Combination therapy with a PSMA ligand conjugate

Abstract

To provide compositions and methods for inhibiting proliferation of prostate-specific membrane antigen (PSMA)-expressing cancer cells.SOLUTION: A method comprises: contacting PSMA-expressing cancer cells with an antiandrogen; and contacting the PSMA-expressing cancer cells with a PSMA ligand-anticancer agent conjugate.SELECTED DRAWING: None

Description

Background Art

[0001] Prostate cancer is the most common malignancy in men in the United States and the second leading cause of cancer death (Non-Patent Document 1). Localized prostate cancer is usually treated with surgery or radiation, and recurrent disease can be temporarily controlled with androgen ablation (Non-Patent Document 2). However, almost all prostate cancers eventually become hormone resistant and then progress rapidly (Non-Patent Document 3). Hormone-resistant or androgen-independent prostate cancer has generally been found to be resistant to conventional chemotherapy. Excluding palliative care, the only approved chemotherapy is docetaxel in combination with prednisone, and more recently, cabazitaxel (Jevtana®) approved by the FDA, which provide a modest (2.4 months) survival benefit (Non-Patent Document 4; Non-Patent Document 5).

Prior Art Documents

Patent Documents

[0002]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

Patent Document 15

Patent Document 16

Patent Document 17

Patent Document 18

Patent Document 19

Patent Document 20

Patent Document 21

Patent Document 22

Patent Document 23

Non-Patent Document

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non - Patent Document 25

Non - Patent Document 26

Non - Patent Document 27

Non - Patent Document 28

Non - Patent Document 29

Non - Patent Document 30

Non - Patent Document 31

Non - Patent Document 32

Non - Patent Document 33

Non - Patent Document 34

Non - Patent Document 35

Non - Patent Document 36

Non - Patent Document 37

Non - Patent Document 38

Non - Patent Document 39

Non - Patent Document 40

Non - Patent Document 41

Non - Patent Document 42

Non - Patent Document 43

Non-Patent Document 44

Non-Patent Document 45

Non-Patent Document 46

Non-Patent Document 47

Non-Patent Document 48

Non-Patent Document 49

Non-Patent Document 50

Non-Patent Document 51

Non-Patent Document 52

Non-Patent Document 53

Non-Patent Document 54

Non-Patent Document 55

Non-Patent Document 56

Non-Patent Document 57

Non-Patent Document 58

Non-Patent Document 59

Non-Patent Document 60

Non-Patent Document 61

Non-Patent Document 62

Non-Patent Document 63

Non-Patent Document 64

Non-Patent Document 65

Non-Patent Document 66

Non-Patent Document 67

Non-Patent Document 68

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention relates to the surprising discovery that at least in part, an androgen and a therapeutic agent such as an anti-cancer agent or a cytotoxic agent (when bound to a PSMA ligand, referred to herein as a "PSMA ligand-anti-cancer agent conjugate" or a "PSMA ligand-cytotoxic agent conjugate", respectively) conjugated to a prostate-specific membrane antigen (PSMA) ligand (referred to herein as a "PSMA ligand conjugate") act synergistically to inhibit the growth of PSMA-expressing cancer cells. Unexpectedly, this synergistic effect occurs in both androgen-dependent cells and androgen-independent cells. Furthermore, it has been discovered that an mTOR inhibitor and a PSMA ligand conjugate also act synergistically to inhibit the growth of PSMA-expressing cancer cells, particularly androgen-independent cells. Additionally, it has been discovered that prednisone and a PSMA ligand conjugate also act synergistically when used in combination. Thus, provided herein are methods, compositions, and kits for the purpose of inhibiting the growth or killing of PSMA-expressing cells (e.g., cancer cells such as prostate cancer cells) or for the purpose of sensitizing or resensitizing cells capable of expressing PSMA, such as androgen-independent PSMA-expressing cells, to androgen therapy.

Means for Solving the Problems

[0005] Some aspects of the present disclosure provide a method for inhibiting the growth of prostate-specific membrane antigen (PSMA)-expressing cancer cells. This method can include contacting a PSMA-expressing cancer cell with a compound that increases the cell surface expression of PSMA and contacting the PSMA-expressing cancer cell with a PSMA ligand-anti-cancer agent conjugate.

[0006] In some embodiments, the compound that increases the cell surface expression of PSMA is an androgen.

[0007] In some embodiments, the androgen blocks the enzyme cytochrome CYP17. In some embodiments, the androgen blocks the enzyme cytochrome CYP17A1.

[0008] In some embodiments, the antiandrogen is an androgen receptor antagonist.

[0009] In some embodiments, the antiandrogen is abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, galeterone, or ortelone. In some embodiments, the antiandrogen is enzalutamide or abiraterone.

[0010] In some embodiments, the antiandrogen is in an amount effective to upregulate PSMA expression.

[0011] In some embodiments, the step of contacting a PSMA-expressing cancer cell with a compound that increases cell surface expression of PSMA, such as an antiandrogen, is simultaneous with the step of contacting the PSMA-expressing cancer cell with a PSMA ligand-anticancer agent conjugate. In some embodiments, the step of contacting a PSMA-expressing cancer cell with a compound such as an antiandrogen and the step of contacting the PSMA-expressing cancer cell with a PSMA ligand-anticancer agent conjugate are sequential.

[0012] In some embodiments, the step of contacting a PSMA-expressing cancer cell with a compound such as an antiandrogen is prior to the step of contacting the PSMA-expressing cancer cell with a PSMA ligand-anticancer agent conjugate.

[0013] In some embodiments, the step of contacting a PSMA-expressing cancer cell with a PSMA ligand-anticancer agent conjugate is performed within one week of the step of contacting the PSMA-expressing cancer cell with a compound such as an antiandrogen.

[0014] In some embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 3 days. In other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 7 days. In still other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 14 days. In still other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 21 days. In still other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 28 days.

[0015] In some embodiments, the PSMA-expressing cancer cells include androgen-independent PSMA-expressing cancer cells.

[0016] In some embodiments, the PSMA-expressing cancer cells include PSMA-expressing cancer cells that are insensitive to anti-androgen therapy.

[0017] In some embodiments, PSMA-expressing cancer cells that are insensitive to anti-androgen therapy are re-sensitized to anti-androgen therapy after the step of contacting the PSMA-expressing cancer cells with a compound such as an anti-androgen.

[0018] In some embodiments, the PSMA-expressing cancer cells are tumor-derived.

[0019] In some embodiments, the PSMA-expressing cancer cells are PSMA-expressing prostate cancer cells. In other embodiments, the PSMA-expressing cancer cells are non-prostate cancer cells with PSMA expression. In still other embodiments, the PSMA-expressing cancer cells are derived from the neovessels of non-prostate cancer or non-prostate tumors.

[0020] In some embodiments, the PSMA-expressing cancer cells are from a subject. In some embodiments, the subject has metastatic castration-resistant prostate cancer that is progressive. In some embodiments, the subject has received prior chemotherapy with one or more taxanes. In some embodiments, the subject has received prior treatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite prior treatment such as with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite treatment such as with one or more anti-androgens. In some embodiments, the subject has not previously received cytotoxic chemotherapy. In some embodiments, the subject has not received prior treatment with one or more anti-androgens. In some embodiments, the subject has not received prior treatment with enzalutamide or abiraterone. In some embodiments, a subject who has not received prior treatment with an anti-androgen such as enzalutamide or abiraterone has metastatic castration-resistant prostate cancer. In some embodiments, the subject is any subject described herein.

[0021] Other aspects of the disclosure provide methods of inhibiting the growth of prostate-specific membrane antigen (PSMA)-expressing cancer cells. The method can include contacting the PSMA-expressing cancer cells with an mTOR inhibitor and contacting the PSMA-expressing cancer cells with a PSMA ligand-anticancer agent conjugate.

[0022] In some embodiments, the mTOR inhibitor is rapamycin.

[0023] In some embodiments, the mTOR inhibitor is in an amount effective to upregulate PSMA expression.

[0024] In some embodiments, the step of contacting the mTOR inhibitor with the PSMA-expressing cancer cells is simultaneous with the step of contacting the PSMA ligand-anticancer agent conjugate with the PSMA-expressing cancer cells.

[0025] In some embodiments, the step of contacting the mTOR inhibitor with the PSMA-expressing cancer cells and the step of contacting the PSMA ligand-anticancer agent conjugate with the PSMA-expressing cancer cells are sequential.

[0026] In some embodiments, the step of contacting the mTOR inhibitor with the PSMA-expressing cancer cells is before the step of contacting the PSMA ligand-anticancer agent conjugate with the PSMA-expressing cancer cells.

[0027] In some embodiments, the PSMA-expressing cancer cells are contacted with the mTOR inhibitor for at least 7 days.

[0028] In some embodiments, the PSMA-expressing cancer cells include androgen-independent PSMA-expressing cancer cells.

[0029] In some embodiments, the PSMA-expressing cancer cells include PSMA-expressing cancer cells that are insensitive to anti-androgen therapy.

[0030] In some embodiments, the PSMA-expressing cancer cells that are insensitive to anti-androgen therapy are resensitized to anti-androgen therapy after the step of contacting the PSMA-expressing cancer cells with the mTOR inhibitor.

[0031] In some embodiments, the PSMA-expressing cancer cells may be tumor-derived.

[0032] In some embodiments, the PSMA-expressing cancer cells are PSMA-expressing prostate cancer cells. In other embodiments, the PSMA-expressing cancer cells are non-prostate cancer cells with PSMA expression. In still other embodiments, the PSMA-expressing cancer cells are derived from the neovessels of non-prostate cancer or non-prostate tumors.

[0033] In some embodiments, the PSMA-expressing cancer cells are derived from a subject. In some embodiments, the subject has metastatic castration-resistant prostate cancer that is progressive. In some embodiments, the subject has received prior chemotherapy with one or more taxanes. In some embodiments, the subject has received prior treatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite prior treatment such as treatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite treatment such as treatment with one or more anti-androgens. In some embodiments, the subject has not previously received cytotoxic chemotherapy. In some embodiments, the subject has not received prior treatment with one or more anti-androgens. In some embodiments, the subject has not received prior treatment with enzalutamide or prior treatment with abiraterone. In some embodiments, a subject who has not received prior treatment with an anti-androgen such as enzalutamide or abiraterone has metastatic castration-resistant prostate cancer. In some embodiments, the subject is any subject described herein.

[0034] In yet other aspects of the present disclosure, provided is a method of inhibiting the proliferation of prostate-specific membrane antigen (PSMA)-expressing cancer cells. The method can include contacting the PSMA-expressing cancer cells with prednisone and contacting the PSMA-expressing cancer cells with a PSMA ligand conjugate. In some embodiments, the method further includes contacting the PSMA-expressing cancer cells with a compound that increases the cell surface expression of PSMA, such as an anti-androgen.

[0035] In some embodiments, the anti-androgen blocks the enzyme cytochrome CYP17. In some embodiments, the anti-androgen blocks the enzyme cytochrome CYP17A1.

[0036] In some embodiments, the anti-androgen is an androgen receptor antagonist.

[0037] In some embodiments, the antiandrogen is abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, galeterone, or ortelone. In some embodiments, the antiandrogen is enzalutamide or abiraterone.

[0038] In some embodiments, the antiandrogen is in an amount effective to upregulate PSMA expression.

[0039] In some embodiments, the step of contacting PSMA-expressing cancer cells with a compound that increases cell surface expression of PSMA, such as prednisone and / or an antiandrogen, is simultaneous with the step of contacting the PSMA-expressing cancer cells with a PSMA ligand conjugate. In some embodiments, the step of contacting PSMA-expressing cancer cells with a compound such as prednisone and / or an antiandrogen and the step of contacting the PSMA-expressing cancer cells with a PSMA ligand conjugate are sequential.

[0040] In some embodiments, the step of contacting PSMA-expressing cancer cells with a compound such as prednisone and / or an antiandrogen is prior to the step of contacting the PSMA-expressing cancer cells with a PSMA ligand conjugate.

[0041] In some embodiments, the step of contacting the PSMA-expressing cancer cells with a PSMA ligand conjugate is performed within one week of the step of contacting the PSMA-expressing cancer cells with a compound such as an antiandrogen.

[0042] In some embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 3 days. In other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 7 days. In still other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 14 days. In still other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 21 days. In still other embodiments, PSMA-expressing cancer cells are contacted with a compound such as an anti-androgen for at least 28 days.

[0043] In some embodiments, the PSMA-expressing cancer cells include androgen-independent PSMA-expressing cancer cells.

[0044] In some embodiments, the PSMA-expressing cancer cells include PSMA-expressing cancer cells that are insensitive to anti-androgen therapy.

[0045] In some embodiments, the PSMA-expressing cancer cells that are insensitive to anti-androgen therapy are resensitized to anti-androgen therapy after the step of contacting the PSMA-expressing cancer cells with a compound such as an anti-androgen.

[0046] In some embodiments, the PSMA-expressing cancer cells are tumor-derived.

[0047] In some embodiments, the PSMA-expressing cancer cells are PSMA-expressing prostate cancer cells. In other embodiments, the PSMA-expressing cancer cells are non-prostate cancer cells with PSMA expression. In still other embodiments, the PSMA-expressing cancer cells are derived from the neovessels of non-prostate cancer or non-prostate tumors.

[0048] In some embodiments, the PSMA-expressing cancer cells are derived from a subject. In some embodiments, the subject has advanced metastatic castration-resistant prostate cancer. In some embodiments, the subject has received prior chemotherapy with one or more taxanes. In some embodiments, the subject has received prior treatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite prior treatment such as with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite treatment such as with one or more anti-androgens. In some embodiments, the subject has not previously received cytotoxic chemotherapy. In some embodiments, the subject has not received prior treatment with one or more anti-androgens. In some embodiments, the subject has not received prior treatment with enzalutamide or abiraterone. In some embodiments, a subject who has not received prior treatment with an anti-androgen such as enzalutamide or abiraterone is a subject who has metastatic castration-resistant prostate cancer. In some embodiments, the subject is any subject described herein.

[0049] Yet another aspect of the disclosure provides a method for specifically delivering a cytotoxic agent to PSMA-expressing cells of a subject. The method can include administering to the subject a compound that increases cell surface expression of PSMA and administering to the subject a conjugate of a PSMA ligand-cytotoxic agent.

[0050] In some embodiments, the compound that increases cell surface expression of PSMA is an anti-androgen.

[0051] In some embodiments, the anti-androgen blocks the enzyme cytochrome CYP17. In some embodiments, the anti-androgen blocks the enzyme cytochrome CYP17A1.

[0052] In some embodiments, the anti-androgen is an androgen receptor antagonist. In some embodiments, the anti-androgen is abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, galeterone, or orteronel. In some embodiments, the anti-androgen is enzalutamide or abiraterone.

[0053] In some embodiments, the compound that increases cell surface expression of PSMA is an mTOR inhibitor. In some embodiments, the mTOR inhibitor is rapamycin.

[0054] In some embodiments, the step of administering the compound and the step of administering the conjugate of the PSMA ligand-cytotoxic agent are simultaneous. In other embodiments, the step of administering the compound and the step of administering the conjugate of the PSMA ligand-cytotoxic agent are sequential. In some embodiments, the step of administering the compound is before the step of administering the conjugate of the PSMA ligand-cytotoxic agent.

[0055] In some embodiments, the step of administering the conjugate of the PSMA ligand-cytotoxic agent is performed within one week of the step of administering the compound.

[0056] In some embodiments, the PSMA-expressing cells are contacted with the compound for at least 3 days. In other embodiments, the PSMA-expressing cells are contacted with the compound for at least 7 days. In still other embodiments, the PSMA-expressing cells are contacted with the compound for at least 14 days. In still other embodiments, the PSMA-expressing cells are contacted with the compound for at least 21 days. In still other embodiments, the PSMA-expressing cells are contacted with the compound for at least 28 days.

[0057] In some embodiments, the PSMA-expressing cells include androgen-independent PSMA-expressing cancer cells.

[0058] In some embodiments, the PSMA-expressing cells include PSMA-expressing cancer cells that are insensitive to anti-androgen therapy.

[0059] In some embodiments, the PSMA-expressing cells that are insensitive to anti-androgen therapy are resensitized to anti-androgen therapy after the step of administering the compound.

