), a centralized database that contains data on the three-dimensional structure of large molecules of proteins and nucleic acids, and can be used by those skilled in the art to identify amino acid positions that can be substituted with unnatural amino acids. Furthermore, when data on the three-dimensional structure is not available, models are created that explore the two-dimensional and three-dimensional structures of the polypeptide. Thus, the identification of amino acid positions that can be substituted with unnatural amino acids is readily possible.

[0115] Exemplary sites for incorporation of unnatural amino acids can be, but are not limited to, sites that are excluded from potential receptor binding regions or regions that bind to binding proteins or binding ligands, that may be fully or partially exposed to solvent, that have minimal or no hydrogen bonding interactions with nearby residues, that may be minimally exposed to nearby reactive residues, and / or areas of high flexibility predicted by the three-dimensional crystal structure of a particular polypeptide with the associated receptor, ligand, or binding protein.

[0116] A variety of unnatural amino acids can be substituted for amino acids at predetermined positions in a polypeptide or incorporated at the predetermined positions. As one example, a particular unnatural amino acid can be selected for incorporation based on examination of the three-dimensional crystal structure of the polypeptide with its associated ligands, receptors and / or binding proteins, and preferences for conservative substitutions.

[0117] In one embodiment, the method described herein comprises incorporating a dolastatin conjugate derivative comprising a first reactive group; and contacting the protein with a molecule comprising a second reactive group, including but not limited to a second protein, polypeptide, or polypeptide analog; an antibody or antibody fragment; and any combination thereof. In an embodiment, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl or dicarbonyl moiety, thereby forming an oxime bond. In an embodiment, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is a hydroxylamine moiety, thereby forming an oxime bond. In an embodiment, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is an oxime moiety, thereby forming an oxime exchange reaction. In an embodiment, the first reactive group is an oxime moiety and the second reactive group is a carbonyl or dicarbonyl moiety, thereby forming an oxime exchange reaction.

[0118] In some cases, the incorporation of a dolastatin conjugate derivative(s) is combined with other additions, substitutions, or deletions in the polypeptide that affect other chemical, physical, pharmacological, and / or biological properties. In some cases, the other additions, substitutions, or deletions may increase the stability of the polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the polypeptide for its receptor, ligand, and / or binding protein. In some cases, the other additions, substitutions, or deletions may increase the solubility of the polypeptide (including, but not limited to, when expressed in E. coli or other host cells). In some embodiments, sites are selected for substitution with naturally encoded or unnatural amino acids in addition to other sites for incorporation of unnatural amino acids to increase the solubility of the polypeptide following expression in E. coli or other host cells. In some embodiments, the polypeptides contain other additions, substitutions or deletions to modulate affinity for associated receptors, binding proteins, and / or receptors, modulate (including but not limited to, increase or decrease) receptor dimerization, stabilize receptor dimers, modulate circulating half-life, facilitate purification, improve certain administration routes, etc. Similarly, non-natural amino acid polypeptides can contain chemical or enzymatic cleavage sequences, protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to, FLAG or poly-His) or other affinity-based sequences (including but not limited to, FLAG, poly-His, GST, etc.), or binding molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification, transport through tissue or cell membranes, release or activation of prodrugs, reduction in size, or other characteristics of the polypeptide.

[0119] In some embodiments, payloads or toxin moieties are used in the anti-PSMA antibody drug conjugates of the present invention. Examples of payloads include, but are not limited to, DNA replication inhibitors, DNA transcription inhibitors, RNA translation inhibitors, cell division inhibitors, cell signaling inhibitors, kinase inhibitors, tubulin polymerase inhibitors, tubulin depolymerization agents, DNA cleavage agents, DNA binders, RNA polymerase inhibitors, auristatins, dolastatins, MMAF, MMAE, MMAD, duocarmycin analogs, pyrobenzodiazepine (PBD) analogs, tubulin analogs, maytansine analogs, amanitin analogs, cryptophycin analogs, epothilone analogs, calicheamicin analogs, doxorubicin analogs, camptothecin analogs.

[0120] In another embodiment of the present invention, a drug payload linker is provided. Antitubulin inhibitors are selected as payloads in combination with cleavable, short cleavable and non-cleavable linkers. As an example, the payload linker combination of the present invention may include, but is not limited to, cleavable, non-cleavable, and short cleavable payload linkers. Cleavable payload linkers may include cleavable dipeptides (including but not limited to Val-Cti, Val-Ala, Val-Lys and Ala-Ala), hydrazine bonds, disulfide bonds or pyrophosphate bonds. Examples of payload linkers used in the anti-PSMA antibody drug conjugates of the present disclosure include non-cleavable MMAE, non-cleavable MMAF, Val-citrulline-acetyl MMAF, short Val-citrulline-acetyl MMAF, or short Val-citrulline-acetyl MMAE. Both MMAE and MMAF were utilized in this study. Non-limiting examples of dolastatin linker derivatives include the following:

[0121] MMAF, which is non-cleavable, has the following structure:

[0122] [ka]

[0123] MMAE, which is non-cleavable, has the following structure:

[0124] [ka]

[0125] MMAF or Val-Citrulline-acetyl (Val-Cit) MMAF, which is cleavable and has the structure:

[0126] [ka]

[0127] A short, cleavable or short Val-Citrulline-Acetyl (Val-Cit) MMAF having the following structure:

[0128] [ka]

[0129] Short, cleavable or short Val-Citrulline-acetyl (Val-Cit) MMAE, having the following structure:

[0130] [ka]

[0131] Table 1 provides drug-linker compounds that can be used with the anti-PSMA antibody or antibody-drug conjugate of the present invention. The synthesis of such payload linkers is well known to those skilled in the art. For example, see Dubowchik et al., Bioconjugate Chem.13: 855-869, (2002); Doronina et al., Nature Biotechnology 21(7): 778-784, (2003); WO 2012 / 166560; WO 2013 / 185117. These are incorporated herein by reference.

