1. Patent Search
  2. Details

ANTI-SLC34A2 ANTIBODIES, ANTIBODY DRUG CONJUGATES, AND METHODS OF USE THEREOF

20230073692 · 2023-03-09

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention relates generally to antibodies that bind SLC34A2, and methods of making and using these anti-SLC34A2 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 SLC34A2, wherein the AB specifically binds human SLC34A2 and cynomolgus monkey SLC34A2, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1 amino acid sequence SYVMH (SEQ ID NO: 43); a VH CDR2 amino acid sequence GVSSSGDSTFYVDSVKG (SEQ ID NO: 44); a VH CDR3 amino acid sequence GGITGAPLVFDI (SEQ ID NO: 45); a VL CDR1 amino acid sequence RASQSISRFLN (SEQ ID NO: 37); a VL CDR2 amino acid sequence VTSSLQS (SEQ ID NO: 38); and a VL CDR3 amino acid sequence QQSYNTPIT (SEQ ID NO: 39).

    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: 67, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 67.

    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′).sub.2 fragment, a scFv, and a scAb.

    4. The isolated antibody of any one of claims 1 to 3, wherein the AB specifically binds human SLC34A2.

    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 (I): ##STR00024## 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.1-6alkyl 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): ##STR00025## 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 an moiety attached to formula (II) wherein the point of attachment is selected from the group consisting of: a chlorine group, a 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 SLC34A2 is a human SLC34A2 and cynomolgus monkey SLC34A2.

    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): ##STR00026## 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.

    21. A conjugated antibody comprising: an antibody or antigen binding fragment thereof (AB) that specifically binds to mammalian SLC34A2, wherein the AB comprises the VH CDR1 amino acid sequence SYVMH (SEQ ID NO: 43); a VH CDR2 amino acid sequence GVSSSGDSTFYVDSVKG (SEQ ID NO: 44), a VH CDR3 amino acid sequence GGITGAPLVFDI (SEQ ID NO: 45); a VL CDR1 amino acid sequence RASQSISRFLN (SEQ ID NO: 37); a VL CDR2 amino acid sequence VTSSLQS (SEQ ID NO: 38); and a VL CDR3 amino acid sequence QQSYNTPIT (SEQ ID NO: 39); and an agent conjugated to the AB, wherein the agent comprises a molecule having a structure of formula (I): ##STR00027## 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: 67, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 67.

    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): ##STR00028## 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): ##STR00029## 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 SLC34A2, 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 SLC34A2 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 SLC34A2 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 SLC34A2 is cancer.

    36. The method of claim 35, wherein the cancer is an ovarian cancer, a lung cancer, a non-small cell lung cancer (NSCLC), a small cell lung cancer (SCLC), a thyroid cancer, an endometrial cancer, a breast cancer, a Her2-negative breast cancer, a triple-negative breast cancer (TNBC), an estrogen receptor-positive breast cancer, or a kidney cancer.

    37. A method of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian SLC34A2 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 SLC34A2, 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

    [0078] FIG. 1 presents the Tms of the two anti-SLC34A2 antibodies.

    [0079] FIG. 2 presents the two anti-SLC34A2 antibodies and ADCs do not bind to other SLC34 Family Members.

    [0080] FIGS. 3a and 3b presents PK studies indicating the stability of the two conjugated anti-SLC34A2 antibodies, OT1 is more stable than OT2.

    [0081] FIG. 4 presents in vitro cytotoxicity of the two conjugated anti-SLC34A2 antibodies in target-positive OVCAR3 cells. OT #1 and OT #2 kill target-positive OVCAR3 cells with high potency, with more than 10× better EC50 compared to the benchmark ADC. OT #1 has less off-target activity than OT #2.

    [0082] FIGS. 5a and 5b presents in vivo toxicity of the two conjugated anti-SLC34A2 antibodies in an Ovarian OVCAR3 mouse model.

    [0083] FIG. 6 presents in vivo toxicity of the two conjugated anti-SLC34A2 antibodies in an Ovarian PDX Model AG-OV37 mouse model

    [0084] FIG. 7 presents OT #1 Inhibits Growth Of Human Lung Cancer Model NCI-H441.

    [0085] FIG. 8 presents OT #1 Inhibits Growth Of Human Lung Cancer Model RERF-LC-Ad1.

    [0086] FIG. 9 presents OT #1 Exhibits Favorable PK Properties In Rats.

    DETAILED DESCRIPTION OF THE INVENTION

    [0087] The present disclosure provides monoclonal antibodies (mAbs) and anti-SLC34A2 drug conjugates that specifically bind SLC34A2.