[0060] In some embodiments, the PSMA-expressing cells are tumor-derived.

[0061] In some embodiments, the PSMA-expressing cells are PSMA-expressing prostate cancer cells. In other embodiments, the PSMA-expressing cells are non-prostate cancer cells with PSMA expression. In still other embodiments, the PSMA-expressing cells are derived from the neovessels of non-prostate cancer or non-prostate tumors.

[0062] In some embodiments, the PSMA-expressing cancer cells are subject-derived. In some embodiments, the subject has progressive metastatic castration-resistant prostate cancer. In some embodiments, the subject has received pre-chemotherapy with one or more taxanes. In some embodiments, the subject has received pretreatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite pretreatment such as with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite treatment such as with one or more anti-androgens. In some embodiments, the subject has not previously received cytotoxic chemotherapy. In some embodiments, the subject has not received pretreatment with one or more anti-androgens. In some embodiments, the subject has not received pretreatment with enzalutamide or abiraterone. In some embodiments, a subject who has not received pretreatment with an anti-androgen such as enzalutamide or abiraterone is a subject who has metastatic castration-resistant prostate cancer. In some embodiments, the subject is any subject described herein.

[0063] Still other aspects of the present disclosure provide compounds that increase cell surface expression of PSMA and conjugates of PSMA ligands - anti - cancer agents or conjugates of PSMA ligands - cytotoxic agents. In still other aspects, compositions are provided that include prednisone and a PSMA ligand conjugate provided herein. In some embodiments, the composition further includes a compound that increases cell surface expression of PSMA.

[0064] In some embodiments, the compound that increases cell surface expression of PSMA is an anti - androgen.

[0065] In some embodiments, the anti - androgen blocks the enzyme cytochrome CYP17. In some embodiments, the anti - androgen blocks the enzyme cytochrome CYP17A1.

[0066] In some embodiments, the anti - androgen is an androgen receptor antagonist. In some embodiments, the anti - androgen is abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, galeterone, or oteronel. In some embodiments, the anti - androgen is enzalutamide or abiraterone.

[0067] In some embodiments, the compound that increases cell surface expression of PSMA is an mTOR inhibitor. In some embodiments, the mTOR inhibitor is rapamycin.

[0068] In some embodiments, the composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0069] Still other aspects of the disclosure provide a kit that may include a container containing a compound that increases cell surface expression of PSMA in PSMA-expressing cells and a container containing a conjugate of a PSMA ligand-anticancer agent or a conjugate of a PSMA ligand-cytotoxic agent. In other aspects, a kit is provided that includes a container containing prednisone and a container containing a PSMA ligand conjugate. In some embodiments, the kit further includes a container containing a compound that increases cell surface expression of PSMA.

[0070] In some embodiments, the compound that increases cell surface expression of PSMA is an antiandrogen.

[0071] In some embodiments, the antiandrogen blocks the enzyme cytochrome CYP17. In some embodiments, the antiandrogen blocks the enzyme cytochrome CYP17A1.

[0072] In some embodiments, the antiandrogen is an androgen receptor antagonist. In some embodiments, the antiandrogen is abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, galeterone, or orteronel. In some embodiments, the antiandrogen is enzalutamide or abiraterone.

[0073] In some embodiments, the compound that increases cell surface expression of PSMA is an mTOR inhibitor. In some embodiments, the mTOR inhibitor is rapamycin.

[0074] In some embodiments, the kit further includes a pharmaceutically acceptable carrier and / or excipient.

[0075] In some embodiments, the compound and / or the PSMA ligand conjugate is in an aqueous medium. In other embodiments, the compound and / or the PSMA ligand conjugate is lyophilized.

[0076] In some embodiments, the kit further comprises a diluent.

[0077] In some embodiments, the kit further comprises instructions for reconstituting the compound and / or the PSMA ligand conjugate. In other embodiments, the kit further comprises instructions for reconstituting prednisone and / or the PSMA ligand conjugate and / or the compound (if such a kit further comprises a compound).

[0078] In some embodiments, the kit further comprises instructions for co-administering the compound and / or the PSMA ligand conjugate. In other embodiments, the kit further comprises instructions for co-administering prednisone and / or the PSMA ligand conjugate. In still other embodiments, the kit further comprises instructions for co-administering a compound comprising prednisone and / or the PSMA ligand conjugate (if such a kit further comprises a compound).

[0079] In some aspects, provided is a method of inhibiting the growth of prostate-specific membrane antigen (PSMA)-expressing cancer cells. In some embodiments, the method comprises contacting PSMA-expressing cancer cells with prednisone and contacting PSMA-expressing cancer cells with a PSMA ligand conjugate. In embodiments of these methods, the PSMA-expressing cancer cells can be any of the cells provided herein. In other embodiments, the PSMA ligand conjugate can be any of the PSMA ligand conjugates provided herein. In other aspects, provided is a composition comprising prednisone and a PSMA ligand conjugate. In yet another aspect, provided is a kit comprising a container containing prednisone and a container containing a PSMA ligand conjugate.

[0080] In some embodiments of any of these methods, compositions, or kits, the PSMA ligand of the conjugate comprises an antibody or antigen-binding fragment thereof that specifically binds to PSMA. In such embodiments, the PSMA ligand is any antibody or antigen-binding fragment provided herein.

[0081] In some embodiments of any of these methods, compositions, or kits, the PSMA ligand of the conjugate comprises a small molecule ligand that specifically binds to PSMA. In such embodiments, the PSMA ligand is any small molecule ligand provided herein.

[0082] In some embodiments of any of these methods, compositions, or kits, the PSMA ligand conjugate comprises MIP-1095 or MIP-1072 conjugated to a cytotoxic radionuclide selected from the group consisting of 123 I 125 I 131 I 124 Br 75 Br 77 and F 18 .

[0083] In some embodiments of any of these methods, compositions, or kits, the anti-cancer agent or cytotoxic agent of the PSMA ligand conjugate is any anti-cancer agent or cytotoxic agent provided herein. In some embodiments, the agent comprises an auristatin, tubulysin, pyrrolobenzodiazepine dimer, calicheamicin, colchicine, ispinesib, combretastatin A4, maytansinoid DM1, maytansinoid DM4, doxorubicin, or a cytotoxic radionuclide. In some embodiments, the auristatin comprises monomethyl auristatin norephedrine or monomethyl auristatin phenylalanine.

[0084] In some embodiments of any of these methods, the method further comprises contacting PSMA-expressing cells with a compound that increases cell surface expression of PSMA. In some embodiments of any of these compositions or kits, the composition or kit further comprises a compound that increases cell surface expression of PSMA. In some embodiments, the compound is an antiandrogen or an mTOR inhibitor. In such embodiments, the antiandrogen can be any antiandrogen provided herein. In some embodiments, the mTOR inhibitor is any mTOR inhibitor provided herein.

[0085] In some embodiments of any of these methods, compositions, or kits, the antiandrogen is abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, galeterone, or ortelone. In some embodiments, the antiandrogen is enzalutamide, abiraterone, or ARN 509.

[0086] In some embodiments of any of the methods provided, the contacting step is simultaneous. In other embodiments, the contacting step is sequential. In some embodiments, the step of contacting prednisone and / or the compound with PSMA-expressing cancer cells is prior to the step of contacting a PSMA ligand conjugate with PSMA-expressing cancer cells.

[0087] In some embodiments of any of these methods, compositions, or kits, the PSMA-expressing cancer cells include androgen-independent PSMA-expressing cancer cells. In some embodiments, the PSMA-expressing cancer cells include PSMA-expressing cancer cells that are insensitive to antiandrogen therapy.

[0088] In some embodiments of any of these methods, the PSMA-expressing cancer cells are from a subject. In such embodiments, the subject can be any subject provided herein.

[0089] In some embodiments of any of the foregoing methods, compositions, or kits, the anti-androgen is enzalutamide, abiraterone, or ARN 509, and the conjugate of the PSMA ligand-anticancer agent is a PSMA ADC.

[0090] In some embodiments of any of the foregoing compositions or kits, the composition or kit further comprises a pharmaceutically acceptable carrier and / or excipient.

[0091] In some embodiments of any of the foregoing kits, any or all components of the kit are in an aqueous medium. In some embodiments of any of the foregoing kits, any or all components of the kit are lyophilized.

[0092] In some embodiments of any of the foregoing kits, the kit further comprises instructions for reconstituting any or all components of the kit. In some embodiments of any of the foregoing kits, the kit comprises instructions for combining any or all components of the kit.

[0093] In some embodiments of any of the foregoing compositions or kits, the composition or kit further comprises a diluent.

[0094] In any of the foregoing aspects or embodiments, the conjugated PSMA ligand can comprise an antibody or an antigen-binding fragment thereof that specifically binds to PSMA. In one embodiment, the PSMA ligand is an antibody or an antigen-binding fragment thereof that binds to the extracellular portion of PSMA. In another embodiment, the PSMA ligand is an antibody or an antigen-binding fragment thereof that binds to the PSMA protein dimer. In yet another embodiment, the PSMA ligand is an antibody or an antigen-binding fragment thereof that preferentially binds to the PSMA protein dimer rather than the PSMA protein monomer. In a further embodiment, the antibody or an antigen-binding fragment thereof binds to the PSMA protein dimer with an affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more greater than that for the PSMA protein monomer. In yet another embodiment, the PSMA protein dimer and the PSMA protein monomer are present in a non-denatured form such as a native conformation.

[0095] In any of the foregoing aspects or embodiments, the antibody or an antigen-binding fragment thereof can be an antibody selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix 4.152.1, or an antigen-binding fragment thereof.

[0096] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can comprise (i) the three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 15, and (ii) the three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 17.

[0097] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can comprise (i) the three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 19, and (ii) the three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 21.

[0098] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can comprise (i) the three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 23, and (ii) the three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 25.

[0099] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can comprise (i) the three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 27, and (ii) the three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 29.

[0100] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can comprise (i) the three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 31, and (ii) the three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 33.

[0101] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can be antibody PSMA 10.3, AB-PG1-XG1-006, AB-PG1-XG1-026, AB-PG1-XG1-051, AB-PG1-XG1-069, AB-PG1-XG1-077, or an antigen-binding fragment thereof.

[0102] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can be an antibody produced by hybridomas of antibody E99, J415, J533, or J591, or ATCC accession numbers HB-12101, HB-12109, HB-12127, or HB-12126, or an antigen-binding fragment thereof.

[0103] In any of the foregoing aspects or embodiments, the antibody or antigen-binding fragment thereof can be an antibody produced by hybridomas of ATCC accession numbers HB12060 (3F5.4G6), HB12309 (3D7-1.1), HB12310 (4E10-1.14), HB12489 (1G3), HB12495 (1G9), HB12490 (2C7), HB12494 (3C4), HB12491 (3C6), HB12484 (3C9), HB12486 (3E6), HB12488 (3E11), HB12485 (3G6), HB12493 (4D4), HB12487 (4D8), HB12492 (4C8B9), HB12664 (3F6), HB12678 (2E4), HB12665 (3C2), HB12672 (2D4), HB12660 (4C8G8), HB12675 (2C4), HB12663 (4C11), HB12661 (1D11), HB12667 (4E8), HB12674 (2G5), HB12620 (4E6), HB12677 (1F4), HB12666 (2E3), HB12662 (3D8), HB12668 (4F8), HB12673 (3D2), HB12676 (1G7), HB12669 (3D4), HB12679 (5G10), or HB12671 (5E9), or an antigen-binding fragment thereof.

[0104] In any of the foregoing aspects or embodiments, the PSMA ligand of the conjugate can include a small molecule ligand that specifically binds to PSMA. In one embodiment, the small molecule ligand binds to or inhibits the enzymatic site of PSMA. In certain embodiments, the small molecule ligand binds to or inhibits the glutamate carboxypeptidase II (CCPII) site on PSMA.

[0105] In any of the foregoing aspects or embodiments, the small molecule ligand can include MIP-1095, MIP-1072, GL2, or DUPA.

[0106] In any of the foregoing aspects or embodiments, the PSMA ligand-anticancer agent conjugate can include EC1069 or EC1719.

[0107] In any of the foregoing aspects or embodiments, the PSMA ligand-anticancer agent conjugate can include BIND-014.

[0108] In any of the foregoing aspects or embodiments, the PSMA ligand conjugate is I 123 、I 125 、I 131 、I 124 Br 75 、Br 77 、and F 18 and includes MIP-1095 or MIP-1072 conjugated to a cytotoxic radionuclide selected from the group consisting of.

[0109] In any of the foregoing aspects or embodiments, the PSMA ligand conjugate is 123 I-MIP-1095 or 123 I-MIP-1072.

[0110] In any of the foregoing aspects or embodiments, the anticancer agent can include auristatin, tubulysin, pyrrolobenzodiazepine dimer, calicheamicin, colchicine, ispinesib, combretastatin A4, maytansinoid DM1, maytansinoid DM4, doxorubicin, or a cytotoxic radionuclide.

[0111] In any of the foregoing aspects or embodiments, the auristatin can include monomethyl auristatin norephedrine or monomethyl auristatin phenylalanine.

[0112] In some embodiments of any method provided, the subject has either not experienced any sensitivity to anti-androgen therapy or has experienced only minimal sensitivity. In some embodiments of any method provided, the subject has experienced a loss of sensitivity to anti-androgen therapy. A subject sensitive to anti-androgen therapy is a subject who experiences a measurable reduction in the subject's cancer, such as a reduction in the number of circulating cancer cells or tumor size, or a reduction in cancer-related symptoms. Sensitivity to anti-androgen therapy can be measured, for example, by determining the level of cancer cell proliferation in the subject after treatment, and in some embodiments, can be compared to the level of cancer cell proliferation before treatment. Sensitivity to anti-androgen therapy can be determined during the course of anti-androgen treatment, and if the level of reduction of the subject's cancer no longer changes appreciably or the subject experiences an undesirable enhancement of cancer progression, the subject can be considered to have experienced a loss of sensitivity to anti-androgen therapy. In some embodiments, an increase in the subject's cancer refers to an increase in the number of cancer cells or tumor size or an increase in cancer-related symptoms. Sensitivity to anti-androgen therapy can be determined by a clinician using routine methods.

[0113] In embodiments of any method provided herein, the subject can be a subject that is either not sensitive at all or only minimally sensitive to anti-androgen therapy. Additionally, in embodiments of any method provided herein, the subject is a subject that has experienced a loss of sensitivity to anti-androgen therapy. Any method and composition provided herein can be used to prime or re-prime such subjects to anti-androgen therapy when administered concomitantly or sequentially with the PSMA ligand conjugate provided herein.

[0114] Any method provided herein can, in some embodiments, include the step of identifying a subject who is completely insensitive or minimally sensitive to anti-androgen therapy or who has experienced a loss of sensitivity to anti-androgen therapy. Any method provided herein can, in some embodiments, include the step of assessing the level of sensitivity of a subject to an anti-androgen. Any method provided herein can, in some embodiments, include the step of administering an anti-androgen until sensitivity to the anti-androgen is lost and the step of administering the anti-androgen concurrently or sequentially with a PSMA ligand conjugate provided herein.

[0115] Any method provided herein can include the step of assessing the progression of a subject's cancer. Progression can be evaluated in several ways. In some embodiments, progression is evaluated by any one or more of the following: determination of PSA level, evaluation of bone metastases, and evaluation of measurable disease. In some embodiments, progression of bone metastases is the appearance of two or more new bone lesions on a bone scan (CT scan, MRI) or the appearance of new lesions on an x-ray. Progression can also be determined by RECIST (Non-Patent Document 6). In other embodiments, cancer progression has occurred if there is an increase in pain, such as bone pain. In any method provided herein, the assessment of progression can include any one or more of the foregoing evaluations.

