ANTI-PSMA ANTIBODIES, ANTIBODY DRUG CONJUGATES, AND METHODS OF USE THEREOF

Abstract

The invention relates generally to antibodies that bind PSMA, and methods of making and using these anti-PSMA antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

Claims

1. An isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PSMA, wherein the AB specifically binds human PSMA and cynomolgus monkey PSMA, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1 amino acid sequence SYDMH (SEQ ID NO: 7); a VH CDR2 amino acid sequence VIWYDGSNKYYADSLKG (SEQ ID NO: 8); a VH CDR3 amino acid sequence VIAARTFYYYGMDV (SEQ ID NO: 9); a VL CDR1 amino acid sequence RSSQSLLHSDGYNYLD (SEQ ID NO: 1); a VL CDR2 amino acid sequence LGSNRAS (SEQ ID NO: 2); and a VL CDR3 amino acid sequence MQALQTPWT (SEQ ID NO: 3).

2. The isolated antibody of claim 1, wherein the AB comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ NOs: 29.

3. The isolated antibody of claim 1 or claim 2, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, a scFv, and a scAb.

4. The isolatedantibody of any one of claims 1 to 3, wherein the AB specifically binds human PSMA.

5. A conjugated antibody comprising the isolated antibody of any one of claims 1 to 4 conjugated to an agent.

6. The conjugated antibody of claim 5, wherein the agent is a toxin or fragment thereof.

7. The conjugated antibody of claim 6, wherein the agent is a microtubule inhibitor.

8. The conjugated antibody of claim 7, wherein the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof.

9. The conjugated antibody of claim 8, wherein the agent comprises a molecule having a structure of formula (1): ##STR00026## wherein R1 is a hydrogen or a C16 alkyl group; wherein R is selected from the group consisting of: a hydrogen, a C.sub.1-6 a linker, or a group X1-Y1-* wherein * is the point of attachment to the nitrogen; and wherein Y1 is an oxycarbonyl group and X1 is a C.sub.1-6 alkyl group, a 9-fluorenylmethyl group, a benzyl group, or a tert-butyl group.

10. The conjugated antibody of any one of claims 5 to 9, wherein the agent is conjugated to the AB via a linker.

11. The conjugated antibody of claim 10, wherein the linker with a structure of formula (II): ##STR00027## wherein R3 is an agent attached to formula (II) where the point of attachment is a nitrogen, sulfur, oxygen, or carbon atom; and wherein R2 is a moiety attached to formula (II) wherein the point of attachment is selected from the group consisting of: a chlorine group, an iodine group, a bromine group, and a thiol group.

12. The conjugated antibody of claim 10, wherein the linker is a cleavable linker.

13. The conjugated antibody of claim 10, wherein the linker is a non-cleavable linker.

14. The conjugated antibody of claim 5, wherein the agent is a detectable moiety.

15. The conjugated antibody of claim 14, wherein the detectable moiety is a diagnostic agent.

16. The conjugated antibody of any one of claims 5 to 15, wherein the mammalian PSMA is a human PSMA and cynomolgus monkey PSMA.

17. The conjugated antibody of claim 5, wherein the agent is conjugated to the AB via a linker, and wherein the linker and the toxin have the structure of formula (III): ##STR00028## wherein R2 is a point of attachment to the AB.

18. The conjugated antibody of any one of claims 5 to 17, wherein the agent is conjugated to a thiol group on the AB.

19. The conjugated antibody of claim 18, wherein the thiol group is a cysteine side chain thiol group.

20. The conjugated antibody of claim 19, wherein the cysteine residue of the conjugated thiol group is at Kabat position 328 of the AB.