[0132] [Table 1-1]

[0133] [Table 1-2]

[0134] [Table 1-3]

[0135] [Table 1-4]

[0136] Generation of anti-PSMA ADCs conjugated to PSMA-targeting ligands In other embodiments of the present invention, targeting ligands are included. Such ligands include ligands that bind to the active site of the PSMA receptor, ligands that are inhibitors of the glutamate carboxypeptidase enzymatic activity of the PSMA receptor, and ligands used for imaging. The present invention also includes ligands that have binding affinities in the subpico to subnanomolar range.

[0137] Anti-PSMA ADCs conjugated to PSMA targeting ligands can be made as described herein. One method of making involves conjugating a toxin-linker to an antibody by a cysteine-maleimide conjugation method, followed by sequential conjugation of a PSMA targeting ligand to a specific site on the antibody based on a proprietary oxime ligation method. Maleimide conjugation is performed by reduction of disulfide bridges between the heavy and light chains of the antibody, facilitated by the addition of a relevant reducing agent. The disulfide bonds of the antibody can be reduced by incubating a reaction mixture adjusted to a pH ranging from 6 to 7.4 consisting of the antibody and 3 to 9 molar-higher reducing agent. The reaction mixture can be incubated at 37°C for 2 hours to obtain a drug:antibody ratio of 3:4. The toxin-conjugated antibody is then buffer exchanged by a reaction mixture containing 10 molar-higher PSMA targeting ligand, the pH of which ranges from 4 to 5. The reaction of the toxin-conjugated antibody with the PSMA targeting ligand can be performed by incubation at room temperature with gentle shaking for 8 to 16 hours. Typically, in this method, the amount of PSMA targeting ligand to antibody ranges from 1.8 to 2. Unconjugated components from the reaction mixture are removed by passing through a cation exchange column and the buffer is exchanged with a proprietary formulation buffer that is isotonic with mouse and human body fluids. Alternatively, the PSMA targeting ligand and toxin can be attached in a single step to a specific site on the antibody by oxime-based conjugation chemistry. This single step attachment can be facilitated by designing a branched linker that includes both the PSMA targeting ligand and the toxin. This single step attachment can be performed by a proprietary click chemistry based on oxime and cyclooctyne. Unconjugated components can be removed from the reaction mixture by passing through a cation exchange purification column. The purified material is then formulated in an isotonic buffer. In an exemplary embodiment, the PSMA targeting ligand (L represents a linker) includes, but is not limited to, the following ligands:

[0138] [ka]

[0139] In an exemplary embodiment, the PSMA ligands of the invention may include compounds having an N-terminal targeting ligand and a C-terminal binding portion or binding moiety. In other exemplary aspects of the invention, the PSMA targeting ligands may include, but are not limited to, the ligands in Table 2 below:

[0140] [Table 2-1]

[0141] [Table 2-2]

[0142] In another exemplary embodiment, the invention includes a PSMA targeting ligand and a linker. The bond between the targeting ligand and the linker and the linker can be, but is not limited to, the following: The bond may be substituted with hydrogen.

[0143] [ka]

[0144] In other embodiments of the invention, the linker can be, but is not limited to, polyethylene glycol (PEG), alkyl, alkene, alkyne, amine, aryl, or amino acid.

[0145] In another exemplary embodiment, the invention includes, but is not limited to, PSMA targeting ligands with the toxins of Table 3.

[0146] [Table 3-1]

[0147] [Table 3-2]

[0148] [Table 3-3]

[0149] [Table 3-4]

[0150] [Table 3-5]

[0151] [Table 3-6]

[0152] [Table 3-7]

[0153] In some embodiments of the present invention, anti-PSMA antibodies, variants or drug conjugates that increase serum half-life, water solubility, bioavailability, therapeutic half-life or circulation time, or modulate immunogenicity or biological activity are desired. One method of achieving such desired properties of the anti-PSMA compositions disclosed herein is through the covalent attachment of the polymer polyethylene glycol (PEG). To maximize the desired properties of polyethylene glycol, the total molecular weight and hydration state of the polymer or polymers attached to the biologically active molecule must be high enough to impart the advantageous properties typically associated with polymer attachment, such as increased water solubility and circulation half-life, without adversely affecting the biological activity of the molecule to which polyethylene glycol is attached.

[0154] Polyethylene glycol derivatives are often conjugated to biologically active molecules through reactive chemical functional groups such as amino acid residues, the N-terminus, and / or carbohydrate moieties. WO 99 / 67291 discloses a method for conjugating proteins with polyethylene glycol. In this publication, at least one amino acid residue of the protein is replaced by a synthetic amino acid, and the protein is contacted with polyethylene glycol under conditions sufficient to achieve conjugation with the protein.

[0155] Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. The sites most suitable for modification by polymer attachment play an important role in receptor binding, and such sites may be necessary for the retention of biological activity of the molecule, and such sites are not suitable for polymer attachment. As a result, promiscuous attachment of polymer chains to such reactive sites of biologically active molecules often leads to a large reduction or even total loss of biological activity of the polymer-modified molecule. Polyethylene glycol attachment can be directed to specific locations within the protein such that the polyethylene glycol moiety does not interfere with the function of the protein. One method of directing polyethylene glycol attachment is to introduce synthetic amino acids into the protein sequence. The protein biosynthetic machinery of the prokaryotic organism Escherichia coli can be altered to efficiently incorporate synthetic amino acids into proteins with high fidelity in response to the amber codon UAG. For example, JW Chin et al., J. Amer. Chem. Soc. 124: 9026-9027, 2002;JW Chin, & PG Schultz, ChemBioChem 3(11):1135-1137, 2002;JW See Chin, et al., PNAS USA 99: 11020-11024, 2002; and L. Wang, & PG Schultz, Chem. Comm.,1: 1-11,2002. Similar methods can be achieved using the eukaryotic yeast S. cerevisiae (e.g., J. Chin et al., Science 301: 964-7, 2003). Using this method, non-naturally encoded amino acids can be incorporated into the anti-PSMA antibodies, variants or drug conjugates of the invention to provide polyethylene glycol attachment sites. See, e.g., WO 2010 / 011735 and WO 2005 / 074650.