[0116] In some embodiments of any of the methods provided herein, the PSMA-expressing cancer cells are from a subject. In some embodiments, the subject has metastatic castration-resistant prostate cancer that is progressive. In some embodiments, the subject has received prior chemotherapy with one or more taxanes. In some embodiments, the subject has received prior treatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite prior treatment such as treatment with one or more anti-androgens. In some embodiments, the subject has prostate cancer that has begun to progress despite treatment such as treatment with one or more anti-androgens. In some embodiments, the subject has not previously received cytotoxic chemotherapy. In some embodiments, the subject has not received prior treatment with one or more anti-androgens. In some embodiments, the subject has not received prior treatment with enzalutamide or prior treatment with abiraterone. In some embodiments, a subject who has not received prior treatment with an anti-androgen such as enzalutamide or abiraterone is a subject who has metastatic castration-resistant prostate cancer. In some embodiments, the subject is any of the subjects described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0117]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Mode for Carrying Out the Invention

[0118] The present invention relates, at least in part, to the combination treatment of PSMA-expressing cancers, such as prostate cancer, specifically advanced metastatic prostate cancer. This treatment can include the administration of at least one agent targeting prostate-specific membrane antigen (PSMA) in combination with an agent targeting the androgen receptor (AR). The present invention is based, at least in part, on the surprising discovery that anti-androgens (e.g., enzalutamide or abiraterone) significantly and reversibly increase PSMA expression, enhancing the activity of PSMA ligand conjugates. In androgen-independent cells, the effects on PSMA expression and conjugate activity were synergistic and were separated from any anti-proliferative effects of the anti-androgen. The synergistic inhibition of tumor cell proliferation was associated with the upregulation of PSMA expression by the anti-androgen even in cells resistant to treatment with the anti-androgen alone. Co-treatment can actually resensitize cells to anti-androgen therapy.

[0119] Both enzalutamide and abiraterone demonstrated potent synergy with PSMA ligand conjugates across a range of concentrations. Synergy was observed in both cells responsive to treatment with antiandrogen alone and those unresponsive. In androgen-dependent LNCaP cells, the pharmacological effects of antiandrogen (e.g., anti-proliferative effects, effects on gene expression, and synergy with PSMA ligand conjugates) were observed across a range similar to that of clinically relevant concentrations. In androgen-independent C4-2 cells, the effects on gene expression and synergy were separated from any evaluable anti-proliferative activity of enzalutamide or abiraterone. Thus, these antiandrogens remained pharmacologically active against C4-2 cells. However, these cells incorporated a compensatory survival mechanism that allowed them to proliferate in the continued presence of androgen receptor (AR) blockers.

[0120] Little to no synergy was observed between antiandrogen and conjugate components (i.e., free MMAE and unmodified PSMA mAb). Free MMAE demonstrated weak synergy from additive effects when combined with antiandrogen. Moderate synergy was present between antiandrogen and docetaxel. Unmodified mAb demonstrated no anti-proliferative activity alone or in combination with antiandrogen. Thus, the potent synergy observed with PSMA ligand conjugates was specific to the conjugate and not attributable to the effects of its components, nor generalizable to microtubule inhibitors as a class.

[0121] PSMA expression was increased 2-fold after 7 days of treatment with enzalutamide and 4-fold after 21 days. The magnitude of PSMA upregulation by enzalutamide or abiraterone approximated that induced by charcoal-stripped serum depleted of a panel of hormones, cytokines, and growth factors (Non-Patent Document 7). These findings suggest near-maximal androgen suppression in our system. The time course of expression was monitored in enzalutamide-treated cells. PSMA expression increased with continuous treatment over 3 weeks but rapidly returned to baseline upon removal of enzalutamide. These findings are relevant for the combined and sequential use of potent antiandrogens and PSMA-targeted therapy.

[0122] In addition, PSMA ligand conjugates demonstrated synergy with PI3K / mTOR pathway inhibitors via diverse mechanisms including increased PSMA expression and disruption of microtubule function. In C4-2 cells, rapamycin showed minimal single-agent activity but enhanced the activity of PSMA ligand conjugates, suggesting that PI3K pathway activation is an adaptive response to ADC treatment in C4-2 cells. Thus, the present invention is also based in part on the surprising finding that mTOR inhibitors such as rapamycin can, in some cases, demonstrate synergy with the activity of PSMA ligand conjugates.

[0123] As used herein, "PSMA ligand conjugate" includes a molecule that specifically binds to PSMA, such as the extracellular domain of PSMA, and is conjugated to a therapeutic agent. The therapeutic agent may be an anti-cancer agent or a cytotoxic agent. Thus, a "PSMA ligand" refers to a molecule that specifically binds to PSMA as described herein. When a PSMA ligand is conjugated to an anti-cancer agent, the PSMA ligand conjugate is also referred to herein as a "PSMA ligand-anti-cancer agent conjugate". When a PSMA ligand is conjugated to a cytotoxic agent, the PSMA ligand conjugate is also referred to herein as a "PSMA ligand-cytotoxic agent conjugate".

[0124] As used herein, "PSMA-expressing cell" refers to a cell that expresses PSMA or is capable of expressing PSMA (e.g., human PSMA). PSMA is a 100 kD type II transmembrane glycoprotein expressed in prostate tissue (Non-Patent Document 8; Patent Document 1). PSMA has been characterized as a type II transmembrane protein with sequence identity to the transferrin receptor (Non-Patent Document 9) and NAALADase activity (Non-Patent Document 10). PSMA is expressed in increased amounts in prostate cancer (Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13; and Non-Patent Document 14). PSMA expression in cancerous prostate is approximately 10-fold higher than in normal prostate. Expression in normal prostate is approximately 10-fold higher than in the brain and 50- to 100-fold higher than in the liver or kidney. In most normal tissues, PSMA expression is not observed.

[0125] PSMA expression increases with disease progression and is highest in metastatic hormone - resistant disease. In addition, PSMA is abundantly expressed on the neovasculature of various non - prostate tumors, including tumors of the bladder, breast, colon, pancreas, sarcoma, melanoma, kidney, liver, lung (e.g., small - cell lung cancer), and kidney, but not on normal blood vessels. Thus, "PSMA - expressing cells" include PSMA - expressing cancer cells such as prostate cancer cells, and PSMA - expressing cells (e.g., endothelial cells) of the neovasculature of some non - prostate cancers or non - prostate tumors.

[0126] As used herein, "androgen - dependent PSMA - expressing cells" refers to PSMA - expressing cells such as cancer cells that respond to anti - androgens. A cell is considered to respond to an anti - androgen herein if its growth can be substantially inhibited by the anti - androgen.

[0127] As used herein, "androgen - independent PSMA - expressing cells" refers to PSMA - expressing cells such as cancer cells that do not respond to anti - androgens, and are also referred to herein as "PSMA - expressing cells insensitive to anti - androgen therapy". A cell is considered not to respond to an anti - androgen herein if its growth is not substantially inhibited by the anti - androgen.

[0128] In some embodiments, PSMA-expressing cells that are insensitive to anti-androgen therapy become sensitized to anti-androgen therapy after the step of contacting the PSMA-expressing cells with a compound that increases cell surface expression of PSMA (e.g., an anti-androgen or an mTOR inhibitor). Such cells can then be further contacted with the PSMA ligand conjugates provided herein, and such contact can be carried out simultaneously or sequentially. Thus, in some embodiments, androgen-independent PSMA-expressing cells (e.g., cancer cells) that would otherwise not respond to an anti-androgen become responsive upon contact with an anti-androgen or an mTOR inhibitor and thus may become sensitized to the anti-androgen, and further contact with a PSMA ligand conjugate can result in inhibition of cell proliferation or cell death. The proliferation of such "sensitized" PSMA-expressing cancer cells can thus be substantially inhibited as a result. Sensitization of androgen-independent PSMA-expressing cells to an anti-androgen can occur, in some embodiments, within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days after contact with a compound that increases cell surface expression of PSMA. In some embodiments, sensitization of androgen-independent PSMA-expressing cells to an anti-androgen can occur in less than 2 days.

[0129] In some embodiments, PSMA-expressing cells are contacted with a compound that increases (e.g., upregulates) the expression of PSMA on the cell surface for a time sufficient to increase the cell surface expression of PMSA. The time sufficient to upregulate the cell surface expression of PSMA can be determined by various protein expression assays, which are known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA) and Western blotting. In some embodiments, the time sufficient to upregulate the cell surface expression of PSMA can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more. In some embodiments, the time sufficient to upregulate the cell surface expression of PSMA can be less than 1 day.

[0130] Examples of PSMA ligands for use provided herein include, but are not limited to, antibodies or antigen-binding fragments thereof and small molecule ligands that specifically bind to PSMA and can act as substrate mimics of the enzyme site on PSMA. Antibodies that specifically bind to PSMA can be referred to herein as "PSMA antibodies." Similarly, small molecule ligands that specifically bind to PSMA can be referred to herein as "PSMA small molecule ligands."

[0131] As used herein, "specific binding" refers to the binding of a molecule (e.g., an antibody) to a predetermined target (e.g., an antigen), in this case PSMA (e.g., human PSMA). In some embodiments, the sequence of PSMA is shown as SEQ ID NO: 1. Typically, the molecule binds with an affinity that is at least 2-fold greater than its binding affinity to non-specific targets (e.g., BSA, casein) that are targets other than PSMA, an isoform or variant of PSMA, or a related target.

[0132] The antibody or antigen-binding fragment thereof of the PSMA ligand conjugate can be any antibody or antigen-binding fragment thereof that binds to PSMA (e.g., specifically binds to an epitope of PSMA). Examples of PSMA antibodies for use provided herein include, but are not limited to, those listed in Table 1. Thus, PSMA antibodies and antigen-binding fragments thereof include PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, 4.248.2, 4.360.3, 4.7.1, 4.4.1, 4.177.3, 4.16.1, 4.22.3, 4.28.3, 4.40.2, 4.48.3, 4.49.1, 4.209.3, 4.219.3, 4.288.1, 4.333.1, 4.54.1, 4.153.1, 4.232.3, 4.292.3, 4.304.1, 4.78.1, and 4.152.1, and antigen-binding fragments thereof.

[0133] In some embodiments, the antibodies are produced by hybridomas designated herein as PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA 3247 PTA-3347), PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1 (PTA-4653).

[0134] These hybridomas comply with and meet the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and have been deposited with the American Type Culture Collection ("ATCC") (address: 10801 University Boulevard, Manassas, Va. 20110-2209), as an International Depository Authority, and have been given a patent deposit name (Table 1).

[0135]

Table 1-1

[0136]

Table 1-2

[0137]

Table 1-3

[0138] In some embodiments, the antibody or antigen-binding fragment thereof of the PSMA ligand conjugate can comprise (i) a heavy chain encoded by a nucleic acid molecule comprising one or more heavy chain coding regions of the nucleotide sequences shown as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and (ii) a light chain encoded by a nucleic acid molecule comprising one or more light chain coding regions of the nucleotide sequences shown as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0139] Furthermore, provided herein is an antigen-binding fragment of the aforementioned antibody for use as a PSMA ligand of the PSMA ligand conjugate provided herein.

[0140] Plasmids encoding the heavy and light chains of antibodies PSMA 10.3, AB-PG1-XG1-006, AB-PG1-XG1-026, AB-PG1-XG1-051, AB-PG1-XG1-069, AB-PG1-XG1-077 were also deposited with the ATCC and are shown in Table 1 above. When used herein, the names of the deposited hybridomas or plasmids can be used interchangeably with the names of the antibodies. It will be apparent to those skilled in the art when the name is intended to refer to an antibody, or when the name refers to a plasmid or hybridoma that encodes or produces an antibody, respectively. Furthermore, the antibody name may be a shortened form of the name shown in Table 1. For example, the antibody AB-PG1-XG1-006 can be referred to as "AB-PG1-XG1-006", "PG1-XG1-006", "XG1-006", or "006". In another example, the antibody PSMA 4.232.3 can be referred to as "PSMA 4.232.3", "4.232.3", "4.232.3", or "4.232". All variations of the antibody name are intended to refer to the same antibody, not different antibodies.

[0141] Also provided are antibodies that are encoded by specific sets of heavy and light chain sequences and are used as PSMA ligands. In some embodiments, provided herein is an antibody (AB-PG1-XG1-006) encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 2 and SEQ ID NO: 8. In other embodiments, provided herein is an antibody (AB-PG1-XG1-026) encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 3 and SEQ ID NO: 9. In yet other embodiments, provided herein is an antibody (AB-PG1-XG1-051) encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 4 and SEQ ID NO: 10. In yet other embodiments, provided herein is an antibody (AB-PG1-XG1-069) encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 5 and SEQ ID NO: 11. In further embodiments, provided herein is an antibody (AB-PG1-XG1-077) encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 6 and SEQ ID NO: 12. In yet other embodiments, provided herein is an antibody (PSMA 10.3) encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 7 and SEQ ID NO: 13. Antigen-binding fragments of these antibodies are also contemplated for use as PSMA ligands in the PSMA ligand conjugates provided herein.

[0142] In some embodiments, the PSMA antibodies provided herein can comprise (i) a heavy chain variable region encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, or SEQ ID NO: 30, and (ii) a light chain variable region encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, or SEQ ID NO: 32. Antigen-binding fragments of these antibodies are also contemplated for use as PSMA ligands in the PSMA ligand conjugates provided herein.

[0143] As used herein, "coding region" refers to the region of a nucleotide sequence that encodes a polypeptide sequence; a coding region can include regions that encode portions of a protein that are later cleaved and removed, such as a signal peptide.

[0144] Those skilled in the art will recognize that the nucleic acids and polypeptides provided herein include the nucleotide sequences and amino acid sequences provided herein. In some examples, the nucleotide sequences and amino acid sequences can include sequences that encode or are a signal peptide. Each of these sequences, with or without the sequence portion that encodes or is a signal peptide, is contemplated herein.

[0145] Furthermore, provided herein are PSMA antibodies comprising specific sets of heavy-chain variable arrays and light-chain variable arrays. In some embodiments, the PSMA antibody comprises an immunoglobulin variable array encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 14 and SEQ ID NO: 16. Similarly, the PSMA antibody can comprise an immunoglobulin variable array comprising the amino acid sequences shown as SEQ ID NO: 15 and SEQ ID NO: 17. In other embodiments, the PSMA antibody comprises an immunoglobulin variable array encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 18 and SEQ ID NO: 20, or comprises an immunoglobulin variable array comprising the amino acid sequences shown as SEQ ID NO: 19 and SEQ ID NO: 21. In yet other embodiments, the PSMA antibody comprises an immunoglobulin variable array encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 22 and SEQ ID NO: 24, or comprises an immunoglobulin variable array comprising the amino acid sequences shown as SEQ ID NO: 23 and SEQ ID NO: 25. In yet other embodiments, the PSMA antibody comprises an immunoglobulin variable array encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 26 and SEQ ID NO: 28, or comprises an immunoglobulin variable array comprising the amino acid sequences shown as SEQ ID NO: 27 and SEQ ID NO: 29. In other embodiments, the PSMA antibody comprises an immunoglobulin variable array encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequences shown as SEQ ID NO: 30 and SEQ ID NO: 32, or comprises an immunoglobulin variable array comprising the amino acid sequences shown as SEQ ID NO: 31 and SEQ ID NO: 33. Antigen-binding fragments of these antibodies are also contemplated for use as PSMA ligands in the PSMA ligand conjugates provided herein.

[0146] In some embodiments, the PSMA antibody is encoded by a nucleic acid molecule that is highly homologous to the aforementioned nucleic acid molecule or an amino acid molecule, or an amino acid molecule. For example, the homologous nucleic acid molecule or amino acid molecule may include a nucleotide sequence or amino acid sequence that is at least about 90% identical to the nucleotide sequence or amino acid sequence provided herein. As another example, the nucleotide sequence and amino acid sequence are at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleotide sequence or amino acid sequence provided herein. Homology can be calculated using various publicly available software well known to those skilled in the art. Representative tools include the BLAST system available from the website of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (NIH).

[0147] In some embodiments, the PSMA antibody comprises (i) a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, or SEQ ID NO: 31, and (ii) a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 17, 21, 25, 29, or 33. As otherwise noted herein, PSMA antigen-binding fragments of the foregoing are also provided.

[0148] In some embodiments, the PSMA antibody comprises (i) three complementarity-determining regions of the heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, or SEQ ID NO: 31, and (ii) three complementarity-determining regions of the light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 17, 21, 25, 29, or 33. As otherwise noted herein, PSMA antigen-binding fragments of the foregoing are also provided.