71. A conjugated antibody comprising: an antibody or antigen binding fragment thereof (AB) that specifically binds to mammalian PSMA, wherein the AB comprises the VH CDR1 amino acid sequence SYDMH (SEQ ID NO: 7); the VH CDR2 amino acid sequence VIWYDGSNKYYADSLKG (SEQ ID NO: 8); the VH CDR3 amino acid sequence VIAARTFYYYGMDV (SEQ ID NO: 9); the VL CDR1 amino acid sequence RSSQSLLHSDGYNYLD (SEQ ID NO: 1); the VL CDR2 amino acid sequence LGSNRAS (SEQ ID NO: 2); and the VL CDR3 amino acid sequence MQALQTPWT (SEQ ID NO: 3); and an agent conjugated to the AB, wherein the agent comprises a molecule having a structure of formula (I): ##STR00029## wherein R1 is a hydrogen or a C.sub.1-6 alkyl group; wherein R is selected from the group consisting of: a hydrogen, a C.sub.1-6 alkyl, a linker, or a group X1-Y1-* wherein * is the point of attachment to the nitrogen; and wherein Y1 is an oxycarbonyl group and X1 is a C.sub.1-6 alkyl group, a 9-fluorenylmethyl group, a benzyl group, or a tert-butyl group.

22. The conjugated antibody of claim 21, wherein the AB comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 29.

23. The conjugated antibody of claim 21 or claim 22, wherein the agent is conjugated to the AB via a linker.

24. The conjugated antibody of claim 23, wherein the linker with a structure of formula (II): ##STR00030## wherein R3 is point of attachment to the molecule of formula (I); and wherein R2 is the point of attachment to the AB.

25. The conjugated antibody of claim 23, wherein the linker and the agent has a structure of formula (III): ##STR00031## wherein R2 is the point of attachment is to the AB.

26. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 4, or the conjugated antibody of any one of claims 5 to 25, and a carrier.

27. The pharmaceutical composition of claim 26 comprising an additional agent.

28. The pharmaceutical composition of claim 27, wherein the additional agent is a therapeutic agent.

29. An isolated nucleic acid molecule encoding the isolated antibody of any one of claims 1 to 4.

30. A vector comprising the isolated nucleic acid molecule of claim 29.

31. A method of producing an antibody by culturing a cell under conditions that lead to expression of the antibody, wherein the cell comprises the nucleic acid molecule of claim 29 or the vector of claim 30.

32. A method of manufacturing an antibody that binds PSMA, the method comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the antibody of any one of claims 1 to 4 under conditions that lead to expression of the antibody; and (b) recovering the antibody.

33. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in which diseased cells express PSMA, comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 4, the conjugated antibody of any one of claims 5 to 25, or the pharmaceutical composition of claim 26 to a subject in need thereof.

34. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease associated with cells expressing PSMA comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 4, the conjugated antibody of any one of claims 5 to 25, or the pharmaceutical composition of claim 26 to a subject in need thereof.

35. The method of claim 33 or claim 34, wherein the disorder or disease associated with cells expressing PSMA is cancer.

36. The method of claim 35, wherein the cancer is a prostate cancer or a metastatic castration-resistant prostate carcinoma.

37. A method of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian PSMA comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 4, the conjugated antibody of any one of claims 5 to 25, or the pharmaceutical composition of claim 26 to a subject in need thereof.

38. A method of inhibiting, blocking, or preventing the binding of a natural ligand or receptor to mammalian PSMA, comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 4, the conjugated antibody of any one of claims 5 to 25, or the pharmaceutical composition of claim 26 to a subject in need thereof.

39. The method of any one of claims 33 to 38, wherein the method comprises administering an additional agent.

40. The method of claim 39, wherein the additional agent is a therapeutic agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1 presents the Tms of the two anti-PSMA antibodies: Fab Tm of cHv75-2a11.G1(L328C)k was 81.0° C., and Fab Tin of cHv75-2a7.G1(C99Y;L128C)k was 73.6° C.

[0080] FIG. 2 presents the binding affinities of the two anti-PSMA antibodies to hPSMA by FACS.

[0081] FIGS. 3a and 3b present the binding affinities of the two anti-PSMA antibodies to cyno and human PSMA by ELISA (FIG. 3a) and FACS (FIG. 3b).

[0082] FIG. 4 presents conjugations of the two anti-PSMA antibodies do not affect antibody's binding affinity to PSMA.

[0083] FIG. 5 presents PK studies indicating the stability of the two conjugated anti-PSMA antibodies.

[0084] FIG. 6a presents exemplary pharmacokinetic properties of ADCs of the present disclosure.

[0085] FIG. 6b present in vitro cytotoxicity of the two conjugated anti-PSMA antibodies in MDA Pca 2b cells.