    [0088] In some embodiments, a target-binding moiety to which compounds of the present disclosure can be conjugated include anti-SLC34A2 antibodies, examples of which are described in the sequences below:

    TABLE-US-00001 TABLE 9 VL CDR Amino Acid Sequences VL CDR1 VL CDR2 VL CDR3 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) [Ag]AB-2 SEQUENCE SEQUENCE SEQUENCE cHv83-3a23.G1 (SEQ ID NO: 37) (SEQ ID NO: 38) (SEQ ID NO: 39) (L328C)k RASQSISRFLN VTSSLQS QQSYNTPIT [Ag]AB-3 SEQUENCE SEQUENCE SEQUENCE cHv83-1b15.G1 (SEQ ID NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42) (L328C)k RASQSIGTFLN VASSLQS QQSYSVPIT

    TABLE-US-00002 TABLE 10 VH CDR Amino Acid Sequences VH CDR1 VH CDR2 VH CDR3 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) [Ag]AB-2 SEQUENCE SEQUENCE SEQUENCE cHv83-3a23.G1 (SEQ ID NO: 43) (SEQ ID NO: 44) (SEQ ID NO: 45) (L328C)k SYVMH GVSSSGDSTFYVDSVKG GGITGAPLVFDI [Ag]AB-3 SEQUENCE SEQUENCE SEQUENCE cHv83-1b15.G1 (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) (L328C)k SHIMY GISSNGLSSYYVDSVKG GGRDRVPAVFDY

    TABLE-US-00003 TABLE 11 VL FR Amino Acid Sequences VL FR1 VL FR2 VL FR3 VL FR4 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) [Ag]AB-2 SEQUENCE SEQUENCE SEQUENCE SEQUENCE cHv83-3a23.G1 (SEQ ID NO: 49) (SEQ ID (SEQ ID NO: 51) (SEQ ID (L328C)k DIQMTQSPSSLSASV NO: 50) GVPSRFSGSGSGTDF NO: 52) GDRVTITC WYQQKPGK TLTISSLQPEDFATY FGQGTR APKVLIY YC LEIKR [Ag]AB-3 SEQUENCE SEQUENCE SEQUENCE SEQUENCE cHv83-1b15.G1 (SEQ ID NO: 53) (SEQ ID (SEQ ID NO: 55) (SEQ ID (L328C)k DIQMTQSPSSLSASI NO: 54) GVPSRFIGSGSGTDF NO: 56) GDRVTITC WYQQKPGK TLTISSLQPEDFATY FGQGTR APKVLIY YC LEIKR

    TABLE-US-00004 TABLE 12 VH FR Amino Acid Sequences VH FR2 VH FR4 VH FR1 (SEQ ID VH FR3 (SEQ ID Antibody (SEQ ID NO:) NO:) (SEQ ID NO:) NO:) [Ag]AB-2 SEQUENCE SEQUENCE SEQUENCE SEQUENCE cHv83-3a23.G1 (SEQ ID NO: 57) (SEQ ID (SEQ ID NO: 59) (SEQ ID (L328C)k EVQLVESGGGLVQPG NO: 58) RFTISRDNSKNTLYL NO: 60) GSLRLSCAASGFTFS WVRQAPG QMGSLRAEDMAVYYC WGQGTM KGLEYVS AR VTVSS [Ag]AB-3 SEQUENCE SEQUENCE SEQUENCE SEQUENCE cHv83-1b15.G1 (SEQ ID NO: 61) (SEQ ID (SEQ ID NO: 63) (SEQ ID (L328C)k EVQLVESGGGWVQPG NO: 62) RFTISRDNSKNLLYV NO: 64) GSLRLSCAASGFTFS WVRQAPG HMGSLKPEDMAMYYC WGQGTL KGLEYVS AR VTVSS

    TABLE-US-00005 TABLE 13  VL Domain Amino Acid Sequences  Variable region (double underline), constant region (dotted underline) Antibody VL (SEQ ID NO:) [Ag] SEQUENCE (SEQ ID NO: 67) AB-2 [00004]embedded image cHv83- [00005]embedded image 3a23.G1 [00006]embedded image (L328C)k [Ag] SEQUENCE (SEQ ID NO: 68) AB-3 [00007]embedded image cHv83- [00008]embedded image 1B15.G1 [00009]embedded image (L328C)k