[0149] In some embodiments, the PSMA antibody includes the antibodies provided in (Patent Document 2), (Patent Document 3), and (Patent Document 4). Those antibodies are incorporated herein by reference. Accordingly, the PSMA antibody includes E99, J415, J533, and J591 monoclonal antibodies; monoclonal antibodies produced by hybridomas having ATCC accession numbers HB-12101, HB-12109, HB-12127, and HB-12126; and monoclonal antibodies produced by hybridomas with ATCC accession numbers HB12060 (3F5.4G6), HB12309 (3D7-1.1), HB12310 (4E10-1.14), HB12489 (1G3), HB12495 (1G9), HB12490 (2C7), HB12494 (3C4), HB12491 (3C6), HB12484 (3C9), HB12486 (3E6), HB12488 (3E11), HB12485 (3G6), HB12493 (4D4), HB12487 (4D8), HB12492 (4C8B9), HB12664 (3F6), HB12678 (2E4), HB12665 (3C2), HB12672 (2D4), HB12660 (4C8G8), HB12675 (2C4), HB12663 (4C11), HB12661 (1D11), HB12667 (4E8), HB12674 (2G5), HB12620 (4E6), HB12677 (1F4), HB12666 (2E3), HB12662 (3D8), HB12668 (4F8), HB12673 (3D2), HB12676 (1G7), HB12669 (3D4), HB12679 (5G10), and HB12671 (5E9). Antigen-binding fragments of these antibodies are also contemplated for use as the PSMA ligand of the PSMA ligand conjugate provided herein.

[0150] Furthermore, provided herein are nucleic acid molecules encoding PSMA antibodies and antigen-binding fragments thereof, as well as vectors comprising the nucleic acid molecules described herein. The provided vectors can be used to transform or transfect host cells to produce PSMA antibodies and antigen-binding fragments thereof having the specificities described herein. In some embodiments, the PSMA antibodies and antigen-binding fragments thereof produced will have the specificities of antibody AB-PG1-XG1-006, AB-PG1-XG1-026, AB-PG1-XG1-051, AB-PG1, XG1-069, AB-PG1-XG1-077, and PSMA 10.3, and antigen-binding fragments thereof. In some embodiments, the vector can comprise an isolated nucleic acid molecule encoding the heavy chain of the antibody listed above, encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequence shown as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In other embodiments, the vector can comprise an isolated nucleic acid molecule encoding the light chain of the antibody listed above, encoded by a nucleic acid molecule comprising one or more coding regions of the nucleotide sequence shown as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In further embodiments, the vector can comprise isolated nucleic acid molecules encoding the heavy and light chains of the antibody listed above. In yet other embodiments, provided herein are plasmids encoding the antibodies or antigen-binding fragments described herein.Such plasmids can be selected from the group consisting of AB-PG1-XG1-006 heavy chain (SEQ ID NO: 2), AB-PG1-XG1-006 light chain (SEQ ID NO: 8), AB-PG1-XG1-026 heavy chain (SEQ ID NO: 3), AB-PG1-XG1-026 light chain (SEQ ID NO: 9), AB-PG1-XG1-051 heavy chain (SEQ ID NO: 4), AB-PG1-XG1-051 light chain (SEQ ID NO: 10), AB-PG1-XG1-069 heavy chain (SEQ ID NO: 5), AB-PG1-XG1-069 light chain (SEQ ID NO: 11), AB-PG1-XG1-077 heavy chain (SEQ ID NO: 6), AB-PG1-XG1-077 light chain (SEQ ID NO: 12), PSMA 10.3 heavy chain (SEQ ID NO: 7), and PSMA 10.3 Kappa (SEQ ID NO: 13).

[0151] As used herein, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains, C H 1, C H 2, and C H 3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or V L ), and a light chain constant region. The light chain constant region is composed of one domain, CL. V H and V L regions can be further subdivided into hypervariable regions named complementarity determining regions (CDRs) interspersed with highly conserved regions named framework regions (FRs). Each V H and V LIt is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0152] As used herein, an "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., PSMA). The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the scope of the term "antigen-binding fragment" of an antibody include: (i) Fab fragments, monovalent fragments consisting of the V L 、V H 、C L 、and C H domains; (ii) F(ab’)2 fragments, divalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragments consisting of V H and CH1 domains; (iv) Fv fragments consisting of the V L and V H domains of a single arm of an antibody; (v) dAb fragments consisting of the V H domain (Non-Patent Document 15); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of the Fv fragment, V and V H are encoded by separate genes, but they are V L and V HDomains pair up and can be linked using recombinant methods by a synthetic linker that enables them to be made as a single protein chain (known as single-chain Fv (scFv); see, e.g., (Non-Patent Document 16); and (Non-Patent Document 17)) that forms a monovalent molecule. Such single-chain antibodies are also intended to be encompassed within the scope of the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained using conventional procedures such as proteolytic fragmentation procedures described in (Non-Patent Document 18), which is incorporated herein by reference, and other techniques known to those of skill in the art. These fragments are screened for utility in the same manner as intact antibodies.

[0153] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PSMA is substantially free of antibodies that specifically bind to antigens other than PSMA). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of PSMA may, in some embodiments, have cross-reactivity with other related antigens (e.g., PSMA species homologs) from other species. Further, an isolated antibody may, in some embodiments, be substantially free of other cellular materials and / or chemical substances.

[0154] The isolated antibodies of the invention include various antibody isotypes such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE. As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region gene (e.g., IgM or IgG1). The antibody can be full-length or can include only antigen-binding fragments such as the antibody constant and / or variable domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgE, or can consist of Fab fragments, F(ab')2 fragments, and Fv fragments.

[0155] The antibody, in some embodiments, may be a monoclonal antibody, a polyclonal antibody, or a mixture of a polyclonal antibody and a monoclonal antibody. Antibodies can be produced by a variety of techniques well known in the art. Procedures for producing polyclonal antibodies are well known. For example, an anti-PSMA polyclonal antibody can be produced by subcutaneously administering PSMA protein to New Zealand white rabbits that have first been bled to obtain pre-immune serum. Typically, one or more adjustments can be made to inject 100 μl of PSMA per site at six different sites. Then, two weeks after the first injection, the rabbits are bled, and boosted regularly with the same antigen three times every six weeks. Serum samples are taken 10 days after each boost. Preferably, the polyclonal antibody is recovered from the serum by affinity chromatography using PSMA, which is used to capture the antibody. This procedure and other procedures for producing polyclonal antibodies are disclosed in (Non-Patent Document 19), which is incorporated herein by reference. Monoclonal antibody production can be achieved by techniques well known in the art as well.

[0156] As used herein, a "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Methods for producing monoclonal antibodies involve obtaining immunocompetent cells, specifically B cells, that have been pre-immunized either in vivo or in vitro with the antigen of interest and that have the ability to produce antibodies suitable for fusion with a B cell myeloma cell line. Mammalian lymphocytes are typically immunized in vivo with the desired protein or polypeptide, for example, with PSMA, in an animal (e.g., a mouse). Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient antibody titer. Once immunized, the animal can be used as a source of antibody-producing lymphocytes. After the last antigen boost, the animal is sacrificed and spleen cells are removed. Mouse lymphocytes form stable fusions with a mouse myeloma cell line (e.g., a BALB / c mouse) at a high rate. Other mouse strains, rabbits, hamsters, sheep, and frogs can also be used as hosts for preparing antibody-producing cells. See (Non-Patent Document 20). For example, as provided herein, mouse strains in which the human immunoglobulin gene has been inserted into the genome (and that are unable to produce mouse immunoglobulins) can be used. Examples include the HUMAB-MOUSE strain created by Medarex / GenPharm International and the XENOMOUSE strain created by Abgenix. Such mice produce human immunoglobulin molecules in response to immunization.

[0157] Those antibody-producing cells in the plasmablast division phase preferentially fuse. Somatic cells can be obtained from the lymph nodes, spleen, and peripheral blood of antigen-stimulated animals, and the selected lymphocytes largely depend on their experimental utility in a particular fusion system. The antibody-secreting lymphocytes are then fused with (mouse) B cell myeloma cells or transformed cells that can be replicated indefinitely in cell culture, thereby creating immortal immunoglobulin-secreting cell lines. The resulting fused cells, i.e., hybridomas, are cultured, and the resulting colonies are screened for the production of the desired monoclonal antibody. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large amounts of antibody. An explanation of the theoretical basis and practical methodology for fusing such cells is shown in (Non-Patent Document 21), which is incorporated herein by reference.

[0158] Alternatively, human somatic cells that produce antibodies, specifically B lymphocytes, are suitable for fusion with myeloma cell lines. B lymphocytes derived from a biopsy sample of an individual's spleen, tonsil, or lymph node can be used in the same manner as more readily available peripheral blood B lymphocytes. These lymphocytes may also be derived from patients diagnosed with prostate cancer or another PSMA-expressing cancer. In addition, human B cells can be directly immortalized by Epstein-Barr virus (Non-Patent Document 22, which is incorporated herein by reference). Although somatic cell hybridization methods are preferred, in principle, other techniques for producing monoclonal antibodies, such as viral transformation or oncogenic transformation of B cells, can be used.

[0159] Myeloma cells suitable for use in the fusion procedure for hybridoma production may be those that do not produce antibodies, have a high fusion efficiency, and have an enzyme deficiency that renders them non-proliferative in a specific selection medium that supports the growth of the desired hybridoma. Examples of such myeloma cell lines that can be used for the production of fused cell lines include, but are not limited to, all mouse-derived, P3-X63 μg8, X63-Ag8.653, NS1 / 1.Ag 4.1, Sp2 / 0-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul; rat-derived, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210; and all human-derived, U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6 (Non-Patent Document 23; Non-Patent Document 24).

[0160] Fusion of mammalian myeloma cells or other fusion partners that can be replicated indefinitely in cell culture is achieved by standard and well-known techniques, such as the use of polyethylene glycol ("PEG") or other fusing agents (see (Non-Patent Document 25), which is incorporated herein by reference).

[0161] In some embodiments, recombinant antibodies are contemplated for the uses provided herein. As used herein, "recombinant antibody" refers to an antibody prepared, expressed, produced, or isolated by recombinant means, such as an antibody isolated from an animal (e.g., a mouse) that is transgenic for the immunoglobulin genes of another species, an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, or an antibody prepared, expressed, produced, or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

[0162] In still other embodiments, the antibody may be a chimeric antibody or a humanized antibody. As used herein, a "chimeric antibody" refers to an antibody in which a murine variable region or hypervariable region is linked to a human constant region or constant framework region and variable framework region. As used herein, a "humanized antibody" refers to an antibody that retains only the antigen-binding CDRs derived from the parent antibody, linked to a human framework region (see (Non-Patent Document 26)). Such chimeric or humanized antibodies that retain the binding specificity of the murine antibody are expected to have reduced immunogenicity when administered in vivo for diagnostic, prophylactic, or therapeutic uses, as provided herein.

[0163] In still other embodiments, the monoclonal antibodies provided herein can be modified to be in the form of bispecific or multispecific antibodies. As used herein, a "bispecific antibody" refers to any agent, such as a protein, peptide, or protein complex or peptide complex, having two different binding specificities that bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. As used herein, a "multispecific antibody" refers to any agent, such as a protein, peptide, or protein complex or peptide complex, having more than two different binding specificities that bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Thus, the antibodies provided herein can be bispecific, trispecific, tetra-specific, or other multispecific antibodies directed to a cell surface antigen such as PSMA and an Fc receptor on an effector cell. A "bispecific antibody" also includes a diabody. A diabody is a bivalent bispecific antibody in which V H and V LA linker is used in which the domains are expressed on a single polypeptide chain and which is too short for pairing to occur between two domains on the same chain, whereby those domains are directed to pair with complementary domains on another chain to generate two antigen-binding sites (see, for example, (Non-Patent Document 27); (Non-Patent Document 28)).

[0164] Bispecific antibodies can be formed from an antigen-binding region specific for the extracellular domain of PSMA and an antigen-binding region specific for effector cells having cytotoxic or tumor-inhibiting activity. The two antigen-binding regions of the bispecific antibody can be either chemically linked or expressed by cells genetically engineered to produce the bispecific antibody. (Generally, see (Non-Patent Document 29)). Suitable effector cells having cytotoxic activity include, but are not limited to, cytotoxic T cells (mainly CD8 + cells) and natural killer cells.

[0165] In some embodiments, the antibodies provided herein are human antibodies. As used herein, "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). The term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences (referred to herein as "humanized antibodies" separately). Human antibodies against PSMA are generated using transgenic mice that retain a human immune system moiety rather than a mouse system. The human PSMA antibodies provided herein specifically bind to cell surface PSMA and / or rsPSMA (recombinant soluble PSMA, i.e., having the sequence of the extracellular portion of PSMA) with sub-nanomolar affinity. The human PSMA antibodies provided herein have a binding affinity of about 1×10-9 Less than M, about 1×10 -10 Less than M, or 1×10 -11 It may have a binding affinity of less than M. In some embodiments, the binding affinity of the human PSMA antibody provided herein is about 5×10 -10 Less than M.

[0166] Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice with respect to most of the heavy and light chain loci of human immunoglobulins. See, for example, (Patent Document 5), (Patent Document 6), (Patent Document 7), (Patent Document 8), (Patent Document 9), and the references cited therein. These contents are incorporated herein by reference. These animals are genetically modified such that there are functional defects in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or part of the human germline immunoglobulin locus such that immunization of these animals results in the production of fully human antibodies against the antigen of interest. Following immunization of these mice (e.g., XENOMOUSE (Abgenix) and HUMAB mouse (Medarex / GenPharm)), monoclonal antibodies are prepared according to standard hybridoma technology. Since these monoclonal antibodies have human immunoglobulin amino acid sequences, they do not induce a human anti-mouse antibody (HAMA) response when administered to humans.

[0167] Preferably, the mice are 6 to 16 weeks old at the time of the first immunization. For example, while other routes of immunization known to those skilled in the art are possible using a purified or concentrated preparation of the PSMA antigen (e.g., recombinant PSMA or PSMA-expressing cells), the mice are immunized by intraperitoneal administration (IP). The PSMA antigen is injected in combination with an adjuvant such as complete Freund's adjuvant, and preferably, after the first injection, booster immunization is performed with the antigen in an adjuvant such as incomplete Freund's adjuvant. The immune response is monitored during the immunization protocol using, for example, plasma samples obtained by retro-orbital bleeding. The plasma is screened by ELISA, and mice having a sufficient titer of anti-PSMA human immunoglobulin are used for fusion. Three days prior to sacrifice and spleen removal, the mice are boost-immunized by intravenous administration of the antigen.

[0168] The antibody or its antigen-binding fragment can be selected based on the following criteria, which are not intended to be exclusive, for example. 1) Binding to live cells expressing PSMA; 2) High affinity binding to PSMA; 3) Binding to a unique epitope on PSMA (to eliminate the possibility that antibodies having complementary activities when used in combination will compete for binding to the same epitope); 4) Opsonization of cells expressing PSMA; 5) Mediation of growth inhibition, phagocytosis, and / or killing of PSMA-expressing cells in the presence of effector cells; 6) Regulation (inhibition or enhancement) of NAALADase, folate hydrolase, dipeptidyl peptidase IV, and / or γ-glutamyl hydrolase activity; 7) Growth inhibition, cell cycle arrest, and / or cytotoxicity in the absence of effector cells; 8) Internalization of PSMA; 9) Binding to a conformational epitope on PSMA; 10) Minimal cross-reactivity with cells or tissues that do not express PSMA; and 11) Preferential binding to the dimeric form of PSMA rather than the monomeric form of PSMA.

[0169] The PSMA antibodies and antigen-binding fragments thereof provided herein typically meet one or more of the aforementioned criteria and, in some instances, all of them.