[0086] FIGS. 7a and 7b presents in vivo toxicity of the two conjugated anti-PSMA antibodies in a LAPC9AI tumor mouse model. FIG. 7a shows tumor regression after administrating ADCs into the subcutaneous human prostate carcinoma model LAPC9AI in CB17 SCID mice. FIG. 7b shows the minimum dose of ADCs for tumor regression (mg/kg).

[0087] FIGS. 8a and 8b presents in vivo toxicity of the two conjugated anti-PSMA antibodies in a LNCaP tumor mouse model. FIG. 7a shows tumor regression after administrating ADCs into the subcutaneous human prostate carcinoma model LNCaP in CB17 SCID mice. FIG. 8b shows the minimum dose of ADCs for tumor regression (mg/kg).

DETAILED DESCRIPTION OF THE INVENTION

[0088] The present disclosure provides monoclonal antibodies (mAbs) and anti-PSMA drug conjugates that specifically bind PSMA.

[0089] In some embodiments, a target-binding moiety to which compounds of the present disclosure can be conjugated include anti-PSMA antibodies, examples of which are described in the sequences below:

TABLE-US-00001 TABLE 1 VL CDR Amino Acid Sequences VL CDR1 VL CDR2 VL CDR3 (SEQ ID (SEQ ID (SEQ ID Antibody NO:) NO:) NO:) [Ag]AB-4 SEQUENCE SEQUENCE SEQUENCE cHv75- (SEQ ID (SEQ ID (SEQ ID 2a11.G1 NO: 1) NO: 2) NO: 3) (L328C)k RSSQSLLH LGSNRAS MQALQTPWT SDGYNYLD [Ag]AB-5 SEQUENCE SEQUENCE SEQUENCE cHv75- (SEQ ID (SEQ ID (SEQ ID 2a7.G1(C99Y; NO: 4) NO: 5) NO: 6) L328C)k RASQGISNWLA AASSLQS QQANSFPLT

TABLE-US-00002 TABLE 2 VH CDR Amino Acid Sequences VL CDR1 VL CDR2 VL CDR3 (SEQ ID (SEQ ID (SEQ ID Antibody NO:) NO:) NO:) [Ag]AB-4 SEQUENCE SEQUENCE SEQUENCE cHv75- (SEQ ID (SEQ ID (SEQ ID 2a11.G1 NO: 7) NO: 8) NO: 9) (L328C)k SYDMH VIWYDGS VIAARTF NKYYA YYYGMDV DSLKG [Ag]AB-5 SEQUENCE SEQUENCE SEQUENCE 75- (SEQ ID (SEQ ID (SEQ ID 2a7.G1 NO: 10) NO: 11) NO: 12) (C99Y; NYWMS NIKKDGS EIQLYLQH L328C)k EKFYVD SVKG

TABLE-US-00003 TABLE 3 VL FR Amino Acid Sequences VL FR1 VL FR2 VL FR3 VL FR4 (SEQ ID (SEQ ID (SEQ ID (SEQ ID Antibody NO:) NO:) NO:) NO:) [Ag]AB-4 SEQUENCE SEQUENCE SEQUENCE SEQUENCE 75- (SEQ ID (SEQ ID (SEQ ID (SEQ ID 2a1l.G1 NO: 13) NO: 14) NO: 15) NO: 16) (L32 DIVMTQS WYLQKS GVPDRFS FGQGTK 8C)k PLSLPVT GQSPQ GSGSGTD VEIKR PGEPASI LLIY FTLKISR SC VEAEDVG VYYC [Ag]AB-5 SEQUENCE SEQUENCE SEQUENCE SEQUENCE cHv75- (SEQ ID (SEQ ID (SEQ ID (SEQ ID 2a7.G1 NO: 17) NO: 18) NO: 19) NO: 20) (C99 DIQMTQ WYQQKPG GVPSRFS FGGGTK Y;L328C)k SPSSVS KAPKLL GSGSGTD VEIKR ASVGGR IY FTLTISN VTITC LQPEDFA SYYC