[0156] Pharmaceutical Compositions In other embodiments of the present invention, the PSMA antibody variant or PSMA antibody drug conjugate further comprises a pharmaceutical composition or formulation. Such pharmaceutical compositions may use various pharma- ceutical acceptable excipients, stabilizers, buffers, and other components for administration to animals. For example, see Remington, The Science and Practice of Pharmacy, 19th ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1995. Since many components (e.g., purified stabilizing components) need to be considered, the identification of a composition or formulation suitable for stability, subject administration, and activity varies for each compound. Suitable salts for incorporation into the composition or formulation include, but are not limited to, sodium chloride, potassium chloride, or calcium chloride. Sodium oxide may be used as a buffer and / or stabilizer. Suitable buffers include phosphate-citrate buffer, phosphate buffer, citrate buffer, L-histidine, L-arginine hydrochloride, bicarbonate buffer, succinate buffer, citrate buffer, and TRIS buffer, either alone or in combination. Surfactants including polysorbates (e.g., polysorbate 80), dodecyl sulfate (SDS), lecithin, either alone or in combination, may also be used.

[0157] In some embodiments of the present invention, the pharmaceutical composition or formulation may be in the form of an aqueous composition, or a reconstituted liquid composition, or in the form of a powder. When in liquid form, the pH of the composition or formulation may range from about 4.0 to about 7.0, or from about 4.5 to about 6.5. However, the pH may be adjusted by one skilled in the art to provide good stability and administration.

[0158] Composition can be further stored in, but not limited to, vial or cartridge, pen delivery device, syringe, intravenous administration tube or intravenous administration bag.In other embodiments, pharmaceutical composition of the present invention can be administered as a single dose, or one or more additional doses can be administered within minutes, days or weeks after the first administration.Additional administration can be intended as necessary to treat, alleviate or prevent cancer, including prostate cancer.

[0159] In some examples, the PSMA antibody, variant or anti-PSMA ADC compositions of the present invention may be used in conjunction with additional therapies or treatments, including, but not limited to, surgery, radiation, cryosurgery, hyperthermia, hormonal treatments, chemotherapy, vaccines and other immunotherapies. In some embodiments, such additional treatments may include therapeutic agents such as chemotherapeutic agents, hormonal agents, antitumor agents, immunostimulants, immunomodulatory agents, corticosteroids, or combinations thereof. In one embodiment, the hormonal agent is enzalutamide.

[0160] In other embodiments, the anti-PSMA ADCs of the invention may be administered with one or more immunostimulants to induce or enhance an immune response. Immunostimulants can stimulate a particular arm of the immune system, such as natural killer (NK) cells that mediate antibody-dependent cellular cytotoxicity (ADCC). Such immunostimulants include, but are not limited to, IL-2, immunostimulatory oligonucleotides (e.g., CpG motifs), α-interferon, γ-interferon, tumor necrosis factor α (TNFα). In other embodiments, the anti-PSMA ADCs of the invention may be administered with one or more immunostimulants, including, but not limited to, cytokines, chemokines (including, but not limited to, SLC5 ELC, MIP3α, MIP3β, IP-IO, MIG, and combinations thereof). Another therapeutic agent may be a vaccine to immunize a subject against PSMA. In some embodiments, such a vaccine includes an antigen, such as a PSMA dimer, and optionally includes one or more adjuvants to induce or enhance an immune response. Many types of adjuvants are known in the art.

[0161] Chemotherapeutic agents or any agent associated with the treatment, amelioration, or prevention of a disease, illness, or cancer in a subject in need of treatment may be administered in combination with an anti-PSMA ADC of the invention. Chemotherapeutic agents may include, for example, but are not limited to, erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium), and the like. Pharm.), fulvestrant (FASLODEX®, AstraZeneca), stents (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SCH66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU5271; Sugen); alkylating agents (thioterephthalate and CYTOXAN®, cyclophosphamide, etc.); alkylsulfonates (busulfan, improsulfan, and piposulfan); antifolates, antineoplastics (pemetrexate (ALIMTA®, Eli Lilly), aziridines (benzodopa, carboquone, meturedopa, and uredopanad); ethylenimines and methylamelamines (including altretamine, triethylenemelamine, triethylphosphoramide, triethylenethiophosphoramide, and trimethylomelanin); acetogenins (especially bullatacin and bullatacinone); camptothenins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8);dolastatins;duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1);eleutherobin;pancratistatin;sarcodictyin;spongistatins;nitrogen mustards (chlorambucil, chlornaphazin, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trophosphamide, uracil mustard, etc.); Trosourea (such as carmustine, chlorozotocin, photemtacin, 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 chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, ca Cutinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcetrubicin, mitoma isin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, roborubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (such as denopterin, methotrexate, trimetrexate); purine analogs (such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine);Pyrimidine analogues (ancitabine, azacitabine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.); androgens (calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.); antiandrogens (e.g., enzalutamide) or androgen deprivation therapy; anti-adrenals (aminoglutethimide, mitotane, trilostane, etc.); folic acid replacement (florinic acid, etc.); aceglatone; aldophosphamide Glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elformitin;elliptinium acetate;epothilones;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidynin;maytansinoids (such as maytansine and ansamitocins);mitoguazone;mitoxantrone;mopidanmol;nitraelin;pentostatin;phenamet;pirarubicin;rosoxantrone;podophyllic acid;2-ethylhydrazine;procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, OR);razoxane;rhizoxin;schizofuran;spinogermanium;tenuazonic acid;triaziquone;2,2',2''-trichlorotriethylamine;trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidine);urethane;vindesine;decarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinoside ("Ara-C");cyclophosphamide;thiotapa;Taxoids (e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free, albumin, nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (such as cisplatin and carboplatin); vinblasine; platina; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; RFS2000, a topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids (such as retinoic acid); capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.;

[0162] The terms "about" are understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used.

[0163] [Example] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereon may be suggested to one skilled in the art and are to be included within the spirit and scope of this application and the appended claims.

[0164] [Example 1] Transient transfection CHO-S cultures were diluted to 0.75 × 10 in FreeStyle CHO medium. 6 The cells were seeded at a concentration of 1.4–1.6 × 10 / mL approximately 16 hours before transfection. 6Cells were transfected the day after reaching a cell number of 1 / mL. At the target cell number, 400 mM para-acetylphenylalanine (pAF) stock was added to a final culture concentration of 1.4 mM.