[0170] In some embodiments, an isolated antibody or antigen-binding fragment thereof can be selected for its ability to bind to live cells expressing PSMA. To demonstrate binding of a monoclonal antibody to live cells expressing PSMA, flow cytometry can be used. For example, a cell line expressing PSMA (grown under standard growth conditions) or prostate cancer cells expressing PSMA are mixed with monoclonal antibodies at various concentrations in PBS containing 0.1% TWEEN 80 and 20% mouse serum and incubated at 37 °C for 1 hour. After washing, the cells are reacted with a fluorescently labeled anti-human IgG secondary antibody (when using a human anti-PSMA antibody) under the same conditions as primary antibody staining. The sample can be analyzed by a fluorescence excitation cell sorter (FACS) device that gates single cells using light and side scatter characteristics. An alternative assay using a fluorescence microscope can be used in addition to, or instead of, the flow cytometry assay. The cells are stained precisely as described above and can be examined by a fluorescence microscope. This method allows visualization of individual cells, but the sensitivity may decrease depending on the density of the antigen. Binding of an antibody or antigen-binding fragment thereof to live cells expressing PSMA can result in inhibition of cell proliferation or mediation of cell lysis. Cell lysis can be complement-mediated or mediated by effector cells. In some embodiments, cell lysis occurs in vivo, preferably in a mammal, and the live cells are tumor cells. Examples of tumors that can be targeted by the PSMA antibodies provided herein (e.g., PSMA antibody conjugates) include any tumor expressing PSMA, such as prostate, bladder, pancreas, lung, colon, kidney, melanoma, and sarcoma. Tumors expressing PSMA include tumors having PSMA-expressing neovessels.

[0171] In some embodiments, the PSMA antibody or antigen-binding fragment thereof binds to PSMA expressed on the cell and is internalized therewith. Thus, a PSMA ligand conjugate comprising a PSMA antibody can be internalized with PSMA expressed on the cell. The mechanism by which this internalization occurs is not important for the practice of the present invention. For example, the antibody or antigen-binding fragment thereof can induce internalization of PSMA.

[0172] In some embodiments, the PSMA antibody or antigen-binding fragment thereof binds to a conformational epitope within the extracellular domain of the PSMA molecule. To determine whether a human PSMA antibody binds to a conformational epitope, each antibody can be tested in assays using native proteins (e.g., non-denaturing immunoprecipitation, flow cytometry analysis of cell surface binding) and denatured proteins (e.g., Western blot, immunoprecipitation of denatured proteins). By comparing the results, it can be determined whether the antibody binds to a conformational epitope. Antibodies that bind to native proteins but not to denatured proteins are those that bind to conformational epitopes and, in some embodiments, are preferred antibodies.

[0173] In other embodiments, the PSMA antibody or antigen-binding fragment thereof binds to a dimer-specific epitope on PSMA. Generally, an antibody or antigen-binding fragment thereof that binds to a dimer-specific epitope binds preferentially to PSMA dimer over PSMA monomer. To determine whether a PSMA antibody specifically binds to PSMA dimer, the antibody can be tested in an assay (e.g., Western blotting following immunoprecipitation) using native dimeric PSMA protein and dissociated monomeric PSMA protein. By comparing the results, it can be determined whether the antibody binds preferentially to the dimer or to the monomer. Antibodies that bind to PSMA dimer but not to monomeric PSMA protein are, in some embodiments, preferred antibodies.

[0174] Other PSMA antibodies or antigen-binding fragments thereof provided herein include antibodies that specifically bind to an epitope on PSMA defined by a second antibody. To determine the epitope, standard epitope mapping methods known in the art can be used. For example, a fragment (peptide) of the PSMA antigen (preferably, a synthetic peptide) that binds to the second antibody can be used to determine whether a candidate antibody binds to the same epitope. In the case of a linear epitope, overlapping peptides of a defined length (e.g., 8 or more amino acids) are synthesized. It is preferred to offset the peptides by only 1 amino acid such that a series of peptides covering 8 amino acids of the PSMA protein sequence are prepared. When fewer peptides are desired, they can be prepared by using a larger offset, e.g., an offset of 2 or 3 amino acids. Additionally, longer peptides (e.g., 9-, 10-, or 11-amino acid lengths) can be synthesized. Binding of the peptides to the antibody can be determined using standard methodologies including surface plasmon resonance (e.g., BIACORE) and ELISA assays. In the case of examining a conformational epitope, larger PSMA fragments can be used as provided herein. Other methods of revealing conformational epitopes using mass spectrometry are described and can be used as provided herein (see, e.g., (Non-Patent Document 30) and the references cited therein). Still other methods for epitope determination are provided in standard laboratory references such as (Non-Patent Document 31). The epitope can be confirmed by introducing point mutations or deletions into a known epitope and then testing the binding to one or more antibodies or antigen-binding fragments thereof to determine which mutations reduce the binding of the antibody or antigen-binding fragment thereof.

[0175] In some embodiments, the PSMA antibody of the PSMA ligand conjugate is a monoclonal antibody or an antigen-binding fragment thereof that binds to a prostate-specific membrane antigen (PSMA) protein dimer, i.e., a PSMA protein dimer that is a homodimer of PSMA protein monomers having the sequence of SEQ ID NO: 1, and this antibody or antigen-binding fragment binds to (i) living cells and (ii) the PSMA protein dimer with an affinity at least 2-fold higher than that for the PSMA monomer, as described in (Patent Document 10) incorporated herein by reference.

[0176] In some embodiments, the PSMA antibody is conjugated to a radioactive molecule. Thus, as an example of such a PSMA ligand conjugate, via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) 177 lutetium ( 177 Lu)-conjugated monoclonal PSMA antibody J591 containing 177 Lu-J591 can be mentioned.

[0177] The PSMA ligand of the PSMA ligand conjugate can be any small molecule ligand that specifically binds to PSMA. Such a small molecule ligand can bind to the PSMA enzyme site in its native conformation. Furthermore, such a small molecule ligand can have any one or more of the characteristics described above for the PSMA antibody ligand.

[0178] In some embodiments, the small molecule ligand is based on a glutamate-urea-lysine heterodimer (e.g., a glutamate-urea-lysine analog) or a glutamate-urea-glutamate-based dimer that specifically binds to the enzyme site on PSMA. In some embodiments, such a small molecule ligand is conjugated to an anti-cancer agent or a radionuclide as a cytotoxic agent (e.g., a cytotoxic radionuclide or a radiotherapy isotope). Thus, as an example of a PSMA ligand conjugate, 123 IMIP-1095( 123(also referred to as IMIP-1466) and 123 Small molecule ligands based on glutamate-urea-amino acids conjugated to a radionuclide via a spacer, such as IMIP-1072 (Molecular Insight Pharmaceuticals, Inc.), can be mentioned. Other examples of PSMA small molecule ligands and PSMA ligand conjugates can be found in (Patent Document 11) and (Patent Document 12), which are incorporated herein by reference. In some embodiments, I 123 is, I 125 I 131 I 124 BR 75 BR 77 and other radioactive halogens selected from the group consisting of F 18 can be substituted.

[0179] 123 The chemical structure of I-MIP-1095 (i.e., 123 I-(S)-2-(3-((S)-1-carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic acid) is as follows.

Chemical formula

[0180] In another embodiment, the PSMA ligand conjugate is 124 I-MIP-1095. In another embodiment, the PSMA ligand conjugate is 131 I-MIP-1095.

[0181] 123 The chemical structure of I-MIP-1072 (i.e., 123 I-(S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)ureido)pentanedioic acid) is as follows.

Chemical formula

[0182] In some embodiments, the small molecule ligand of the PSMA ligand conjugate is the GL2 molecule described in (Patent Document 13). Any small molecule ligand provided herein that includes the GL2 molecule can be conjugated to a therapeutic agent via nanoparticles (e.g., polymer-based, lipid-based, and / or nucleic acid-based nanoparticles). In such embodiments, the nanoparticles can contain a therapeutic agent. Thus, in some embodiments, the PSMA ligand conjugate includes a small molecule ligand conjugated to a nanoparticle containing an anti-cancer agent or a cytotoxic agent. Examples of such PSMA ligand conjugates include, but are not limited to, BIND-014 (Bind Biosciences, Inc.) described in (Patent Document 13). These PSMA ligands and PSMA ligand conjugates are incorporated herein by reference.

[0183] The PSMA small molecule ligand, in some embodiments, is one of the following compounds I, II, III, and IV:

Chemical formula

[0184] The PSMA small molecule ligand, in other embodiments,

Chemical formula

Chemical formula

[0185] The PSMA small molecule ligand, in yet other embodiments,

Chemical formula

[0186] In some embodiments, a PSMA small molecule ligand that is a substrate that binds to or mimics an enzyme site on PSMA includes 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA). In some embodiments, such small molecule ligands are conjugated to a chemotherapeutic agent such as tubulysin hydrazide (TubH). The synthesis and use of an example of such a PSMA ligand conjugate (EC1069) is described in (Non-Patent Document 32); (Non-Patent Document 33). EC1719 is another example of a PSMA ligand conjugate that includes TubH. EC1069 and EC1719 can target the PSMA receptor expressed on prostate cancer cells with a chemotherapeutic agent (Endocyte). Analogs of EC1069 and EC1719 that link DUPA and TubH can also target the PSMA receptor expressed on prostate cancer cells. Thus, analogs of EC1069 and EC1719 are also contemplated herein. Accordingly, the terms "EC1069" and "EC1719" encompass EC1069, EC1719, and their analogs. The linkers of these analogs can be, in some embodiments, peptides containing D-amino acids or peptides to which a sugar moiety, amide, or ester is attached. Thus, an example of a linker is D-γ-Glu D-Asp-D-Phe-D-Cys (FIG. 10). As provided herein, other linkers can be used.

[0187] Conjugation of one or more therapeutic agents to a PSMA ligand can involve many chemical mechanisms such as, for example, covalent bonding, affinity binding, intercalation, coordination bonding, electrostatic bonding, and complex formation. Conjugation includes encapsulation and is intended to refer to any mechanism by which one component can be associated with another component. Conjugation can be direct conjugation of a therapeutic agent to a PSMA ligand or can be indirect, such as via a linker, polymer, particle, etc., which is a linker, polymer, particle, etc. to which the therapeutic agent is attached.

[0188] The covalent linkage can be achieved either by direct condensation of existing side chains or by incorporation of an external cross-linking molecule. Many divalent or polyvalent agents are useful for coupling protein molecules to other proteins, peptides, or amine functional groups, etc. For example, the literature is rich in coupling agents such as carbodiimide, diisocyanate, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various coupling agents known in the art, but rather to illustrate more common coupling agents.

[0189] In some embodiments, when the PSMA ligand is an antibody, it is contemplated to first derivatize the antibody and then couple the therapeutic agent to the derivative product. Suitable cross-linking agents used in this method include, for example, SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate and SMPT, 4-succinimidyl-oxycarbonyl-methyl-(2-pyridyldithio)toluene.

[0190] In some embodiments, when the agent is a protein toxin, it can be fused to a PSMA ligand by genetic methods to form a hybrid immunotoxin fusion protein. The fusion protein can include additional peptide sequences, such as a peptide spacer that functionally links the PSMA ligand and the toxin, provided that it does not have an undue effect on the targeting or toxin activity of the fusion protein. As is well known in the art, in some embodiments, these proteins can be linked by a peptide linker or spacer, such as a glycine-serine spacer peptide, or a peptide hinge. Thus, for example, when the PSMA ligand is a PSMA antibody, the C-terminus of the PSMA antibody can be fused to the N-terminus of the protein toxin molecule to form an immunotoxin that retains the binding properties of the PSMA antibody. Other fusion procedures are known to those skilled in the art. To express the fusion immunotoxin, the nucleic acid encoding the fusion protein is inserted into an expression vector that stably expresses the fusion protein in mammalian cells, such as CHO cells, according to standard methods. The fusion protein can be isolated and purified from cells or culture supernatants using standard methods, such as a PSMA affinity column.

[0191] Examples of anti-cancer agents for use provided herein include, but are not limited to, cytotoxic agents, chemotherapeutic agents, and agents that act on tumor neovasculature. Cytotoxic agents include, but are not limited to, cytotoxic radionuclides, chemical toxins, and protein toxins. Examples of cytotoxic radionuclides or radiotherapy isotopes include, for example, 225 Ac, 211 At, 212 Bi, 213 Bi, 212 Pb, 224 Ra, 223 alpha-emitting isotopes such as Ra. Examples of cytotoxic radionuclides or radiotherapy isotopes include, for example, 186 Rh, 188 Rh, 177 Lu, 90 Y, 131 I,67 Cu, 64 Cu, 153 Sm, 166 Examples of beta-emitting radionuclides include Ho and the like. In some examples, the cytotoxic radionuclide may emit Auger electrons and / or low-energy electrons, and the isotopes 123 I, 124 I, 125 I, 131 I, 75 Br, 77 Br, and 18 include F.

[0192] Radionuclides are typically coupled to an antibody or an antigen-binding fragment thereof by chelation. For example, in the case of metallic radionuclides, a bifunctional chelator is commonly used to link the isotope to the antibody or other protein of interest. Typically, the chelator is first bound to the antibody, and then the chelator-antibody conjugate is contacted with the metallic radioisotope. Several bifunctional chelators have been developed for this purpose, including amino acids of the diethylenetriaminepentaacetic acid (DTPA) series described in (Patent Document 14), (Patent Document 15), and (Patent Document 16), which are incorporated herein by reference. As another example, a hydroxamic acid-based bifunctional chelating agent is described in (Patent Document 17), the content of which is incorporated herein by reference. Another example is a chelating agent named p-SCN-Bz-HEHA (1,4,7,10,13,16-hexaazacyclo-octadecane-N,N’,N’’,N’’’,N’’’’,N’’’’’-hexaacetic acid) (Non-Patent Document 34), which is 225 an effective chelator for radioactive metals such as Ac. Yet another example is DOTA (1,4,7,10-tetraazacyclododecane N,N’,N’’,N’’’-tetraacetic acid), which is a bifunctional chelating agent that can be used in a two-step method of labeling and then conjugating (see (Non-Patent Document 35)).

[0193] Examples of chemical toxins or chemotherapeutic agents include, but are not limited to, members of the enediyne family of molecules such as calicheamicin and esperamicin. Also included as chemical toxins or chemotherapeutic agents are pyrrolobenzodiazepine (PBD) dimers (e.g., SJG-136, SG2000, SG2202, SG2285 as described in Non-Patent Document 36), calicheamicin, colchicine, ispinesib (a novel small molecule inhibitor of kinesin spindle protein), combretastatin (e.g., combretastatin A4), maytansinoid DM4 (N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl) maytansine) and maytansinoid DM1 (mertansine) and other maytansine derivatives, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatin, etoposide, bleomycin, and / or 5-fluorouracil. Other anti-tumor agents include dolastatin (Patent Documents 18 and 19) and its derivatives. Examples of dolastatin and its derivatives include dolastatin 10 (dolavaline-valine-dolisoleucine-dolaproline-dolaphenine) and its derivative auristatin PHE (dolavaline-valine-dolisoleucine-dolaproline-phenylalanine-methyl ester) (Non-Patent Documents 37; Non-Patent Document 38), aurastatin E (e.g., monomethyl auristatin norephedrine), aurastatin F (e.g., monomethyl auristatin phenylalanine), etc. Also included as toxins are toxic lectins, plant toxins such as ricin toxins, abrin toxins, modeccin toxins, botulinum toxins, and diphtheria toxins. Other chemotherapeutic agents are known to those of skill in the art and can be used as provided herein.

[0194] Examples of agents that act on tumor blood vessels include, but are not limited to, tubulin binders such as tubulysin and its derivatives (e.g., anti-tubulin agents) (Non-Patent Document 39), combretastatin A4 (Non-Patent Document 40), angiostatin and endostatin (reviewed in Non-Patent Document 41, which is incorporated herein by reference), and interferon-inducible protein 10 (Patent Document 20). Several other angiogenesis inhibitors are contemplated and include the following: 2ME2, angiostatin, angiozyme, anti-VEGF RhuMAb, Apra (CT-2584), avicine, benefin, BMS275291, carboxyamidotriazole, CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC 412), CM101, combretastatin A-4 prodrug, EMD 121974, endostatin, flavopiridol, genistein (GCP), IM-862, ImmTher, interferon alpha, interleukin 12, gefitinib (ZD1839), marimastat, metastat (Col-3), neovastat, octreotide, paclitaxel, penicillamine, photofrin, photopoint, PI-88, prinomastat (AG-3340), PTK787 (ZK22584), RO317453, solimastat, squalamine, SU 101, SU 5416, SU-6668, slazisat (FCE 26644), slamine (metalate), tetrathiomolybdate, thalidomide, TNP-470, and vitaxin. Further angiogenesis inhibitors are described in Non-Patent Document 42, which is incorporated herein by reference. Such agents are contemplated for use with the PSMA ligand conjugates provided herein.