TABLE-US-00004 TABLE 4 VH FR Amino Acid Sequences VH FR1 VH FR2 VH FR3 VH FR4 (SEQ ID (SEQ ID (SEQ ID (SEQ ID Antibody NO:) NO:) NO:) NO:) [Ag]AB-4 SEQUENCE SEQUENCE SEQUENCE SEQUENCE cHv75- (SEQ ID (SEQ ID (SEQ ID (SEQ ID 2a11.G1 NO: 21) NO: 22) NO: 23) NO: 24) (L3 QVQLVES WVRQA RFTISRD WGQGTT 2SC)k GGGVVQP PGKGL NSKNTLY VTVSS GRSLRLS EWVA LQMNSLR CAASGFT AEDTAVY FS YCAR [Ag]AB-5 SEQUENCE SEQUENCE SEQUENCE SEQUENCE 75- (SEQ ID (SEQ ID (SEQ ID (SEQ ID 2a7.G1 NO: 25) NO: 26) NO: 27) NO: 28) (C99Y; EVQLVE WVRQA RFTISR WGQGTL L328C)k SGGGLV PGKGL DNAKNS VTVSS QPGGSL EWVA LYLQIN RLSCAA SLRAED SGITFS TAMYYC AR

TABLE-US-00005 TABLE 5 VL Domain Amino Acid Sequences Variable region (double underline), constant region (dotted underline) Antibody VL (SEQ ID NO:) [Ag]A SEQUENCE B-4 (SEQ ID NO: 29) cHv75 [00004] — [00005] 2a11.G [00006] 1(L328 [00007] C)k [Ag]A SEQUENCE B-5 (SEQ ID NO: 30) cHv75 [00008] — [00009] 2a7.G1 [00010] (C99Y; [00011] L328 C)k

TABLE-US-00006 TABLE 6 VH Domain Amino Add Sequences Variable region (double underline), constant region (dotted underline) Antibody VH (SEQ ID NO:) [Ag]A SEQUENCE B-4 (SEQ ID NO: 31) cHv7 [00012] 5- [00013] 2all. [00014] G1(L [00015] 328C) [00016] k [00017] [00018] [Ag]A SEQUENCE B-5 (SEQ ID NO: 32) cHv7 [00019] 5- [00020] 2a7.G [00021] 1(C99 [00022] Y;L3 [00023] 28C)k [00024] [00025]

TABLE-US-00007 TABLE 7 VL Nucleic Acid Sequences Antibody Nucleotide sequences [Ag]A SEQUENCE B-4 (SEQ ID NO: 33) cHv7 GATATTGTGATGACTCAGTC 5- TCCACTCTCCCTGCCCGTCA 2a11. CCCCTGGAGAGCCGGCCTCC G1 ATCTCCTGCAGGTCTAGTCA (L328C) GAGCCTCCTGCATAGTGATG k GATACAACTATTTGGATTGG TACCTGCAGAAGTCAGGGCA GTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTT CAGTGGCAGTGGATCAGGCA CAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGA TGTTGGGGTTTATTACTGCA TGCAAGCTCTACAAACTCCG TGGACGTTCGGCCAAGGGAC CAAGGTGGAAATCAAACGGA CTGTCGCTGCACCATCTGTC TTCATCTTCCCGCCATCTGA TGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTG CTGAATAACTTCTATCCCAG AGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAA TCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAG CAAAGCAGACTACGAGAAAC ACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAG CTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGT [Ag]A SEQUENCE B-5 (SEQ ID NO: 34) cHv7 GACATCCAGATGACCCAGTC 5- TCCTTCTTCCGTGTCTGCAT 2a7.G CTGTAGGAGGCAGAGTCACC 1(C99 ATCACTTGTCGGGCGAGTCA Y;L3 GGGTATTAGCAACTGGTTAG 28) CCTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAACTCCT GATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCA AGGTTCAGCGGCAGTGGATC TGGGACAGATTTCACTCTCA CCATCAGCAACCTGCAGCCT GAAGATTTTGCAAGTTACTA TTGTCAACAGGCTAACAGTT TCCCCCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAA ACGGACTGTCGCTGCACCAT CTGTCTTCATCTTCCCGCCA TCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGT GCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCC TCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGA CAGCAAGGACAGCACCTACA GCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGA GAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGT GT