[0165] Polyethylenimine / DNA (PEI / DNA) complexes were prepared as follows: DNA (1.42 μg / 1x10 6 The DNA / RPMI mixture was incubated for 2 min at room temperature. PEI stock (1 mg / mL) was added to the DNA solution in a 3:1 ratio (PEI / DNA (μg / μg)). The mixture was incubated for 5 min at room temperature.

[0166] The mixture was gently added to the cell culture and mixed. The flask was transferred to a 32°C incubator. Western blot analysis was performed 6 days after transfection. The supernatant was collected 7 days after transfection.

[0167] [Example 2] Humanization of antibodies The parental murine J591 (Liu et al., Cancer Research, 57, 3629-36354, 1997) antibody was humanized by selecting human frameworks based on sequence identity with the murine framework sequences. The light and heavy chain CDRs of the murine antibody were grafted onto the human frameworks, respectively, and analyzed for binding to PSMA antigen. Humanized variants were generated by pairing four human heavy chain frameworks with six light chain frameworks. The variants were transiently expressed in HEK293 cells, and the supernatants were tested for binding to PSMA antigen expressed in LnCap cells by FACS. Table 4 shows the selected heavy chain variable region sequences and four light chain variable region sequences used in further testing. In binding analysis, the four humanized full-length variants, shown as humanized anti-PSMA variants 1, 2, 3 and 4 (Table 4), retained binding affinity (nanomolar range binding affinity) comparable to the chimera (Table 4). As disclosed in Table 4, humanized anti-PSMA variant 1 comprises the heavy chain sequence of SEQ ID NO: 8 and the light chain sequence of SEQ ID NO: 9. Humanized anti-PSMA variant 2 comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 11. Humanized anti-PSMA variant 2 comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 11. Humanized anti-PSMA variant 3 comprises the heavy chain sequence of SEQ ID NO: 12 and the light chain sequence of SEQ ID NO: 13. Humanized anti-PSMA variant 4 comprises the heavy chain sequence of SEQ ID NO: 14 and the light chain sequence of SEQ ID NO: 15.

[0168] [Table 4-1]

[0169] [Table 4-2]

[0170] [Table 4-3]

[0171] [Table 4-4]

[0172] Example 3: Recombinant expression of anti-PSMA antibodies A lead humanized anti-PSMA antibody was selected and recombinantly expressed in CHO cells by both transient transfection (described in Example 1) and stable bulk pool methods to test the expression of the humanized sequence. In the transient transfection and stable bulk pool approaches, proprietary technologies were constructed in expression vectors and CHO host cells, respectively (see, e.g., International Publication No. 2018 / 223108). The unnatural amino acid para-acetylphenylalanine (pAF) was incorporated into the PSMA antibody at position A114 (Kabat numbering scheme; see Kabat et al., NIH Publication No. 369-847, 1993; A114 position is underlined in Table 4). A114 is the first amino acid residue in the constant region of the heavy chain.

[0173] Example 4: Production of antibody-drug conjugates (ADCs) Antibodies can be purified using chromatography-based methods well known to those skilled in the art, including Protein A affinity capture and cation exchange polishing. Antibodies harvested from cell culture media are captured on a Protein A affinity column and eluted under acidic conditions. The antibody preparation is polished by titrating to pH 5 and applying to a cation exchange column. After cation exchange, the antibody can be used to buffer exchange with a conjugation buffer.

[0174] In the present invention, humanized anti-PSMA antibodies are conjugated with toxins (MMAE or MMAF) via small molecule hydrophilic linkers. The drug-payload linker is covalently attached to pAF on the anti-PSMA heavy chain to form an anti-PSMA-ADC. The PSMA antibody is conjugated with the drug-payload linker by incubation at 28°C in the presence of 135 mM acetic acid hydrazide (acting as a catalyst). Before exchanging the anti-PSMA ADC into formulation buffer (50 mM histidine, 150 mM sodium chloride, 2.5% trehalose pH 6), the reaction mixture was subjected to a cation exchange chromatography column to remove unconjugated antibody, unconjugated drug-payload linker, aggregates and impurities. The antibody-drug conjugate was eluted from the column under a salt gradient that resulted in a monodisperse peak.

[0175] Analytical characterization of the monodisperse peaks from cation exchange chromatography indicates that the major species was observed within 8 h, which was the anti-PSMA ADC with a drug-to-antibody ratio (DAR) in the range of 1.8-2. In quality control evaluations performed on the antibodies and ADCs of the invention, all preparations made by this strategy contained less than 3% aggregates and endotoxin levels less than 5EU / mg. The preparations were found to be of excellent quality material.

[0176] Fifteen anti-PSMA ADCs were generated using two lead humanized variants and a chimeric antibody utilizing five payload linkers disclosed herein (non-cleavable MMAE, non-cleavable MMAF, cleavable MMAF, short cleavable MMAF, and short cleavable MMAE). Anti-PSMA ADCs generated using humanized variants 1 and 2 disclosed in Table 4 were selected for further testing in the following examples.

[0177] [Example 5] In vitro efficacy test The in vitro efficacy of anti-PSMA ADCs is determined using C4-2, a representative cell line for metastatic castration-resistant prostate cancer (mCRPC). Cell cultures are incubated with the antibody-drug conjugates for 96 hours at 37°C. Cell viability is assessed using Cell Titer. Glo® (Promega, USA). Antiproliferative activity was measured using potency, IC 50 (concentration that achieves 50% of maximum activity) and cell death and E max (maximal growth inhibition level). Table 5 summarizes the cell killing and in vitro efficacy of anti-PSMA ADCs.

[0178] [Table 5]

[0179] The cell killing activity assay suggests that the payload-linker combination is important for developing a potent antibody drug conjugate. The potent antibody drug conjugate showed sub-nanomolar efficacy with cell killing of more than 75%. The antibody drug conjugate with MMAF payload had a higher cell killing rate and efficacy than the antibody drug conjugate with MMAE payload. The in vitro cell killing test suggests that the anti-PSMA ADC with MMAF payload was an order of magnitude more effective than the anti-PSMA ADC with MMAE payload.