[0195] In some embodiments, the PSMA ligand conjugate is a PSMA antibody-drug conjugate. Non-limiting examples of PSMA antibody-drug conjugates are described in (Patent Document 21) and (Patent Document 22), and each such example thereof is incorporated herein by reference. In some embodiments, the PSMA antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof that specifically binds to PSMA, and is conjugated to an auristatin such as a dolastatin 10 derivative, specifically, MMAE (also referred to herein as monomethyl auristatin E or monomethyl auristatin norephedrine) or MMAF (also referred to herein as monomethyl auristatin F or monomethyl auristatin phenylalanine).

[0196] The antibody or antigen-binding fragment thereof can, in some embodiments, be conjugated to MMAE or MMAF by a compound of the following formula (Formula 1): -A n -Y m -Z m -X n -W n -(wherein A is a carboxylic acid acyl unit; Y is an amino acid; Z is an amino acid; X and W are each a self-immolative spacer; n is an integer of 0 or 1; m is an integer of 0 or 1, 2, 3, 4, 5, or 6). In some embodiments, the ADC is represented by the formula (Formula 2): L-{A n -Y m -Z m -X n -W n -D} p (wherein L is an antibody or an antigen-binding fragment thereof that binds to PSMA, D is MMAE or MMAF, and p is an integer of 1, 2, 3, 4, 5, 6, 7, or 8). The other components are as described above. In one embodiment, the carboxylic acid unit "A n " is linked to the antibody or antigen-binding fragment via a sulfur atom from the antibody or antigen-binding fragment.

Chemical formula

[0197] In one embodiment, A is [Chemical Formula] (wherein q is from 1 to 10). Thus, in one embodiment, the conjugate is [Chemical Formula] (wherein L, Y, Z, X, W, D, n, m, q, and p are as defined above).

[0198] In another embodiment, A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl, m-succinimidobenzoyl, 4-(p-succinimidophenyl)-butyryl, 4-(2-acetamido)benzoyl, 3-thiopropionyl, 4-(1-thioethyl)-benzoyl, 6-(3-thiopropionylamide)-hexanoyl, or maleimidocaproyl. In a further embodiment, A is maleimidocaproyl. Representative examples of various carboxylic acid acyl units and methods for their synthesis and conjugation are described in (Patent Document 23), the entire content of which, specifically with examples and methods for their synthesis and conjugation, is incorporated herein by reference.

[0199] In another embodiment, Y is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or proline. In yet another embodiment, Y is valine. In a further embodiment, Z is lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro group, histidine, ornithine, ornithine protected with acetyl or formyl, or citrulline. In yet another embodiment, Z is citrulline. In one embodiment, Y m -Z m is valine-citrulline. In another embodiment, Y m -Z m is a protein sequence that can be selectively cleaved by a protease.

[0200] In a further embodiment, X is a compound having the formula

Chemical formula

Chemical formula

[0201] In some embodiments, W is

Chemical formula

[0202] In one embodiment, the compound of formula 1 is maleimidocaproyl. Maleimidocaproyl has been used to conjugate two specific auristatins to an anti-CD30 mAb (AC10) (Non-Patent Document 43). Maleimidocaproyl reacts with a thiol group to form a thioether.

[0203] MMAE or MMAF can be conjugated to an antibody or an antigen-binding fragment thereof using methods known in the art (see, e.g., Non-Patent Document 44) or as described herein. In some embodiments, two or more MMAE or MMAF molecules are conjugated to an antibody or an antigen-binding fragment thereof. In other embodiments, one, two, three, four, five, six, seven, or eight MMAE or MMAF molecules are conjugated to an antibody or an antigen-binding fragment thereof. In yet other embodiments, at least two, three, four, or five MMAE or MMAF molecules are conjugated to an antibody or an antigen-binding fragment thereof. In further embodiments, two, three, four, or five MMAE or MMAF molecules are conjugated to an antibody or an antigen-binding fragment thereof.

[0204] In some embodiments, the PSMA ligand conjugate is a PSMA antibody (or an antigen-binding fragment thereof)-maleimidocaproyl-valine-citrulline-p-aminobenzyl oxycarbonyl-monomethyl auristatin norephedrine, a PSMA antibody (or an antigen-binding fragment thereof)-maleimidocaproyl-valine-citrulline-p-aminobenzyl carbamate-monomethyl auristatin norephedrine, a PSMA antibody (or an antigen-binding fragment thereof)-maleimidocaproyl-monomethyl auristatin norephedrine, a PSMA antibody (or an antigen-binding fragment thereof)-maleimidocaproyl-valine-citrulline-p-aminobenzyl oxycarbonyl-monomethyl auristatin phenylalanine, a PSMA antibody (or an antigen-binding fragment thereof)-maleimidocaproyl-valine-citrulline-p-aminobenzyl carbamate-monomethyl auristatin phenylalanine, or a PSMA antibody (or an antigen-binding fragment thereof)-maleimidocaproyl-monomethyl auristatin phenylalanine. In any of the foregoing, the PSMA antibody or an antigen-binding fragment thereof may be any of the antibodies or antigen-binding fragments provided herein.

[0205] PSMA antibody-drug conjugates have been found to have particularly high levels of selectivity, for example, when comparing the killing of non-PSMA-expressing cells to the killing of PSMA-expressing cells. Thus, in some embodiments, the PSMA antibody-MMAE conjugate or the PSMA antibody-MMAF conjugate has a selectivity for C4-2 cells or LNCaP TM cells over PC-3 TM cells of at least 250. In other embodiments, this selectivity is at least 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 17500, 20000 or more. In some embodiments, this selectivity is between 250-500, 500-750, 750-1000, 1000-2000, 2000-5000, 5000-10000, 10000-15000, or 15000-20000. "Selectivity," as defined herein, is the ratio of the IC TM value of the PSMA antibody-MMAE conjugate or the PSMA antibody-MMAF conjugate in C4-2 cells or LNCaP TM cells (PSMA-expressing cells) to that in PC-3 50 cells (non-PSMA-expressing cells).

[0206] PSMA antibody-drug conjugates mediate PSMA-expressing cell-specific killing at very low concentrations, such as at or near picomolar concentrations, in some embodiments. PSMA antibody-drug conjugates exhibit an IC -10 of less than about 1×10 -11 M, less than about 1×10 -12 M, or less than about 1×10 50 M in some embodiments. In some embodiments, the IC 50 is about 1.5×10 -11is achieved at a concentration of less than M. In other embodiments, the PSMA antibody-drug conjugate has an IC between 10-210, 40-210, 60-210, or 65-210 picomolar (pM). 50 is shown. In yet other embodiments, the PSMA antibody-drug conjugate has an IC of about 10, 40, 60, or 80 pM. 50 is shown. In yet other embodiments, the PSMA antibody-drug conjugate has an IC of about 11, 42, 60, or 83 pM. 50 is shown.

[0207] The level of cell death can be determined by any of the methods provided herein or otherwise known in the art.

[0208] "Anti-androgen", as used herein, refers to an agent that blocks (e.g., inhibits) the action of androgen hormones and molecules regulated by androgens. Adrenergic receptor antagonists are considered anti-androgens herein. The term "anti-androgen" includes anti-androgens, anti-androgen analogs, and anti-androgen derivatives. In prostate cancer, anti-androgens block the activity of testosterone, thereby usually delaying the growth of prostate cancer. In some embodiments, the anti-androgen blocks the enzyme cytochrome P450 17A1 encoded by the CYP17A gene. Anti-androgens may be steroidal or non-steroidal (also referred to as "pure"). Examples of anti-androgens for use provided herein include, but are not limited to, abiraterone (ZYTIGA®), enzalutamide (XTANDI®), nilutamide (NILANDRON®), flutamide (EULEXIN®), bicalutamide (CASODEX®), and ortelone (TAK-700, Tokai Pharmaceuticals, Inc.). Potent anti-androgens such as, for example, enzalutamide, abiraterone, ARN 509 (Aragon Pharmaceuticals, Inc.), and galeterone (TOK-001 or VN / 124-1, Tokai Pharmaceuticals, Inc.), which are commonly used in advanced metastatic castration-resistant prostate cancer and affect the expression of numerous molecules regulated by androgens, such as PSMA expression.

[0209] As used herein, "potent" anti-androgens or "second-generation" anti-androgens include anti-androgens that retain activity in a cellular environment of increased androgen receptor expression relative to wild-type expression. Potent anti-androgens include those that are active in subjects with castration-resistant prostate cancer. Potent anti-androgens also include anti-androgens that bind to the androgen receptor with a higher relative affinity than commonly used clinical anti-androgens (e.g., bicalutamide, nilutamide, flutamide). Examples of potent anti-androgens include, but are not limited to, enzalutamide, abiraterone, ARN-509, galeterone, and diarylthiohydantoin RD162 and MDV310, 3β-hydroxyandrost-5,16-diene (HAD), and androst-1,4-diene-3,17-dione-17-ethylene ketal (OAK) (incorporated herein by reference (Non-Patent Document 45)). For example, abiraterone inhibits the CYP17A1 enzyme. As another example, enzalutamine is an androgen receptor antagonist. As yet another example, galeterone is a selective CYP17 inhibitor and an androgen receptor antagonist. Methods that can be used to identify potent anti-androgens are described, for example, in (Non-Patent Document 46) incorporated herein by reference. Any anti-androgen described herein can be used in any method provided herein.

[0210] Compounds that increase cell surface expression of PSMA may be mTOR inhibitors. Examples of mTOR inhibitors for use provided herein include, but are not limited to, rapamycin, everolimus (AFINITOR®, ZORTRESS®), and temsirolimus (TORISEL®). The term "mTOR inhibitor" includes mTOR inhibitors, mTOR inhibitor analogs, and mTOR inhibitor derivatives.

[0211] Further provided herein are compositions, such as pharmaceutical compositions, comprising a compound that increases cell surface expression of PSMA, a PSMA ligand conjugate, or a combination of the two. In some embodiments, the composition is administered to a subject (e.g., a mammal such as a human). In some embodiments, a composition comprising a PSMA ligand conjugate is administered to a subject, and a separate composition comprising a compound that increases cell surface expression of PSMA is administered to the subject either sequentially or simultaneously. Alternatively, in some embodiments, a composition comprising both a PSMA ligand conjugate and a compound that increases cell surface expression of PSMA is administered to a subject.

[0212] In some embodiments, a composition comprising a compound that increases cell surface expression of PSMA is first administered to a subject, and then immediately, within 1 hour, within several hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, or within more than 1 week, a composition comprising a PSMA ligand conjugate is administered to the subject. Any of the compositions can be administered once or more than once (e.g., 2 times, 3 times, etc.).

[0213] As used herein, a "compound that increases cell surface expression of PSMA" refers to a compound that increases cell surface expression of PSMA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200% or more compared to normal cell surface expression of PSMA-expressing cells or compared to cell surface expression in the absence of such a compound. For example, an anti-androgen can, in some embodiments, increase cell surface expression of PSMA on cancer cells by at least 20% or more.

[0214] In some embodiments, the composition comprises a compound and / or a PSMA ligand conjugate that increases the cell surface expression of PSMA, in combination with a physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer. As used herein, "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" includes any and all salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. "Pharmaceutically acceptable carrier" as used herein refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. The term "carrier" refers to a natural or synthetic organic or inorganic component that facilitates the application of the active ingredient in combination therewith. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion).

[0215] In some embodiments, the composition can be administered to a subject in a pharmaceutically acceptable amount and in a pharmaceutically acceptable composition. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the biological activity effectiveness of the active ingredient (e.g., PSMA ligand, anti-cancer agent, cytotoxic agent, anti-androgen, mTOR inhibitor). Such compositions can contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents such as adjuvants, chemokines, and immunopotentiating agents including cytokines. When used in medicine, the salts should be pharmaceutically acceptable, but it is not excluded that salts that are not pharmaceutically acceptable can be advantageously used to prepare their pharmaceutically acceptable salts.

[0216] The salts retain the desired biological activity of the parent compound and do not exert any undesired toxicological effects (see, for example, (Non-Patent Document 47)). Examples of such salts include acid addition salts and base addition salts. Examples of acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and those derived from non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Examples of base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc. The pharmaceutical composition can contain a suitable buffer containing acetic acid in the salt; citric acid in the salt; boric acid in the salt; and phosphoric acid in the salt.

[0217] In some embodiments, the composition can contain a suitable preservative such as benzalkonium chloride; chlorobutanol; parabens, and / or thimerosal.

[0218] In some embodiments, the composition can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. All methods include the step of combining the active compound(s) (e.g., a PSMA ligand, an anticancer agent, a cytotoxic agent, and / or a compound that upregulates the cell surface expression of PSMA) with a carrier that constitutes one or more accessory ingredients. In some embodiments, the composition is prepared by uniformly and intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then shaping the product if necessary.

[0219] Compositions suitable for parenteral administration advantageously comprise a sterile aqueous or non-aqueous preparation of a PSMA ligand conjugate and / or a compound that increases the cell surface expression of PSMA (e.g., anti-androgen, mTOR inhibitor), which is preferably isotonic with the recipient's blood. This preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension of a non-toxic diluent or solvent acceptable for parenteral use, such as a solution of 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic saline. In addition, sterile, non-volatile oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating non-volatile oil containing synthetic monoglycerides or diglycerides can be used. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in (Non-Patent Document 48). Any of the compositions provided herein can be sterile.

[0220] Active compounds (e.g., PSMA ligands, anti-cancer agents, cytotoxic agents, and / or compounds that upregulate the cell surface expression of PSMA) can be prepared with carriers that protect the compound from rapid release, such as depot formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented and are generally known to those skilled in the art. See, for example, (Non-Patent Document 49).

[0221] The composition can be administered by any conventional route, including by injection or by slow-release infusion over time. Administration may be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavitary, intratumoral, or transdermal. When the composition is used therapeutically, preferred routes of administration include intravenous and pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. In general, such systems should utilize components that do not significantly impair the biological properties of the antibody, such as paratope binding ability (see, e.g., Non-Patent Document 50, incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for manufacturing antibody aerosol agents without undue experimentation.

[0222] The compositions provided herein can, in some embodiments, be administered in an effective amount. An "effective amount" is the amount of an active compound (e.g., a PSMA ligand conjugate and / or a compound that increases cell surface expression of PSMA) that, alone or together with further doses, produces a desired response, e.g., inhibits cell proliferation of PSMA-expressing cells and / or kills PSMA-expressing cells. In the case of cancer, this may include, for example, merely temporarily delaying the progression of the cancer, but more preferably, it includes permanently halting the progression of the cancer. This can be monitored by routine methods. The desired response to the treatment of cancer or other disease or condition may also be a delay in onset, or prevention of the onset of cancer or other disease or condition.

[0223] Such effective amounts will, of course, depend on such factors as the particular condition being treated (e.g., cancer expressing PSMA), the severity of the condition, the parameters of the individual patient including age, health, size and weight, the duration of the treatment, the nature of any combination therapy (if any), the specific route of administration, and factors within the knowledge and expertise of the medical practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with merely routine experimentation. It is generally preferred to use the maximum dosage of the individual components or combinations thereof, i.e., the maximum safe dosage based on sound medical judgment. However, it will be understood by those of ordinary skill in the art that a patient / subject may assert a lower dosage or tolerance for medical reasons, psychological reasons, or substantially any other reason.

[0224] The compositions provided herein, in some embodiments, are sterile and contain an effective amount of a PSMA ligand conjugate and / or a compound that increases cell surface expression such as PSMA, in a weight or volume unit suitable for administration to a patient / subject to provide a desired response. This response can be measured, for example, by determining the physiological effect of the composition, such as tumor regression or alleviation of symptoms. Other assays are known to those of ordinary skill in the art and can be used to measure the response level.

[0225] The dosage of the composition to be administered to a subject can be selected according to various parameters, specifically according to the method of administration used and the condition of the subject. Other factors include the desired duration of treatment. If the response of the subject is insufficient at the initial applied dosage, higher dosages (or effective higher dosages by a different, more local delivery route) can be used to the extent tolerated by the patient.