TABLE-US-00008 TABLE 8 VH Nucleic Acid Sequences Antibody Nucleotide sequences [Ag]A SEQUENCE B-4 (SEQ ID NO: 35) cHv7 CAGGTGCAGCTGGTGGAGTC 5- TGGGGGAGGCGTGGTCCAGC 2a11. CTGGGAGGTCCCTGAGACTC G1(L TCCTGTGCAGCGTCTGGATT 328C) CACCTTCAGTAGCTATGACA k TGCACTGGGTCCGCCAGGCT CCAGGCAAGGGGCTGGAGTG GGTGGCAGTTATTTGGTATG ATGGAAGTAATAAATACTAT GCAGACTCCTTGAAGGGCCG ATTCACCATCTCCAGAGACA ATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTCAG AGCCGAGGACACGGCTGTGT ATTACTGTGCGAGGGTTATA GCAGCTCGTACCTTCTACTA CTACGGTATGGACGTCTGGG GCCAAGGGACCACGGTCACC GTCTCCTCAGCATCCACCAA GGGCCCATCGGTCTTCCCCC TGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGG CGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGC ACCCAGACCTACATCTGCAA CGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAAA GTTGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCAC CGTGCCCAGCACCTGAACTC CTGGGGGGACCGTCAGTCTT CCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGG CGTGGAGGTGCATAATGCCA AGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCT GCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGG TGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAA CCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCC AGCGACATCGCCGTGGAGTG GGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTATA GCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAA CGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA [Ag]A SEQUENCE B-5 (SEQ ID IIO: 36) cHv7 GAGGTGCAGCTGGTGGAGTC 5- TGGGGGAGGCTTGGTCCAGC 2a7.G CTGGGGGGTCCCTGAGACTC 1(99 TCCTGTGCAGCCTCTGGAAT Y;L3 CACCTTTAGTAATTATTGGA 28C) TGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGACTGGAGTG GGTGGCCAACATAAAGAAAG ATGGAAGTGAGAAATTCTAT GTGGACTCTGTGAAGGGCCG ATTCACCATCTCCAGAGACA ACGCCAAGAACTCACTGTAT CTGCAAATCAACAGCCTGAG AGCCGAGGACACGGCTATGT ATTACTGTGCGAGAGAAATA CAGCTATACCTGCAGCACTG GGGCCAGGGCACCCTGGTCA CCGTCTCCTCAGCATCCACC AAGGGCCCATCGGTCTTCCC CCTGGCACCCTCCTCCAAGA GCACCTCTGGGGGCACAGCG GCCCTGGGCTGCCTGGTCAA GGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGG GCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGA AAGTTGAGCCCAAATCTTGT GACAAAACTCACACATGCCC ACCGTGCCCAGCACCTGAAC TCCTGGGGGGACCGTCAGTC TTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCT CCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCA AGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGG AGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCAC CGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGC CTGCCCAGCCCCCATCGAGA AAACCATCTCCAAAGCCAAA GGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTA TAGCAAGCTCACCGTGGACA AGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACA ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCGGGTAA A

EXAMPLES

EXAMPLE 1. Generation and Characterization of Anti-PSMA Antibodies

[0090] To express PSMA antibodies recombinantly in transfected Chinese hamster ovary (CHO) cells, PSMA antibody variable heavy and light chain sequences were cloned into plasmids constructs upstream of the human heavy chain IgG1 and human light chain Igκ constant regions respectively. The complete PSMA antibody human heavy chain and light chain cassettes were cloned downstream of a promoter/enhancer in a cloning vector. A polyadenylation site was included downstream of the antibody coding sequence. The recombinant PSMA antibody expressing constructs were transfected into CHO cells. The protein A purified PSMA antibodies secreted from recombinant CHO cells were evaluated for binding to cell surface PSMA by flow cytometry and by biacore.

[0091] The purified antibodies were subsequently characterized by SDS-PAGE, SEC, CE-SDS, DSC, binding affinity determination, and paralog/homolog binding assessment. FIG. 1 showed DSC assays indicating the Tms of the two anti-PSMA antibodies: Fab Tm of cHv75-2a11.G1(L328C)k was 81.0° C., and Fab Tm of cHv75-2a7.G1(C99Y;L328C)k was 73.6° C. FIG. 2 indicated both antibodies exhibiting good binding affinities on hPSMA. Human and cyno PSMA share 97% homology. The two anti-PSMA antibodies, cHv75-2a1 (L328C)k and cHv75-2a11.G1(1,328C)k, showed similar binding affinities on cyno and human PSMA by ELISA & Biacore (FIG. 3) and FACS (FIG. 3b), but not mouse PSMA.