[0180] This study also demonstrates that the activity of antibody-drug conjugates is affected by the type of linker. Antibody-drug conjugates with cleavable linkers had higher cell killing rates and efficacy than antibody-drug conjugates with noncleavable linkers for the same type of payload. As shown in Table 5, in vitro cell killing studies show that cleavable linkers were an order of magnitude more effective than noncleavable linkers in anti-PSMA ADCs with either MMAE or MMAF payloads. This suggests that potent anti-PSMA ADCs can be made by the appropriate combination of cytotoxic payloads and linkers. The combination of MMAE payloads and noncleavable linkers showed poor efficacy and killing. Furthermore, the killing of target cells by anti-PSMA antibody ADCs is achieved by targeted delivery of cytotoxic agents by anti-PSMA antibodies. Targeted delivery of cytotoxic agents is facilitated by binding of the anti-PSMA antibody component of the antibody-drug conjugate to PSMA, a receptor or antigen expressed on the cell surface, which is internalized into the cell by the receptor. Studies using prostate cancer cell lines that do not express PSMA, such as PC-3, suggest that potent anti-PSMA ADCs do not exert any cell killing activity. PC-3 cell lines were incubated with potent anti-PSMA ADCs for 96 hours and did not die. This suggests that the anti-PSMA ADCs of the present invention are target specific. No bystander killing effect of the antibody drug conjugates was observed. The lack of bystander killing effect of the antibody drug conjugates of the present invention is important to eliminate non-specific off-target toxicity due to toxic components, which is often observed in antibody drug conjugates containing MMAE as a cytotoxic payload. Furthermore, the target specific activity of the anti-PSMA ADCs of the present invention makes them valuable in the treatment of metastatic prostate cancer. Observations from these studies suggest that any cancerous lesion beyond the primary cancerous prostate tissue expressing PSMA can be treated with the anti-PSMA ADCs of the present invention.

[0181] Furthermore, the cell killing activity of anti-PSMA ADCs depends on the number of receptors expressed on the cell surface. Prostate cancer cell lines with receptor copy numbers of more than 60,000 can be efficiently killed by the anti-PSMA ADCs of the present invention. For example, in vivo studies were performed using anti-PSMA ADCs containing MMAF payloads with non-cleavable linkers. In vitro cell killing studies demonstrated killing of cell lines with receptor copy numbers greater than 60,000 by immunohistochemistry (IHC) and quantitative indirect immunofluorescence (QiFi) analysis (Table 6).

[0182] [Table 6]

[0183] The cell-killing activity of the anti-PSMA ADC of the present invention depends on the receptor copy number of PSMA, which is an indication of the treatment benefit in treating prostate cancer.The receptor copy number of PSMA is usually higher in cancerous prostate tissues compared to the expression of PSMA in tissues without cancer.The difference in expression of PSMA receptor copy number between cancerous prostate tissues and normal tissues can facilitate the reduction and / or elimination of target-related non-prostate tissue-related toxicity.This analysis suggests that anti-PSMA ADC is less active in tissues that express low amounts of PSMA receptor copy number than cancerous prostate tissues that express high amounts of PSMA receptor copy number.Therefore, the background toxicity of antibody-drug conjugates can be significantly reduced.

[0184] Example 6: Pharmacokinetic (PK) profile of drug payload-linker The systemic stability of the anti-PSMA ADCs herein can be evaluated by conducting pharmacokinetic studies in both mice and rats in which the antibody-drug conjugate is administered as an intravenous (iv) bolus. The pharmacokinetic evaluation suggests that the stability of the intact antibody-drug conjugate depends on the stability of the linker. Studies conducted in mice at a single dose of 1 mg / kg suggest that anti-PSMA ADCs with MMAF payloads linked by either non-cleavable or short cleavable (Val-citrulline-acetyl) linkers remain intact throughout the duration of the study. The half-life of intact anti-PSMA ADCs with MMAF payloads linked by either non-cleavable or short cleavable linkers is 10 days (Figure 1, top two panels). The systemic stability of the short cleavable linkers is independent of the payload. The linker in the anti-PSMA ADC with an MMAE payload linked by a short cleavable linker also remains intact throughout the duration of the study, with the half-life of the intact antibody-drug conjugate being as long as 10 days (Figure 1, right panel). In contrast, however, the linker in the anti-PSMA ADC with an MMAF payload linked by a cleavable (Val-Citrulline-PEG1) linker is cleaved early, shortening the half-life of the intact antibody-drug conjugate to approximately 4 days (Figure 1, bottom left panel). F1 represents detection of antibody-drug conjugate in serum with antibody. F3 represents detection of antibody-drug conjugate in serum with toxin-payload linker.

[0185] The systemic stability of the anti-PSMA ADC with noncleavable MMAF was also evaluated in rats at doses of 1 mg / kg and 5 mg / kg. The study revealed that the antibody-drug conjugate was stable, with the linker and cytotoxic agent remaining intact throughout the duration of the study (Figure 2).

[0186] The pharmacokinetic profile of the anti-PSMA ADC with non-cleavable MMAF was compared to that of an anti-PSMA antibody without a cytotoxic agent (denoted as naked antibody) in rats. Evaluation of the pharmacokinetic profile suggests that the clearance of the anti-PSMA ADC with non-cleavable MMAF is similar to that of the anti-PSMA antibody (Figure 3). This suggests that the intrinsic stability of the antibody is not affected by p-acetylphenylalanine-based site-specific conjugation and drug loading.

[0187] The stability of antibody-drug conjugates is essential to reduce toxicity from cytotoxic agents. Preventing cleavage of cytotoxic agents from antibody-drug conjugates during systemic circulation is an important way to reduce toxicity. The anti-PSMA ADCs of the present disclosure, with non-cleavable and short cleavable linkers, are significantly more stable in systemic circulation, and the cytotoxic agent remains attached to the antibody. This is important because most antibody-drug conjugates are unstable, and the drug payload falls off from the linker and / or conjugation site (binding site), thereby resulting in significant non-target-related toxicity from the cytotoxic agent. The therapeutic use of antibody-drug conjugates is currently limited due to such toxicity. Most antibody-drug conjugates in clinical trials have revealed dose-limiting toxicity that is believed to be due to the cytotoxic agent. Due to such toxicity, the therapeutic use of antibody-drug conjugates is limited, and to date, only four antibody-drug conjugates have been approved for therapeutic use. Treatment of prostate cancer with antibody-drug conjugates targeting PSMA has proven difficult in the field so far due to severe toxicity and lack of therapeutic index. The pharmacokinetic data suggest that the anti-PSMA ADCs of the invention can overcome limitations due to non-specific toxicity from cytotoxic agents by preventing cleavage of the drug payload from the linker and / or attachment site through a unique combination of stable conjugation chemistry and stable linker. The stable conjugation chemistry utilized in generating the novel anti-PSMA ADCs of the invention can be achieved by formation of a ketoxime bond between a non-natural / unnatural amino acid (e.g., p-acetylphenylalanine) incorporated into the antibody at a specific position and a drug-payload linker derivatized with a hydroxylamine group.