    TABLE-US-00006 TABLE 14  VH Domain Amino Acid Sequences  Variable region (double underline), constant region (dotted underline) Antibody VH (SEQ ID NO:) [Ag] SEQUENCE (SEQ ID NO: 67) AB-2 [00010]embedded image cHv83- [00011]embedded image 3a23.G1 [00012]embedded image (L328C)k [00013]embedded image [00014]embedded image [00015]embedded image [00016]embedded image [Ag] SEQUENCE (SEQ ID NO: 68) AB-3 [00017]embedded image cHv83- [00018]embedded image 1b15.G1 [00019]embedded image (L328C)k [00020]embedded image [00021]embedded image [00022]embedded image [00023]embedded image

    TABLE-US-00007 TABLE 15 VL Nucleic Acid Sequences Anti- body Nucleotide sequences [Ag] SEQUENCE AB-2 (SEQ ID NO: 69) cHv83- GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA 3a23.G1 TCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT (L328C) CAGAGCATTAGCAGGTTTTTAAATTGGTATCAGCAGAAA k CCAGGGAAAGCCCCTAAGGTCCTGATCTATGTTACATCC AGTTTACAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGT GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGT TACATACCCCTATCACCTTCGGCCAAGGGACACGACTGG AGATTAAACGGACTGTCGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCT CTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGG GTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAG TCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGT [Ag] SEQUENCE AB-3 (SEQ ID NO: 70) cHv83- GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA 1b15.G1 TCTATAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT (L328C) CAGAGCATTGGCACCTTTTTAAATTGGTATCAACAAAAA k CCAGGGAAAGCCCCTAAGGTCCTGATCTATGTTGCATCC AGTTTGCAAAGTGGGGTCCCATCAAGGTTCATTGGCAGT GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGT TACAGTGTTCCGATCACCTTCGGCCAAGGGACACGACTG GAGATTAAACGGACTGTCGCTGCACCATCTGTCTTCATC TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGT

    TABLE-US-00008 TABLE 16 VH Nucleic Acid Sequences Anti- body Nucleotide sequences [Ag] SEQUENCE AB-2 (SEQ ID NO: 71) cHv83- GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGC 3a23.G1 CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATT (L328C) CACCTTCAGTAGTTATGTTATGCACTGGGTCCGCCAGGCT k CCAGGGAAGGGACTGGAATATGTTTCAGGTGTTAGTAGTA GTGGGGATAGCACATTTTATGTAGACTCTGTGAAGGGCAG ATTCACCATCTCCAGAGACAATTCCAAGAACACGCTTTAT CTTCAAATGGGCAGCCTGAGAGCTGAGGACATGGCTGTGT ATTACTGTGCGAGAGGGGGTATAACTGGAGCTCCACTGGT TTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCT TCAGCATCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCT TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCTGCCCAG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGC TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA [Ag] SEQUENCE AB-3 (SEQ ID NO: 72) cHv83- GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTGGGTCCAGC 1b15.G1 CGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATT (L328C) CACCTTCAGTAGTCATATTATGTACTGGGTCCGCCAGGCT k CCAGGGAAGGGACTGGAATATGTTTCGGGTATTAGCAGTA ATGGACTTAGCTCATATTATGTTGACTCTGTGAAGGGCAG ATTCACCATCTCCAGAGACAATTCCAAGAATTTACTGTAT GTTCATATGGGCAGCCTGAAACCTGAGGACATGGCTATGT ATTACTGTGCGAGAGGGGGCCGGGATAGAGTGCCAGCTGT CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC TCCGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCT TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCTGCCCAG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGC TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

    EXAMPLES

    Example 1. Generation and Characterization of Anti-SLC34A2 Antibodies

    [0089] To express SLC34A2 antibodies recombinantly in transfected Human embryonic kidney 293 cells, SLC34A2 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 SLC34A2 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 SLC34A2 antibody expressing constructs were transfected into 293 cells. The protein A purified SLC34A2 antibodies secreted from recombinant 293 cells were evaluated for binding to cell surface SLC34A2 by flow cytometry and by Biacore.

    [0090] The purified antibodies were subsequently characterized by SDS-PAGE, SEC, CE-SDS, Differential Scanning Calorimetry (DSC), binding affinity determination, and paralog/homolog binding assessment. FIG. 1 showed DSC assays indicating the Tms of the two anti-SLC34A2 antibodies: Fab Tm of cHv83-3a23.G1(L328C)k was 77.6° C., and Fab Tm of cHv83-1b15.G1(L328C)k was 71.4° C., and OT #1 has superior in vitro thermal stability compared to OT #2. Table 1 shows the two anti-SLC34A2 antibodies bind to recombinant SLC34A2 with high affinity, and cross-reactive in human, cyno, and rats. FIG. 2 shows the anti-SLC34A2 antibodies and ADCs are specifically binding to SLC34A2, but not other SLC34 family proteins.