EXAMPLE 2. Conjugation and Characterization of Anti-PSMA Antibodies

[0092] Flow cells 1 and 2 (FC1 and FC2) were coupled to Protein A/G using the automated immobilization wizard (500 nM Protein A/G in pH 4.5 glycine buffer). Final RUs were 1127 and 1272 respectively.

[0093] The strategy was to immobilize the mAbs (25 nM) on FC2 followed by injection of Human PSMA dimer (DT1596) or Cyno PSMA dimer (DT1618) on FC1 and FC2 diluted in HBS-EP buffer at various concentrations (500 nM-31.25 nM).

[0094] FC1 was used as reference channel.

[0095] Table A showed ADC Yield of the two antibodies.

TABLE-US-00009 TABLE A % ADC ADC Name DAR Aggregate Yield (%) cHv75-2a7.G1(C99Y; 1.86 1.13 93.3 L328C)k-AGL-01332-93 cHv75-2a11.G1(L328C)k- 1.93 0.93 99.6 AGL-01332-93

[0096] FIG. 4 compared binding affinities of anti-PSMA antibodies and conjugated antibodies to PSMA, indicating that conjugations do not affect PSMA binding.

[0097] AGS75 ADC was dosed at 5 mg/kg as a single intravenous bolus injection on day 0 into female CD17/SCID non-tumor bearing mice. Blood samples were collected at different time points starting 2 minutes post injection up to 21 days after dosing. Serum was collected immediately after complete clotting and stored frozen until the analysis.

[0098] The PK ECL followed a standard sandwich ELISA technique, with PSMA protein being used as the capture protein. In brief, assay plates (Standard MSD plates) were coated with 50 μl of PSMA at a concentration of 1 μg/ml and incubated overnight at 4° C. On day 2, the coating solution was washed with PBS/0.05% Tween20 wash buffer using the plate washer. 150 μL of blocking buffer was added and incubated at room temp for 1 hour followed by 3 washes with 300 μl/well of PBS/0.05% Tween20 using the plate washer. Serially diluted standard and serum study samples are pipetted into the wells. The 12-point standard curve in 1% Mouse; serum samples tested at 1:16200 dilution, ran in duplicates (50 μl/well). The controls were also added in duplicate. The plates were covered and incubated for 1 hour at room temp, then washed 3 times to remove the excess unbound substances and 50 μl/well of MSD Anti-Human IgG3 SulfoTag detection antibody for total protein and 50 μl/well of SG15.22 for ADC added in assay buffer. The plates were covered, incubated for 1 hour at room temp, washed 3 times. For total assay, 150 μl/well of MSD Read buffer (diluted to 2×with D.I. water) is added to the wells. The plates were then read on the MSD Meso Sector S600. For ADC assay, 50 μl/well of diluted MSD Streptavidine sulfa-tag was added. The plates were covered and incubated for 1 hour at room temp, then washed 3 times to remove the excess unbound detection antibody and 150 μl/well of MSD Read buffer (diluted to 2×with D.I. water) is added to the wells. The plates were then read on the MSD Meso Sector S600 and analyzed via MSD Discovery Workbench software.

[0099] FIG. 5 showed stability of ADCs. Table B and FIG. 6a showed Pharmacokinetic Study of AGS-75 ADC, in CB17/SCID non-tumor bearing mice. Minimal deconjugation and long half-lives were observed with both ADCs, and 2a7 and 2a11 exhibit similar exposure and PK properties.