[0188] [Example 7] In vivo efficacy test The antitumor efficacy of anti-PSMA ADCs with noncleavable MMAF and short cleavable MMAF was tested in tumor xenograft models in mice engrafted with prostate cancer cell lines. C4-2, a metastatic castration-resistant prostate cancer (mCRPC)-associated cell line, was obtained from ATCC and propagated in vitro according to ATCC instructions. C4-2 human cancer xenografts were propagated in male immunodeficient mice with cells implanted subcutaneously. Mice were weighed and tumor volumes were measured using electronic calipers. Individual tumor volumes were calculated as length × width × width / 2. Mean volume of 500 mm 3 Mice bearing 100 ng / kg vascularized tumors (determined by appearance) were randomized into treatment groups and administered intravenously 100 mg / kg of individual body weight of anti-PSMA ADC at 0.1, 1, 5, and 10 mg / kg.

[0189] This study suggests that anti-PSMA ADCs with non-cleavable MMAF and anti-PSMA ADCs with short cleavable MMAF are effective in preventing tumor growth. Anti-PSMA ADCs at 5 mg / kg and 10 mg / kg were effective in inhibiting tumor growth for 37 days after administration. Anti-PSMA ADCs at 1 mg / kg were effective in tumor congestion until regrowth was observed 35 days after administration. No significant difference from the control was observed at the 0.1 mg / kg dose. Body weight analysis showed that treated animals also gained weight as normal healthy mice. This means that there is no obvious toxicity associated with anti-PSMA ADCs. Figures 4A and 4B show representative results of tumor volume and body weight of anti-PSMA ADCs of the present invention with non-cleavable MMAF. Figures 5A and 5B show representative results of tumor volume and body weight of anti-PSMA ADCs of the present invention with short cleavable MMAF.

[0190] [Example 8] Pharmacokinetic study of non-cleavable MMAF Pharmacokinetics (PK) was evaluated in tumor and non-tumor bearing mice after a single dose of anti-PSMA ADC with non-cleavable MMAF. Mice were injected with 1 mg / kg or 5 mg / kg of non-cleavable MMAF ADC by bolus intravenous injection. Serum was collected and analyzed for the presence of non-cleavable MMAF ADC. Similar PK profiles were observed for anti-PSMA ADC concentrations in non-tumor and tumor bearing mice at the dose levels evaluated (1 mg / kg and 5 mg / kg) (Figure 6A). Pharmacokinetics (PK) was also evaluated in cynomolgus monkeys following single dose administration of the anti-PSMA ADC using various concentrations of uncleavable MMAF: 1.17 mg / kg, 3.86 mg / kg, 9.36 mg / kg, 11.7 mg / kg, 23.3 mg / kg, and 35.0 mg / kg (Figure 6B). The anti-PSMA ADC was administered 15-20 minutes by intravenous infusion (intended route of administration in clinical practice). Samples were collected for 28 days following administration. Bioanalysis of the intact anti-PSMA ADC was performed using a qualified Meso The PK profiles were performed using the Scale Discovery method. Overlapping PK profiles were observed for the intact ADC and the full antibody, confirming that the anti-PSMA ADC of the present invention is extremely stable in the systemic circulation. A nonlinear increase in exposure was observed at low doses (1.17 mg / kg to 3.86 mg / kg), which may be attributed to possible target-mediated excretion. Further increases in dose resulted in a relatively linear increase in exposure with increasing dose. Clearance was slow, ranging from 0.118 mL / h / kg to 0.304 mL / h / kg, and the volume of distribution was small, ranging from 44.2 mL / kg to 58.2 mL / kg. The half-life was long, ranging from 129 hours to 346 hours, with a general trend toward longer half-life with increasing dosing. It should be noted that the extreme stability of the anti-PSMA ADC in the systemic circulation minimizes off-target toxicity associated with the release of free payload from the ADC and promotes a long half-life, thereby allowing for a patient-compatible dosing regimen of once every 3 or 4 weeks.

[0191] [Example 9] Anti-PSMA ADC combination therapy The anti-PSMA ADCs of the invention may be used in combination with any agent for treating, alleviating, ameliorating, or preventing prostate cancer in a subject, including but not limited to hormone therapy or chemotherapy. For example, the hormone therapy enzalutamide has been demonstrated to increase cell surface PSMA expression by approximately three-fold in vitro (Murga et al., Prostate 15;75(3):242-54, 2015). Furthermore, enzalutamide-treated mice bearing human PDX prostate tumors showed further inhibition of tumor growth when combined with the auristatin payload MMAE and the PSMA ADC (DiPippo et al., Prostate 15;76(3):325-34, 2016). Based on the increased PSMA expression noted in the art, the present The inventors studied the in vivo efficacy of combination therapy of an anti-PSMA ADC of the invention having an MMAF payload with enzalutamide.

[0192] The anti-tumor efficacy of the anti-PSMA ADC of the present invention in combination with the hormonal therapy enzalutamide was tested in a tumor xenograft model in mice implanted with prostate cancer cell lines. C4-2, a metastatic castration-resistant prostate cancer (mCRPC)-associated cell line, was obtained from ATCC and propagated in vitro according to ATCC instructions. C4-2 human cancer xenografts were propagated in male immunodeficient mice with cells implanted subcutaneously. A second model was also tested using male immunodeficient mice bearing patient-derived xenograft (PDX) TM00298 (Jackson Laboratories, CA) prostate tumors.