    TABLE-US-00009 TABLE 1 Anti- SLC34A2 antibody binding affinities on recombinant SLC34A2 Rat 1E Rat 1E Rat 1E OVCAR3 Human SLC34A2 Cyno SLC34A2 Murine SLC34A2 Antibody Bmax Kd (nM) Bmax Kd (nM) Bmax Kd (nM) Bmax Kd (nM) cHv83- 500207 2.278 68788 0.2543 92072 0.1703 11322 0.09035 3a23.G1(L328C)k cHv83- 538044 3.787 129552 1.027 59732 0.9671 15000 0.06311 1b15.G1k(L328C)k cHmLYS-1c3.g1k N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.

    Example 2. Conjugation and Characterization of Anti-SLC34A2 Antibodies

    [0091] Table 2 showed ADC Yield of the two antibodies. OT #1 conjugates to AGL-01332-93 consistently, while OT #2 displays atypical over-conjugation behavior, including high H2.

    TABLE-US-00010 TABLE 2 ADC Yield of the two anti-SLC34A2 antibodies AGL-01332-93 (BrAc) AGL-01332-93Cl (Nal activated) % Antibody n avg DAR Range n avg DAR Range Aggregation OT1: cHv83- 3 1.98 1.90-2.11 2 1.82 1.80-1.84 1.70 3a23.G1(L328C)k OT2: cHv83- 1 2.43 2 2.19 2.17-2.2  1.30 1b15.G1(L328C)k

    [0092] Table 3 showed the binding affinity comparison between the anti-SLC34A2 antibodies and conjugated antibodies to SLC34A2, indicating that conjugation of the OT1 does not affect SLC34A2 binding.

    TABLE-US-00011 TABLE 3 Anti- SLC34A2 antibodies and ADCs binding affinity on recombinant SLC34A2 Rat1E cells expressing SLC34A2 Epitope Human Cynomolgus Rat Antibody bin (nM) (nM) (nM) cHv83-3a23.G1(L328C)k 1  0.5526 0.1698 0.6888 cHv83-3a23.G1(L328C)k- 1  0.4108 0.2268 0.686 AGL-01332-93 (OT#1) cHv83-1b15.G1(L328C)k 2a 0.7214 0.6703 0.2456 cHv83-1b15.G1(L328C)k- 2a 1.759 1.203 0.9127 AGL-01332-93 (OT#2)

    [0093] The PK ECL followed a standard sandwich ELISA technique, with SLC34A2 protein being used as the capture protein. In brief, assay plates (Standard MSD plates) were coated with 50 μl of SLC34A2 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 IgG 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 sulfo-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.

    [0094] FIG. 3 showed stability of ADCs. Table 4. showed Pharmacokinetic Study of AGS-83 ADC in CB17/SCID non-tumor bearing mice. Minimal deconjugation and long half-lives were observed with both ADCs, and OT #1 exhibit better exposure and PK properties over OT #2.

    TABLE-US-00012 TABLE 4 PK Properties of two anti- SLC34A2 ADC candidates. Dose C.sub.max T.sub.1/2 AUC.sub.last AUC.sub.inf CL Vss ADC (mg/kg) Assay (μg/mL) (day) (day*μg/mL) (day*μg/mL) (mL/day/kg) (mL/kg) OT#1: cHv83- 5 ADC 128 ± 15.3 14.6 ± 1.09 672 ± 67.2 1020 ± 148 4.98 ± 0.674 101 ± 7.24 3a23.G1(L328C)k- TAb 142 ± 8.60 15.8 ± 1.60 821 ± 79.7 1350 ± 187 3.76 ± 0.492 83.6 ± 7.15  AGL-01332-93 OT#2: cHv83- 5 ADC 112 ± 6.42  8.27 ± 0.926 436 ± 31.1 .sup. 522 ± 54.4 9.67 ± 0.954 107 ± 6.10 1b15.G1(L328C)k- TAb 121 ± 8.32 10.2 ± 1.72 539 ± 23.1 .sup. 705 ± 72.0 7.15 ± 0.671 101 ± 6.25 AGL-01332-93

    Example 3. In Vitro Cytotoxicity of Conjugated Anti-SLC34A2 Antibodies

    [0095] To assess in vitro cytotoxicity with SLC34A2 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.

    [0096] FIG. 4 indicated OT #1 demonstrates potent in vitro cytotoxicity and is more specific than OT #2.