[0226] The synergistic effects obtained by various combinations of the PMSA ligand conjugate and the compounds provided herein, such as anti-androgens or mTOR inhibitors, can result in effective dosages that are less than the dosages required for efficacy when any of the components of the combination are used alone. A further advantage of the dosage reduction effect is that a broader safety profile can be achieved, i.e., the additional benefit of fewer adverse side effects can be obtained.

[0227] For example, the dosage of the PSMA ligand conjugate and / or the compound that increases the cell surface expression of PSMA can range from about 10 μg / kg to about 100,000 μg / kg. Depending on the composition, the dosage can be delivered continuously, such as by a sustained pump, or intermittently. The desired time intervals for multiple administrations of a particular composition can be determined by one of ordinary skill in the art without undue experimentation. Other protocols for composition administration, where the dosage, administration schedule, administration site, administration method, etc. are different from those described above, are known to those of ordinary skill in the art.

[0228] In some embodiments, the dosage of the PSMA ligand conjugate is administered intravenously. In such embodiments, the dosage of the PSMA ligand conjugate can be from about 1.0 mg / kg to 2.5 mg / kg. For example, in some embodiments, the dosage of the PSMA ligand conjugate administered intravenously is 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, or 2.5 mg / kg. In some embodiments, the dosage of the PSMA ligand conjugate administered intravenously is from about 1.0 mg / kg to 2.3 mg / kg. In one embodiment, the PSMA ligand conjugate is a PSMA ADC, and the PSMA ADC is provided at a dosage of from about 1.8 mg / kg to 2.3 mg / kg.

[0229] The length of time for which the PSMA ligand conjugate is administered intravenously can vary. In some embodiments, the PSMA ligand conjugate can be administered intravenously for 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, or 90 minutes.

[0230] In some embodiments, the PSMA ligand conjugate is administered intravenously at an interval of once a week, once every two weeks, or once every three weeks, etc., until a total of 4, 6, or 8 doses, for example. In some embodiments, the PSMA ligand conjugate is administered intravenously more than twice a week.

[0231] In some embodiments, the PSMA ligand conjugate can be administered intravenously for about 60 minutes once every three weeks at a dose of about 1.0 mg / kg to 2.3 mg / kg until a total of 8 doses.

[0232] In some embodiments, the compounds provided herein, such as anti-androgens, are administered in the form of oral capsules. In such embodiments, the dose of such a compound can be from about 40 mg to about 160 mg (e.g., 20 mg or 40 mg per capsule). For example, in some embodiments, the dose of such a compound administered orally is 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, or 160 mg. In some embodiments, the oral dose of such a compound is administered once or twice a day for about 21 days to about 28 days. In some embodiments, the dose of such a compound is 80 mg (e.g., two oral capsules, 40 mg each) to 160 mg (e.g., four oral capsules, 40 mg each) and is administered once a day (OD) for 28 days. In some embodiments, such a compound is enzulatamide at a dose of 80 mg (e.g., two oral capsules, 40 mg each) to 160 mg (e.g., four oral capsules, 40 mg each) and is administered once a day (OD) for 28 days.

[0233] In some embodiments, the oral dosage of the compounds provided herein, such as anti-androgens, can be from about 500 mg to about 1000 mg. For example, in some embodiments, the dosage of such compounds administered orally is 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg. In some embodiments, the oral dosage of such compounds is administered once or twice a day for about 21 to about 28 days. In some embodiments, the dosage of such compounds is 500 - 1000 mg and is administered once a day (OD) for 28 days. In some embodiments, such compounds are abiraterone or abiraterone acetate at a dosage of 500 mg - 1000 mg and are administered once a day (OD) for 28 days.

[0234] In some embodiments, the mTOR inhibitor is administered at an I.V. dosage of about 5 - 25 mg at intervals of once a week. In another embodiment, the mTOR inhibitor is administered at an I.V. dosage of 5 - 10 mg at intervals of once a week. In one embodiment, the mTOR inhibitor is temsirolimus (Toresal®) which is administered at 25 mg I.V. over 30 - 60 minutes at intervals of once a week.

[0235] Generally, the dosage of the radionuclide delivered by the PSMA ligand conjugates provided herein can range from about 0.01 mCi / kg to about 10 mCi / kg. In some embodiments, the dosage of the radionuclide ranges from about 0.1 mCi / kg to about 1.0 mCi / kg. In one embodiment, the PSMA ligand conjugate is provided at an I.V. dosage of about 1 - 10 GBq 131 is I-MIP-1095. In another embodiment, 131 I-MIP-1095 is provided in a dosage range of 2 - 8 GBq. In yet another embodiment, the average dose is about 5 GBq. The optimal dose for a given isotope can be determined experimentally by simple routine titration experiments well known to those skilled in the art.

[0236] For example, for purposes of testing or veterinary treatment, administering the compositions provided herein to mammals other than humans is carried out under substantially the same conditions as described above.

[0237] The compositions provided herein (e.g., comprising a PSMA ligand conjugate and / or a compound that increases cell surface expression of PSMA) have diagnostic and therapeutic utility in vitro and in vivo. For example, these compounds can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to effect the treatment, prevention, or diagnosis of cancer or other diseases or conditions. As used herein, the term "subject" is intended to include humans and non-human animals. Preferred subjects include human patients having a disorder characterized by expression of PSMA, typically abnormal expression (e.g., overexpression).

[0238] The compositions provided herein can, in some embodiments, be used in combination with other therapeutic treatment modalities. Such other therapies include surgery, radiation, cryosurgery, hyperthermia, hormone therapy, chemotherapy, vaccines, and other immunotherapies.

[0239] Subjects with prostate cancer, in some embodiments, have received, are receiving, or are scheduled to receive hormonal therapy. Thus, in some embodiments, the compositions provided herein can be administered to a subject after, together with, or before hormonal therapy, such as in the case of prostate cancer. Examples of hormonal therapy for prostate cancer include, but are not limited to, the following: luteinizing hormone-releasing hormone agonists (e.g., leuprolide, goserelin, and buserelin), which can stop the testes from producing testosterone; antiandrogens (e.g., flutamide, bicalutamide, enzalutamide, and nilutamide) discussed elsewhere herein, which can block the action of androgens such as testosterone; drugs that can prevent the adrenal glands from producing androgens (e.g., ketoconazole and aminoglutethimide); orchiectomy, which is a surgical procedure that removes one or both testes, the main source of male hormones such as testosterone, to reduce the amount of hormone produced; and estrogen, which can prevent the testes from producing testosterone.

[0240] Multiple subjects can benefit from the methods and compositions provided herein. In some embodiments, such subjects have progressive metastatic castration-resistant prostate cancer despite having castrate levels of serum testosterone (e.g., less than 50 mg / dL) and having received pre-chemotherapy with docetaxel. In other embodiments, the subject has metastatic castration-resistant prostate cancer, has received pretreatment with taxane chemotherapy, and administration of abiraterone and / or enzalutamide is ongoing. In yet other embodiments, the subject has progressive metastatic castration-resistant prostate cancer despite having castrate levels of serum testosterone and has received pretreatment with abiraterone and / or enzalutamide but has not received pretreatment with cytotoxic chemotherapy. In yet other embodiments, the subject has progressive metastatic castration-resistant prostate cancer despite having castrate levels of serum testosterone and has received one pretreatment with abiraterone and / or enzalutamide. In further embodiments, the subject has progressive metastatic castration-resistant prostate cancer despite having castrate levels of serum testosterone and has not received pretreatment with abiraterone and / or enzalutamide. In further embodiments, the subject has metastatic castration-resistant prostate cancer that is asymptomatic or minimally symptomatic despite having castrate levels of serum testosterone and has not received pretreatment with abiraterone and / or enzalutamide. In some embodiments, the subject has stable metastatic castration-resistant prostate cancer and is receiving treatment with abiraterone and / or enzalutamide. In other embodiments, the subject has biochemically recurrent prostate cancer and has previously received primary therapy (e.g., radical prostatectomy (e.g., open, laparoscopic, or robot-assisted) or radiation therapy (e.g., dose-escalated three-dimensional conformal RT, intensity-modulated RT, brachytherapy, or combinations thereof).In yet other embodiments, the subject has localized high-risk prostate cancer (e.g., prostate-specific antigen (PSA) greater than 10 nanograms per milliliter (ng / ml); PSA velocity greater than 2 ng / ml per year (defined as an increase in PSA of greater than 2 ng / ml over the previous 12 months); Gleason score of 7 (4+3) or greater; or Gleason score 6 if either PSA is greater than 10 ng / ml or PSA velocity is greater than 2 ng / ml / year) and is a candidate for prostatectomy.

[0241] Further provided herein is a kit comprising the composition(s). In some embodiments, the kit includes a container containing a compound that increases cell surface expression of PSMA and a container containing a PSMA ligand conjugate (or a component thereof). The kit can further contain at least one additional reagent provided herein. In some embodiments, the kit can include a carrier compartmentalized to tightly enclose therein one or more containers or a series of containers, such as test tubes, vials, flasks, bottles, syringes, etc. One container or a series of containers can contain one or more compounds that increase cell surface expression of PSMA. Another container or a series of containers can, in some embodiments, contain a PSMA ligand conjugate (or a component thereof). The components of the kit can be packaged in an aqueous medium or in lyophilized form. The components of the conjugate can be supplied in fully conjugated form, in intermediate form, or as separate moieties to be conjugated by the user of the kit.

[0242] The kit can also, in some embodiments, include a diluent, and / or instructions for reconstituting the lyophilized form of the PSMA ligand conjugate and / or the compound for increasing cell surface expression of PSMA, or instructions for diluting the aqueous components of the kit. The kit can also include instructions for using the compound for increasing cell surface expression of PSMA and / or the PSMA ligand conjugate in combination.

[0243] The present invention will be described in more detail by the following examples, which should in no way be construed as further limitations. The entire contents of all references cited throughout this application (including reference papers, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated by reference into this specification. However, the citation of any reference is not intended to admit that the reference is prior art.

Example

[0244] Example 1: PSMA ADC exhibits synergistic effects with anti-androgens and rapamycin For the inhibitors, the anti-proliferative activities were tested individually and in combination over a series of concentrations in a matrix format. The inhibition data were compared with the Bliss prediction, and the difference between the experimental and predicted results was calculated for each point in the matrix. A positive difference indicates an effect exceeding additivity, i.e., a synergistic effect. A negative difference indicates antagonism. As follows, the statistical significance of the results was evaluated.

[0245] The mean inhibition data and Bliss differences for PSMA antibody-drug conjugates (PSMA ADC) in combination with enzalutamide, abiraterone, or rapamycin are shown in FIG. 2. The heat map shows the Bliss differences as positive (green), negative (red), or nearly zero (yellow). Frames are used to indicate statistically significant Bliss differences and the corresponding percent inhibition. PSMA ADC showed similar single-agent activity against LNCaP and C4-2 cells, with IC 50 values of approximately 200 pM in each case. These results are consistent with previous reports (Non-Patent Documents 51, 52). Enzalutamide, abiraterone, and rapamycin each inhibited the growth of LNCaP cells (FIG. 2A), but showed only minimal anti-proliferative effects against C4-2 cells when used as single agents (FIG. 2B).

[0246] Despite the lack of direct antiproliferative activity in C4-2 cells, enzalutamide, abiraterone, and rapamycin all significantly enhanced the activity of PSMA ADC. The Bliss difference reached nearly 40% and was statistically significant across a range of inhibitor concentrations and inhibition levels (Figure 2B). No significant antagonism was observed for these combinations under any conditions. Statistically significant synergy was also observed in LNCaP cells for the combinations of PSMA ADC / enzalutamide and PSMA ADC / abiraterone (Figure 2A). However, compared to C4-2 cells, its spread and magnitude were more limited. Similarly, in LNCaP cells, the Bliss difference was also positive across the concentration matrix for the combination of PSMA ADC / rapamycin. However, none of the values reached statistical significance.

[0247] Mean inhibition data and Bliss differences are shown in Figure 9 for PSMA ADC in combination with the antiandrogens ARN-509 or TOK-001. The heatmap shows Bliss differences that are positive (green), negative (red), or approximately 0 (yellow). ARN-509 and TOK-001 each inhibited the growth of LNCaP cells (Figure 9A), but showed minimal antiproliferative effects against C4-2 cells when used as single agents (Figure 9B). Despite the lack of direct antiproliferative activity in C4-2 cells, ARN-509 and TOK-001 enhanced the activity of PSMA ADC.

[0248] To determine whether the observed synergy was specific to the combination of PSMA ADC / antiandrogen, small molecule ligands were tested in combination with antiandrogens. Mean inhibition data and Bliss differences are shown in Figure 11 for the conjugate of a small molecule PSMA ligand-antitumor agent (EC1069) in combination with enzalutamide. The heatmap shows Bliss differences that are positive (green), negative (red), or approximately 0 (yellow) for synergy.

[0249] As a first step in analyzing the mechanism of the synergistic effect, anti-androgen and rapamycin were used in combination with free MMAE and unmodified PSMA mAb. Free MMAE showed sub-nanomolar potency (IC 50 , approximately 0.5 nM) against both LNCaP and C4-2 cells, as expected (Non-Patent Document 52). When paired with enzalutamide or abiraterone, free MMAE showed additive activity in LNCaP cells and additive to weak synergistic activity on C4-2 (Figure 3). Similar results were obtained when docetaxel, another microtubule inhibitor, was used in combination with enzalutamide. Examples of statistically significant synergy for the docetaxel / enzalutamide combination were sporadic (Figure 3).

[0250] The rapamycin / MMAE combination showed additive effects in LNCaP and moderate synergy in C4-2 cells (Figure 4). Thus, rapamycin did not show strong synergy with MMAE compared to PSMA ADC on both cell lines. Nevertheless, the rapamycin / MMAE combination appears to be as effective as or more potent than the enzalutamide / MMAE or abiraterone / MMAE combinations in C4-2 cells. Weak to moderate synergy was observed between rapamycin and enzalutamide in both cell lines. In C4-2 cells, non-inhibitory concentrations of enzalutamide enhanced the weak anti-proliferative activity of rapamycin (Figure 4).

[0251] Unmodified PSMA mAb was inactive both alone and in combination with enzalutamide, abiraterone, or rapamycin (Figure 5). Due to the extremely limited activity of the individual drugs or combinations, the assays were performed only twice in some cases. Thus, the robust synergy observed with the combination of PSMA ADC and anti-androgen is specific to this conjugate and not reproduced in any of its individual components.

[0252] As a control, a mock combination was prepared and the PSMA ADC was added to the assay plate by two separate additions to evaluate synergy. As expected, the mock combination did not show statistically significant synergy or antagonism in either cell line.

[0253] Example 2: Anti-androgens reversibly increase PSMA expression in a time- and dose-dependent manner In further studies, treatment-induced changes in PSMA expression were investigated as a potentially promising correlate of synergy. Flow cytometry and Western blotting were used to evaluate cell surface and total PSMA, respectively.

[0254] Enzalutamide and abiraterone increased the cell surface levels of PSMA in a dose-dependent manner in both cell lines (Figures 6A - D). PSMA expression was approximately doubled 7 days after treatment at anti-androgen concentrations above 1 mM. In contrast, rapamycin increased PSMA expression only in C4-2 cells (Figures 6E - F). Low nanomolar concentrations of rapamycin were sufficient to induce more than a two-fold increase in PSMA expression in C4-2 cells.

[0255] To investigate the kinetics of expression, C4-2 cells were cultured in enzalutamide (1 mM) for 3 weeks and then cultured for an additional 8 days in the absence of drug. Cell surface PSMA was measured by flow cytometry. PSMA expression increased steadily over time in the presence of enzalutamide. After 21 days, PSMA expression on the treated cells was 4-fold higher than that on untreated cells passaged in parallel. PSMA expression returned to baseline levels within 7 days of culture in the absence of enzalutamide (Figure 6G).

[0256] Treatment-induced upregulation of PSMA was also revealed by Western blotting (Figure 7). When the magnitude of the increase was determined by serial dilution of the lysates and semi-quantitative analysis of the Western blot, it was approximately 5-fold for enzalutamide-treated LNCaP cells (not shown). Enzalutamide, abiraterone, and rapamycin upregulated PSMA to a similar extent in C4-2 cells. The magnitude of PSMA expression approached, but rarely exceeded, that seen in cells cultured with charcoal-treated serum in the presence of antiandrogen. Overall, flow cytometry and Western blot data showed a consistent pattern of treatment-induced changes in PSMA expression.