[0193] Mice were weighed and tumor volumes were measured using electronic calipers. Individual tumor volumes were calculated as length × width × width / 2. Mean volume 500 mm 3Mice bearing vascularized tumors (determined by appearance) were randomized into treatment groups and administered intravenously per individual body weight. Anti-PSMA ADC was administered intravenously at 0.5, 1, and 3 mg / kg per week for 4 weeks in the C4-2 model. Anti-PSMA ADC was administered intravenously at 1 and 3 mg / kg per week for 4 weeks in the PDX model. Enzalutamide was administered orally at 10 mg / kg daily for 28 days in both prostate models. In both prostate models, one group of mice received a combination treatment of 1 mg / kg anti-PSMA ADC + 10 mg / kg enzalutamide (Figures 7 and 8). Mice in the PDX model were also treated with a combination treatment of 3 mg / kg anti-PSMA ADC + 10 mg / kg enzalutamide (Figure 8).

[0194] Tumor inhibition was dose-responsive in the enzalutamide-resistant model at 3 mg / kg and 1 mg / kg anti-PSMA ADC. Complete inhibition was achieved at 3 mg / kg with 80% TGI. A partial response was observed at 1 mg / kg with 37% TGI. Body weight analysis showed that treated animals also gained weight as normal healthy mice, indicating that there was no obvious toxicity associated with anti-PSMA ADC. Figures 7A and 8A show representative results of tumor volume of anti-PSMA ADC of the present invention with non-cleavable MMAF. Figures 7B and 8B show representative results of body weight of anti-PSMA ADC of the present invention with non-cleavable MMAF.

[0195] This study demonstrated improved efficacy with the anti-PSMA ADC of the present invention in combination with enzalutamide. As a single agent, enzalutamide increased the level of cell surface PSMA in prostate cancer cells. The combination of anti-PSMA ADC and enzalutamide had a greater %TGI than monotherapy. This study suggests that anti-PSMA ADCs with non-cleavable MMAF and anti-PSMA ADCs with short cleavable MMAF were effective in preventing tumor growth with synergistic effects in the presence of enzalutamide.

[0196] Example 10: Human clinical trials of the safety and / or efficacy of anti-PSMA antibody-drug conjugates (ADCs) in treating prostate cancer Objective: To compare the safety and pharmacokinetics of administration compositions containing the anti-PSMA ADCs of the invention.

[0197] Study Design: The study will involve a Phase I, single or multi-center, open-label, randomized dose escalation study followed by a Phase II study in patients with prostate cancer. Patients must not have been exposed to PMSA derivatives prior to study entry. Patients must not have received cancer treatment within 2 weeks of study initiation. Treatments include the use of chemotherapeutic agents, hematopoietic growth factors, and biotherapeutics such as monoclonal antibodies. Patients must have recovered (grade 0 or 1) from all toxicities from previous treatments. All subjects will undergo safety evaluations and all blood draws for pharmacokinetic analysis will be performed as scheduled. All studies will be performed with Institutional Ethics Committee approval and patient consent.

[0198] Phase I: Patients receive anti-PSMA ADC intravenously in a dosing cycle once every 3 weeks. The dose of anti-PSMA ADC may be maintained or adjusted depending on toxicity as assessed by the following outline: Treatment is repeated every 3 weeks in the absence of unacceptable toxicity. The dose of anti-PSMA ADC is escalated in a cohort of 3-6 patients until the maximum tolerated dose (MTD) of anti-PSMA ADC is determined. The MTD is determined as the dose just before 2 out of 3 patients or 2 out of 6 patients experience dose-limiting toxicity. Dose-limiting toxicity is determined based on the definitions and criteria defined in the National Cancer Institute (NCI) Common Terminology for Adverse Events (CTCAE) Version 3.0 (August 9, 2006).

[0199] Phase II: Patients receive anti-PSMA ADC at the MTD determined in Phase I, as in Phase I. Treatment is repeated every 3 weeks for several cycles in the absence of disease progression or unacceptable toxicity. After completing a course of study treatment, patients who demonstrate a complete or partial response may receive additional doses. Patients who remain stable for more than 2 months after completing 6 courses of study treatment may receive an additional 6 courses upon disease progression, provided they meet the original eligibility criteria.

[0200] Blood Collection: Blood is collected serially by direct venipuncture before and after dosing of the anti-PSMA ADC. Venous blood samples (5 mL) for determination of serum concentrations are collected approximately 10 minutes before dosing and approximately 0.5 hours, 4 hours, 1 day, 2 days, 4 days, 7 days, 14 days, and 21 days after dosing. Each serum sample is divided into two equal portions. All serum samples are stored at -20°C. Serum samples are shipped on dry ice.

[0201] Pharmacokinetics: Patients will have plasma / serum samples collected for pharmacokinetic assessments prior to treatment initiation and at 0.5 hours, 4 hours, 1 day, 2 days, 4 days, 7 days, 14 days, and 21 days after study initiation. Pharmacokinetic parameters will be calculated using the latest version of Phoenix WinNonlin software using model-independent methods. The following pharmacokinetic parameters will be determined: peak serum concentration (C max ); Time to reach maximum serum concentration (t max ); Area under the curve from start to last blood draw (AUC last ) the area under the concentration-time curve (AUC); and the terminal elimination half-life (t 1 / 2 ). Elimination rate constants are estimated by linear regression of concentration-time log-linear plots of the terminal linear region of the continuous data. Means, standard deviations (SD), and coefficients of variation (CV) of pharmacokinetic parameters are determined for each treatment. Ratios of parameter means (stored formulation / non-stored formulation) are determined.