    Example 4. In Vivo Efficacy of Conjugated Anti-SLC34A2 Antibodies in a OVCAR3 Tumor Mouse Model

    [0097] Two to five pieces of OVCAR3 Ovarian tumors were implanted subcutaneously per female 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 AGS83 ADC intravenously at 2 mg/kg, and 5 mg/kg for each treatment group. Tumor size was determined by external caliper measurement twice a week.

    [0098] 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%.

    [0099] FIG. 5 showed OT #1 And OT #2 Exhibit Potent, Dose-Dependent TGI In Ovarian Model OVCAR3. In OVCAR3, both OT leads induced tumor regression at 5 mg/kg; both OT leads induced TGI that were statistically greater than the benchmark ADC at 2 mg/kg.

    Example 5. In Vivo Efficacy of Conjugated Anti-SLC34A2 Antibodies in an Ovarian PDX Model AG-OV37 Mouse Model

    [0100] Two to five pieces of AG-OV37 Ovarian tumors were implanted subcutaneously per female 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 AGS83 ADC intravenously at 2 mg/kg, and 5 mg/kg for each treatment group. Tumor size was determined by external caliper measurement twice a week.

    [0101] 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%.

    [0102] FIG. 6 showed OT #1 Exhibits More Potent TGI Than OT #2 And Benchmark ADC In Ovarian PDX Model AG-OV37. In AG-OV37, both OT leads induced statistically greater TGI than the benchmark ADC at both 2 and 5 mg/kg; OT #1 induced regression at 2 mg/kg but OT #2 and the benchmark ADC did not.

    Example 6. In Vivo Efficacy of Conjugated Anti-SLC34A2 Antibodies in a Human Lung Cancer NCI-H441 Mouse Model

    [0103] Two to five pieces of NCI-H441 Human Lung cancer cell line were implanted subcutaneously per CB17/SCID 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 AGS83 ADC intravenously at 5 mg/kg for each treatment group. Tumor size was determined by external caliper measurement twice a week.

    [0104] 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)×100/6. The percent of tumor regression was defined as (Treated baseline-Treated)/Treated baseline×100%.

    [0105] FIG. 7 showed OT #1 Inhibits Growth Of Human Lung Cancer Model NCI-H441. In NCI-H441, a single 5 mg/kg dose of OT #1 induced significant tumor growth inhibition and 16% regression compared to the isotype control.

    Example 7. In Vivo Efficacy of Conjugated Anti-SLC34A2 Antibodies in a Human Lung Cancer RERF-LC-Ad1 Mouse Model

    [0106] Two to five pieces of RERF-LC-Ad1 Human Lung cancer cell line were implanted subcutaneously per 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 AGS83 ADC intravenously at 2.5 mg/kg or 5 mg/kg for each treatment group. Tumor size was determined by external caliper measurement twice a week.

    [0107] 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%.

    [0108] FIG. 8 showed OT #1 Inhibits Growth Of Human Lung Cancer Model RERF-LC-Ad1. In RERF-LC-Ad1, OT #1 induced dose-dependent, statistically significant tumor growth inhibition at 2.5 and 5 mg/kg weekly dosing.

    Example 8. Non-GLP Multiple-Dose Toxicity Study of Conjugated Anti-SLC34A2 Antibodies in Rats

    [0109] Objective: Assess toxicity and toxicokinetics of cHv83-3a23.G1(L328C)k-AGL-01332-93 (OT #1)

    TABLE-US-00013 Study Design Dose Group Level Concentration Volume Number of Animals Group Color Route (mg/kg) (mg/mL) (mL/kg) .sup.a Males 1 White IV 0 0 5 5.sup.b + 6.sup.c 2 Yellow IV 10 2 5 5.sup.b + 6.sup.c 3 Green IV 20 4 5 5.sup.b + 6.sup.c 4 Blue IV 40 8 5 5.sup.b + 6.sup.c

    [0110] Measures include In-life: clinical observations, body weight, food consumption, clinical pathology, urinalysis, BA/TK, and Post-mortem: gross pathology, organ weights, anatomic pathology.

    [0111] Study design: 4-week study with no recovery period, with Dose levels: 0, 10, 20, 40 mg/kg

    [0112] Results: Body weight loss (˜10%) on Day 29 and clinical observations of swelling (neck) at 40 mg/kg only. Effects on red cell mass, liver toxicity biomarkers, plasma proteins, and histopathology of bone marrow, kidney, liver, spleen considered potentially reversible (reversibility was not assessed). In general, findings at 40 mg/kg were of greater severity/incidence compared to 10 and 20 mg/kg. ADC localization correlated with areas of blood vasculature and not with histopathology findings. FIG. 9 showed OT #1 Exhibits favorable PK Properties in rats.

    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.