[0257] Example 3: Treatment-Induced Changes in Components of the AR, PSA, and PI3K Pathways Levels of AR, PSA, total Akt, phospho-Akt (S473), total S6, and phospho-S6 were evaluated by Western blotting before and after treatment. Actin was examined as a measure of total cell loading. As expected, antiandrogen-induced upregulation of PSMA was accompanied by downregulation of PSA (Figure 7). In contrast, rapamycin increased PSA expression. This result is consistent with previous observations for rapamycin or its analogs (Non-Patent Documents 53 and 54). The simultaneous upregulation of PSMA and PSA by rapamycin is in contrast to the opposing effects of antiandrogen on PSMA and PSA. The effect of treatment on AR protein levels was mild (Figure 7).

[0258] Akt activation was a common response to anti-androgen treatment or rapamycin treatment in LNCaP cells. However, the effect of treatment on Akt activation in C4-2 cells was limited. Similarly, anti-androgens increased the phospho-S6 level in LNCaP cells but not in C4-2 cells. These findings are consistent with mTOR-mediated adaptation to anti-androgen treatment in LNCaP cells. Rapamycin-mediated activation of Akt is known to occur by disruption of a negative feedback loop containing insulin receptor substrate 1 (Non-Patent Documents 55, 56, 57, 58). As expected, rapamycin effectively abolished S6 phosphorylation in both LNCaP and C4-2, indicating that rapamycin is pharmacologically active in both cell lines (Figure 7).

[0259] Example 5: Additive to Weak Synergistic Effects for Combinations with Other Drugs In the exploratory study, additional compound groups were screened for activity alone and in combination with PSMA ADC. These agents included the Akt inhibitor MK-2206 (Non-Patent Document 59); the PI3K inhibitor GDC-0941 (Non-Patent Document 60); prednisone; and the kinesin spindle protein (ksp) Eg5 inhibitor SB-743921 (Non-Patent Document 61). MK-2206 and GDC-0941 are selective inhibitors of upstream components of the PI3K / mTOR pathway. Prednisone is a component of abiraterone-based and docetaxel-based treatment regimens in prostate cancer. SB-743931 was included in the inventors' study based on gene expression profiling in the cBio cancer genomics database (Non-Patent Document 62), and co-expression of genes (P = 0.000004) for PSMA and Eg5 in primary and metastatic prostate cancer tumors was shown. Each of these compounds showed additive to weak synergistic activity when combined with PSMA ADC (Figure 8). That is, the Bliss difference showed a tendency towards synergy for most conditions and reached statistical significance in a few isolated cases. No significant antagonism was observed for any combination. Similar results were obtained when prednisone was replaced with its active metabolite, prednisolone.

[0260] Table 2 (Figure 19) shows an overview of the synergistic effects observed in this study. Drugs: For each drug combination, the spread of the synergistic effect is represented as the percentage of evaluable conditions that resulted in a statistically significant synergistic effect across the matrix of drug concentrations. The drug combinations were reordered from the greatest to the least spread of the synergistic effect observed in LNCaP cells. The PSMA ADC / enzalutamide combination and the PSMA ADC / abiraterone combination showed the most extensive synergistic effect in LNCaP cells. Next were the combinations of prednisone or SB-743921 with PSMA ADC. For the PSMA ADC / SB-743921 combination, the Bliss difference could not be evaluated because some drug-drug combinations resulted in inhibition exceeding 100%, so the data should be interpreted with caution. It is necessary to further test this combination in a more focused concentration range. Most combinations did not show a significant synergistic effect in LNCaP cells.

[0261] The synergistic effect was more extensive in C4-2 cells. For the PSMA ADC / enzalutamide combination and the PSMA ADC / abiraterone combination, as was the case for the combination of PSMA ADC or MMAE with rapamycin, synergistic conditions occurred frequently in C4-2 cells. Other combinations (e.g., the combination of MK-2206 or GDC0941 with PSMA ADC) also showed a significant synergistic effect in C4-2 cells but not in LNCaP cells (Table 2 (Figure 19)). Combinations that did not show a significant synergistic effect in either LNCaP cells or C4-2 cells included the PSMA mAb and the PSMA ADC mock combination.

[0262] Materials and Methods Materials LNCaP cells were obtained from the American Type Culture Collection (ATCC). C4-2 is an androgen-independent subclone of LNCaP (Non-Patent Document 63). LNCaP and C4-2 are characterized by the T877A mutation of AR and the loss of PTEN expression (Non-Patent Documents 64 and 65). These cell lines enable the study of androgen dependence in a homogeneous genetic background. Both cell lines were passaged in RPMI1640 (Mediatech Inc.) supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 μM non-essential amino acids, 1% penicillin / streptomycin, and 10% fetal bovine serum (FBS, Life Technologies). Cells were used within 10 passages after being withdrawn from the shared bank. PSMA ADC was prepared as described (Non-Patent Document 51), and enzalutamide and abiraterone (parent drugs) were obtained from MedKoo Biosciences (Figure 1). Prednisone and prednisolone were purchased from Sigma; rapamycin was manufactured by EMD Millipore. GDC0941, MK-226, and SB-743921 were manufactured by Selleck Chemicals. The following antibodies were obtained from Santa Cruz Biotechnology: anti-AR (sc-7305, 441) and anti-PSA (sc-7638, C-19). Two mouse anti-PSMA mAbs were used. Namely, MAB544 (Maine Biotechnology) for Western blotting and 3.9 (Progenics) for flow cytometry. The following antibodies were obtained from Cell Signaling: total Akt (#2920S), phospho-Akt (S473, #4051L), total S6 (#2317S), and phospho-S6 (#2211L). The anti-actin antibody (MAB1501) was manufactured by Millipore. The isotype-specific secondary IRDye antibody was manufactured by LI-COR Biosciences.

[0263] Cell viability assay Cells were seeded at a density of 1×10 3 cells in 25 μL in a 384-well white microplate (Perkin Elmer) and cultured overnight. The next day, the cells were treated with one or two drugs in a total volume of 50 μL. The cultures were incubated at 37 °C for 7 hours. Cell viability was evaluated on day 0 and day 7 using CellTiter-Glo (Promega) and an Analyst GT luminescence reader (Molecular Devices) for luminescence at 560 nm. The percent inhibition of proliferation was calculated as (V u -V t ) / (V u -V0) × 100. Here, V u , V t , and V0 represent the survival value of untreated cells on day 7, the survival value of treated cells on day 7, and the survival value of cells before treatment on day 0, respectively. A value of 100% reflects complete inhibition of proliferation (V t = V0). Values above 100% reflect cytotoxic compounds or combinations, where V t < V0.

[0264] Flow cytometry For short-term treatment (≤7 days), cells were seeded onto a 24-well plate (BD Falcon) overnight at a density of 200,000 cells per 500 μL. The next day, the wells were replaced with 500 μL of fresh medium containing the inhibitor and incubated at 37 °C until the designated time point. For long-term treatment (>7 days), cells were seeded onto a 24-well plate (BD Falcon) overnight at a density of 500,000 cells per flask in 5 mL of medium. The next day, it was replaced with 5 mL of treated or untreated fresh medium. Thereafter, every week, the cells were detached with Cell Dissociation solution (Sigma), washed with fresh medium, counted using Vi-CELL XR (Beckman Coulter), divided into two T-25 flasks, and cultured with or without the inhibitor. Before analysis, the cells were detached, counted, and suspended in PBS (calcium / magnesium-free PBS(-), Life Technologies) containing 0.3% bovine serum albumin (BSA, Sigma) and 0.1% sodium azide (VWR). Cells (100,000 in 100 μL) were placed into a round-bottom 96-well plate (Falcon) and incubated with 1 μg / mL of anti-PSMA mAb 3.9 conjugated to phycoerythrin (PE) for 30 minutes at room temperature. The cells were then washed twice with 200 μL of PBS / BSA / azide buffer and read using a FACSCalibur instrument (BD Biosciences). Mouse IgG2b-PE conjugate (Abcam) with irrelevant specificity was included as an isotype control.

[0265] Western blotting 300,000 cells were incubated overnight in 4 mL of RPMI 1640 medium with 10% FBS in a 6-well plate (BD Falcon), and then an inhibitor was added. The plate was further incubated at 37 °C for 7 days, washed once with 5 mL of cold PBS(-), and placed on ice. The cells were lysed with 130 μL of RIPA lysis buffer (Santa Cruz Biotechnology), and then centrifuged at 14,000 × g for 10 minutes at 4 °C. For the supernatant, the protein concentration was quantified using a BCA kit (Pierce / Thermo), separated under reducing conditions using a NuPAGE Novex 4-12% Bis-Tris gel (Life Technologies), and transferred to nitrocellulose using an iBlot 7-Minute Blotting System (Life Technologies). The membrane was blocked using Odyssey Blocking Reagent (LI-COR Biosciences), incubated with primary and secondary antibodies, and visualized using an Odyssey infrared imager (LI-COR Biosciences).

[0266] Synergy calculation and statistical methods Unless otherwise indicated, for drug combinations, independent cell viability assays were repeated 3 - 7 times. Inhibition data were fitted to a four-parameter logistic equation using GraphPad Prism. Potential non-additive effects were evaluated by the Bliss independence method (Non-Patent Documents 66, 67, 68). Briefly, the predicted Bliss value for the combination (F c ) was calculated as F c = F a + F b -(F a × F b ). Here, F a and F b represent the observed values of the growth inhibition rates caused by compounds A and B used alone. The Bliss difference is the difference between the experimentally observed inhibition and the predicted value (F c) was calculated by subtracting. The mean Bliss difference was calculated for each point in the drug-drug combination matrix, converted to a percentage, and the statistical significance from a null value of 0 was evaluated using a two-sided t-test at a significance level of 0.05. When the growth inhibition rate exceeded 100%, such values were outside the Bliss framework, so no statistical evaluation was performed. Additionally, Bliss differences of less than 5% were considered to have limited biological relevance and were excluded from the consideration of synergistic effects.

[0267]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Claims

**Claim 1** An anti-androgen selected from abiraterone, enzalutamide, nilutamide, flutamide, bicalutamide, ARN 509, or galeterone, and a conjugate of a prostate-specific membrane antigen (PSMA) ligand and a cytotoxic agent for inhibiting the growth of PSMA-expressing cancer cells containing cells insensitive to anti-androgen therapy alone, the composition comprising the composition, wherein the PSMA ligand of the conjugate comprises an antibody or antigen-binding fragment thereof that specifically binds to PSMA. **Claim 2** The antibody or antigen-binding fragment thereof is PSMA 3.7, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, Abgenix 4.152.1, AB-PG1-XG1-006, AB-PG1-XG1-026, AB-PG1-XG1-051, AB-PG1-XG1-069, AB-PG1-XG1-077, and An antibody produced by a hybridoma of ATCC deposit numbers HB-12101 (E99), HB-12109 (J415), HB-12127 (J533), HB-12126 (J591), HB12060 (3F5.4G6), HB12309 (3D7-1.1), HB12310 (4E10-1.14), HB12489 (1G3), HB12495 (1G9), HB12490 (2C7), HB12494 (3C4), HB12491 (3C6), HB12484 (3C9), HB12486 (3E6), HB12488 (3E11), HB12485 (3G6), HB12493 (4D4), HB12487 (4D8), HB12492 (4C8B9), HB12664 (3F6), HB12678 (2E4), HB12665 (3C2), HB12672 (2D4), HB12660 (4C8G8), HB12675 (2C4), HB12663 (4C11), HB12661 (1D11), HB12667 (4E8), HB12674 (2G5), HB12670 (4E6), HB12677 (1F4), HB12666 (2E3), HB12662 (3D8), HB12668 (4F8), HB12673 (3D2), HB12676 (1G7), HB12669 (3D4), HB12679 (5G10), or HB12671 (5E9) The composition according to claim 1, which is an antibody selected from the group consisting of, or an antigen-binding fragment thereof

3. The antibody or its antigen-binding fragment comprises (i) three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 15, and (ii) three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 17, or (i) three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 19, and (ii) three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 21, or (i) three complementarity-determining regions of the heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 23, and (ii) three complementarity-determining regions of the light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 25, or (i) three complementarity-determining regions of a heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 27, and (ii) three complementarity-determining regions of a light-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 29, or The composition according to claim 1, comprising: (i) three complementarity-determining regions of a heavy-chain variable region comprising the amino acid sequence shown as SEQ ID NO: 31, and (ii) three complementarity-determining regions of a light-chain variable region comprising the amino acid sequence shown as SEQ ID NO:

33.

4. The composition according to any one of claims 1 to 3, wherein the cytotoxic agent comprises auristatin, tubulysin, pyrrolobenzodiazepine dimer, calicheamicin, colchicine, ispinomycin, combretastatin A4, maytansinoid DM1, maytansinoid DM4, doxorubicin, or a cytotoxic radionuclide.

5. contacting prostate-specific membrane antigen (PSMA)-expressing cancer cells with an antiandrogen; contacting the PSMA-expressing cancer cells with a conjugate of a PSMA ligand and a cytotoxic agent The composition according to any one of claims 1 to 4, for use in a method comprising:

6. The composition according to any one of claims 1 to 5, further comprising a pharmaceutically acceptable carrier and / or excipient.

7. The composition according to any one of claims 1 to 6, wherein the antiandrogen is in an amount effective to upregulate PSMA expression.

8. The composition according to any one of claims 5 to 7, wherein the step of contacting the PSMA-expressing cancer cells with the antiandrogen and the step of contacting the PSMA-expressing cancer cells with the conjugate of the PSMA ligand and the cytotoxic agent are simultaneous.

9. The composition according to any one of claims 5 to 7, wherein the step of contacting the PSMA-expressing cancer cells with the antiandrogen and the step of contacting the PSMA-expressing cancer cells with the conjugate of the PSMA ligand and the cytotoxic agent are sequential.

10. The composition according to claim 9, wherein the step of contacting the PSMA-expressing cancer cells with the antiandrogen is prior to the step of contacting the PSMA-expressing cancer cells with the conjugate of the PSMA ligand and the cytotoxic agent.

11. The composition according to any one of claims 5 to 10, wherein the PSMA-expressing cancer cells are PSMA-expressing prostate cancer cells.

12. The composition according to any one of claims 5 to 10, wherein the PSMA-expressing cancer cells are derived from a subject.

13. The composition according to claim 12, wherein the subject has progressive metastatic castration-resistant prostate cancer.

14. The composition according to claim 12 or 13, wherein the subject has received prior chemotherapy with at least one taxane.

15. The composition according to any one of claims 12 to 14, wherein the subject has received pretreatment with one or more antiandrogens.

16. The composition according to any one of claims 12 to 15, wherein the subject has prostate cancer that has progressed despite pretreatment.

17. The composition according to claim 12 or 13, wherein the subject has not previously received cytotoxic chemotherapy.

18. A container containing an antiandrogen, A container containing a conjugate of a PSMA ligand - cytotoxic agent And comprising the antiandrogen and conjugate according to any one of claims 1 to 17, a kit for inhibiting the growth of PSMA-expressing cancer cells containing cells insensitive to antiandrogen therapy alone.

19. The composition according to any one of claims 4 to 17, wherein the cytotoxic radionuclide emits Auger electrons and / or low-energy electrons.

20. The cytotoxic radionuclide is I １２３ , I １２４ , I １２５ , I １３１ , Br ７５ , Br ７７ , and F １８ The composition according to claim 19, comprising

21. The composition according to any one of claims 4 to 17, wherein the cytotoxic radionuclide comprises an alpha-emitting isotope.

22. The cytotoxic radionuclide is ２２５ Ac, ２１１ At, ２１２ Bi, ２１３ Bi, ２１２ Pb, ２２４ Ra, ２２３ The composition according to claim 21, comprising Ra.

23. The composition according to any one of claims 4 to 17, wherein the cytotoxic radionuclide comprises a beta-emitting isotope.

24. The cytotoxic radionuclide is １８６ Rh, １８８ Rh, １７７ Lu, ９０ Y, １３１ I, ６７ Cu, ６４ Cu, １５３ Sm, １６６ The composition according to claim 23, comprising Ho.

Images