[0202] Patient Response to Combination Therapy: Patient response will be assessed by imaging with x-rays, CT scans and MRI, with imaging performed prior to the start of the study and at the end of the first cycle, with additional imaging performed every 4 weeks or at the end of subsequent cycles. Imaging modalities will be selected based on cancer type and feasibility / availability, with the same imaging modality being utilized for similar cancer types and throughout the course of each patient's study. Response rates will be determined using the RECIST scale (Therasse et al, J.Natl. Cancer Inst. 2000 Feb 2;92(3):205-16; http: / / ctep.cancer.gov / forms / TherasseRECISTJNCI.pdf). Patients will also undergo cancer / tumor biopsies to assess phenotypic changes in cancer precursor cells and changes in clonogenic growth by flow cytometry, Western blotting and IHC, and genetic changes in cells by FISH. Patients will be followed up periodically for 4 weeks after completion of study treatment. [Brief description of the drawings]

[0203] [Figure 1] 1 shows a pharmacokinetic study conducted in mice with anti-PSMA antibody drug conjugates (ADCs) using non-cleavable MMAF, short cleavable MMAF, cleavable MMAF and short cleavable MMAE. [Diagram 2] 1 shows a pharmacokinetic study conducted in rats with an anti-PSMA ADC that exhibits non-cleavable MMAF at various concentrations. [Diagram 3] Pharmacokinetic comparison of unconjugated anti-PSMA antibody (naked antibody) compared to ADC using MMAF, which is non-cleavable. [Figure 4A] 1 shows single dose responses in the C4-2 xenograft prostate model on tumor growth by non-cleavable MMAF anti-PSMA ADC at various doses. [Figure 4B]1 shows single dose responses in the C4-2 xenograft prostate model in terms of body weight change with non-cleavable MMAF anti-PSMA ADC at various doses. [Figure 5A] Shows single dose responses in the C4-2 xenograft prostate model on tumor growth with non-cleavable MMAF anti-PSMA ADC at various doses. [Figure 5B] Shows single dose response in the C4-2 xenograft prostate model in terms of body weight change with non-cleavable anti-PSMA ADC MMAF at various doses. [Figure 6A] Pharmacokinetic studies in tumor and non-tumor bearing mice following a single dose of an anti-PSMA ADC with non-cleavable MMAF. [Figure 6B] Pharmacokinetic studies in cynomolgus monkeys (FIG. 6B) following a single dose of anti-PSMA ADC with non-cleavable MMAF are shown. [Figure 7A] Shows the repeat dosing response in the C4-2 xenograft prostate model in terms of tumor growth at various doses of MMAF, a non-cleavable anti-PSMA ADC, and MMAF + enzalutamide, a non-cleavable anti-PSMA ADC. [Figure 7B] Shows the repeat dosing response in the C4-2 xenograft prostate model in terms of body weight change at various doses of MMAF, a non-cleavable anti-PSMA ADC, and MMAF + enzalutamide, a non-cleavable anti-PSMA ADC. [Figure 8A] Shows repeat dosing response in a PDX prostate model in terms of tumor growth at various doses of MMAF, a non-cleavable anti-PSMA ADC, and MMAF + enzalutamide, a non-cleavable anti-PSMA ADC. [Figure 8B] Shows repeat dosing response in a PDX prostate model in terms of body weight change at various doses of MMAF, a non-cleavable anti-PSMA ADC, and MMAF + enzalutamide, a non-cleavable anti-PSMA ADC.

Claims

1. An anti-prostate-specific membrane antigen (anti-PSMA) antibody comprising a heavy chain variable region sequence selected from SEQ ID NO: 1 or 6, and a light chain variable region sequence selected from SEQ ID NO: 2, 3, 4, 5 or 7.

2. The anti-PSMA antibody described in claim 1, wherein the anti-PSMA antibody comprises a heavy chain sequence selected from SEQ ID NO: 8, 10, 12, 14 or 16, and a light chain sequence selected from SEQ ID NO: 9, 11, 13, 15 or 17.

3. An anti-PSMA antibody described in claim 1 or 2, wherein the heavy chain sequence includes an amino acid that is not naturally encoded.

4. The anti-PSMA antibody of claim 3, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

5. The non-naturally encoded amino acid is para-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, p-acylphenylalanine, o-acylphenylalanine, m-aminophenylalanine, p ... arylphenylalanine, p-OMephenylalanine, o-OMephenylalanine, m-OMephenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine tyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L- 5. The anti-PSMA antibody of claim 4, which is Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, or p-propargyloxy-phenylalanine.

6. The anti-PSMA antibody of claim 5, wherein the non-naturally encoded amino acid is para-acetylphenylalanine.

7. An antibody-drug conjugate comprising the anti-PSMA antibody of any one of claims 1 to 6 conjugated to at least one drug linker selected from Tables 1 to 3, An antibody-drug conjugate, wherein the conjugation occurs via a non-naturally encoded amino acid incorporated into the heavy chain sequence.

8. The antibody-drug conjugate described in claim 7, wherein the drug linker comprises a cytotoxic agent.

9. The antibody-drug conjugate of claim 8, wherein the cytotoxic agent is a dolastatin, a dolastatin derivative or a dolastatin analog.

10. The antibody-drug conjugate of claim 9, wherein the dolastatin, dolastatin derivative or dolastatin analog is a monomethylauristatin selected from monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE).

11. The monomethylauristatin is a cleavable MMAE or MMAF, a non-cleavable MMAE or MMAF, or a short cleavable MMAE or MMAF. The antibody-drug conjugate of claim 10.

12. A pharmaceutical composition comprising an anti-PSMA antibody of any one of claims 1 to 6 or an antibody-drug conjugate of any one of claims 7 to 11 for a method of reducing or inhibiting tumor growth or progression in a PSMA-expressing cancer or cancer cells, comprising: The method includes contacting the PSMA-expressing cancer or cancer cells with an effective amount of the anti-PSMA antibody or antibody-drug conjugate pharmaceutical composition.

13. The method of claim 12, further comprising contacting the PSMA-expressing cancer or cancer cells with an effective amount of a therapeutic agent.

13. The pharmaceutical composition of claim 12.

14. The pharmaceutical composition of claim 13, wherein the therapeutic agent is a chemotherapeutic agent, a hormonal agent, an antitumor agent, an immunostimulant, an immunomodulatory agent, a corticosteroid, or a combination thereof.

15. The pharmaceutical composition described in claim 14, wherein the hormonal agent is enzalutamide.

16. A pharmaceutical composition for treating cancer comprising an anti-PSMA antibody described in any one of claims 1 to 6 or an antibody-drug conjugate described in any one of claims 7 to 11.

17. A nucleic acid encoding any one of SEQ ID NOs: 1 to 17.

18. A vector comprising the nucleic acid of claim 17.

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