TABLE-US-00010 TABLE B AUC.sub.last AUC.sub.inf CL C.sub.max (day * (day * (mL/ V.sub.ss (μg/ t.sub.1/2 μg/ μg/ day/ (mL/ ADC Assay mL) (day) mL) ml) kg) kg) cHv75- ADC 133 15.5 812 1351 83.1 3.70 2a7.G1(C99Y; TAb 140 16.5 927 1599 74.7 3.12 L328C) k-AGL-01332-93 cHv75- ADC 141 11.5 783 1081 77.1 4.62 2a11.G1(L328C) TAb 164 11.7 919 1282 65.8 3.90 k-AGL-01332-93

EXAMPLE 3. In vitro Cytotoxicity of Conjugated Anti-PSMA Antibodies

[0100] To assess in vitro cytotoxicity with PSMA directed 01332-93 ADC, MDA Pca 2b cells were plated at 5000 cells/well in F-12 media (Gibco) with supplements in 96 well plates. After overnight culture at 37 degrees ADC were titrated into the cultures starting at 5 ug/mL. Cells were cultured with ADC for 6 days and cell viability was assessed by Cell Titre Glo (Promega) assay after 10′ incubation. Luminescence was determined on a Synergy plate reader (BioTek). % Survival vs. ADC concentration curves and EC50s were calculated with Graph Pad Prism software.

[0101] FIG. 6b indicated both ADCs exhibited similar and potent cytotoxicity.

EXAMPLE 4. In vivo Efficacy of Conjugated Anti-PSMA Antibodies in a LAPC9AI Tumor Mouse Model

[0102] Two to five pieces of LAPC9AI or LNCaP tumors were implanted subcutaneously per male CB17/SCID or NSG mice 4-6 weeks of age. When the average tumor volumes reached approximately 200 mm.sup.3, mice were size matched and randomized into treatment and control groups before giving a single dose of AGS75 ADC intravenously at 2.5 mg/kg, 4 mg/kg and 6 mg/kg for each treatment group. Tumor size was determined by external caliper measurement twice a week.

[0103] A statistical analysis of the tumor volume data was performed using the Kruskal-Wallis test and the implementation of the Kruskal-Wallis test was carried out using the parametric ANOVA F-test on the ranks of the data. The percent tumor growth inhibition in each treated group versus a control group was calculated as [(Control−Control baseline)−(Treated−Treated baseline)]/(Control−Control baseline)×100%. The percent of tumor regression was defined as (Treated baseline-Treated)/Treated baseline×100%.

[0104] FIG. 7a showed tumor regression after administrating ADCs into the subcutaneout human prostate carcinoma model LAPC9AI in CB17 SCID mice. FIG. 7h showed the minimum dose of ADCs for tumor regression (mg/kg).

EXAMPLE 5. In Vivo Efficacy of Conjugated Anti-PSMA Antibodies in a LNCaP Tumor Mouse Model

[0105] Two to five pieces of LAPC9AI or LNCaP tumors were implanted subcutaneously per male CB17/SCID or NSG mice 4-6 weeks of age. When the average tumor volumes reached approximately 200 mm.sup.3, mice were size matched and randomized into treatment and control groups before giving a single dose of AGS75 ADC intravenously at 2.5 mg/kg, 4 mg/kg and 6 mg/kg for each treatment group. Tumor size was determined by external caliper measurement twice a week.

[0106] A statistical analysis of the tumor volume data was performed using the Kruskal-Wallis test and the implementation of the Kruskal-Wallis test was carried out using the parametric ANOVA F-test on the ranks of the data. The percent tumor growth inhibition in each treated group versus a control group was calculated as [(Control−Control baseline)−(Treated−Treated baseline)]/(Control−Control baseline)×100%. The percent of tumor regression was defined as (Treated baseline−Treated)/Treated baseline×100%.

[0107] FIG. 8a showed tumor regression after administrating ADCs into the subcutaneout human prostate carcinoma model LNCaP in CB17 SCID mice. FIG. 8b showed the minimum dose of ADCs for tumor regression (mg/kg).

EXAMPLE 6. In Vivo Toxicity Study of Conjugated Anti-PSMA Antibodies in a Cynomolgos Model

[0108] To determine on-target and off-target toxicity profile of cHv75-2a11.G1(L328C)k-AGL-01332-93 and potential recovery.

[0109] Groups: control, cHv75-2a11.G1(L1328C)k-AGL-01332-93 at 9 & 12 mg/kg

[0110] Animals on study: n=4/dose level (2/gender), 1/gender/dose level at each the dosing and recovery phase necropsies

[0111] endpoints: body weight, food consumption, clinical observations, clinical pathology, urinalysis, BA/TK samples

[0112] Postmortem endpoints: gross necropsy, anatomic pathology (terminal and recovery)

Other Embodiments

[0113] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following.