Combination therapy with T cell-engaging molecules for the treatment of prostate cancer

Abstract

The present invention relates to a method for treating prostate cancer with a combination of an antiandrogen compound and a T-cell engaging molecule that specifically binds to human prostate specific membrane antigen (PSMA) and human CD3. In particular, the present invention relates to a method for treating prostate cancer, including metastatic castration-resistant prostate cancer, in a patient in need thereof, comprising administering to the patient one or more cycles of an antiandrogen compound in combination with a PSMA-targeted T-cell engaging molecule, wherein administration of a first dose of the antiandrogen compound is delayed relative to administration of a first therapeutic dose of the PSMA-targeted T-cell engaging molecule during the first cycle.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 322,138, filed March 21, 2022, the specification of which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on Mar. 20, 2023, is named A10004-WO01-SEC_ST26.xml and is 206,177 bytes in size.

[0003] The present invention relates to the field of cancer immunology and biopharmaceuticals. In particular, the present invention relates to a method for treating prostate cancer, particularly castration-resistant prostate cancer, by administering a T cell engaging molecule that specifically binds to human prostate specific membrane antigen (PSMA) and human cluster of differentiation 3 (CD3) in combination with an antiandrogen compound according to a specific administration schedule. [Background technology]

[0004] Prostate cancer is the second most common malignancy (after lung cancer) diagnosed in men worldwide. In 2018, 1,276,106 new cases of prostate cancer were reported worldwide, and prostate cancer caused 358,989 deaths (3.8% of all deaths caused by cancer in men) (Rawla, World J Oncol., Vol. 10: 63-89, 2019).

[0005] Metastasis is the leading cause of morbidity and mortality in prostate cancer. Since 1941, patients diagnosed with metastatic prostate cancer have undergone continuous androgen deprivation therapy (ADT) in the form of surgical castration, chemical castration including luteinizing hormone releasing hormone agonist (LHRH) modulating compounds, and / or antiandrogen therapy. Metastatic prostate cancer often develops resistance to ADT ("castration resistance") due to increased intratumoral steroid production, altered steroid transporter expression, increased androgen receptor expression (e.g., androgen receptor amplification), and other mechanisms (Galletti et al., Cancer Treat Rev., Vol. 57:16-27, 2017). Since 2010, several therapies have been approved to treat these patients with metastatic castration-resistant prostate cancer (mCRPC). Two novel hormonal therapies, enzalutamide and abiraterone, have shown significant survival benefit in mCRPC patients. In first-line mCRPC, abiraterone plus prednisone improved median OS from 30.3 to 34.7 months compared with placebo plus prednisone. Similarly, enzalutamide improved OS in the first-line mCRPC setting (35.3 vs. 31.3 months) (Sartor and de Bono, N Engl J Med., Vol. 378: 645-657, 2018). Radium-223 showed a survival benefit (14.9 vs. 11.3 months) in patients with bone metastases when combined with best standard of care including older hormone therapy, radiation therapy, and bisphosphonates. Sipuleucel-T, an autologous cellular immunotherapy, increased median survival by 4.1 months compared with placebo, but no PSA or radiographic responses were observed. Cabazitaxel, a tubulin-binding taxane, increased median survival by 2.4 months compared with mitoxantrone, but many study participants did not complete treatment due to toxicity. Recently approved therapies have shown survival benefits for patients with mCRPC, but drug resistance often complicates the disease course and contributes to recurrence and mortality. The effects of approved therapies are generally not durable and may be associated with limited tolerability.See, e.g., de Bono et al., Lancet, Vol. 376:1147-54, 2010; Scher et al., N Engl J Med, Vol. 367:1187-97, 2012; Parker et al., N Engl J Med, Vol. 369:213-23, 2013; and Ryan et al., Lancet Oncol, Vol. 16:152-60, 2015.

[0006] New PSMA-targeted therapies have shown activity in patients with mCRPC (Hofman et al., Lancet Oncol., Vol. 19(6): 825-833, 2018). PSMA is a 100 kDa type II integral membrane glycoprotein that is expressed primarily on prostate epithelial cells (Christiansen et al., Prostate, Vol. 55: 9-19, 2003; Israeli et al., Cancer Res., Vol. 53: 227-230, 1993). PSMA is expressed in the prostate and in a limited number of tissues, including a subset of renal proximal tubules, some cells of the intestinal brush border membrane, and rare cells in the liver and colonic crypts (O'Keefe et al., Prostate, Vol. 58:200-210, 2004; Chang et al., Cancer Res., Vol. 59:3192-3198, 1999; Troyer et al., Int J Cancer, Vol. 62:552-558, 1995; Israeli et al., Cancer Res., Vol. 54:1807-1811, 1994; Lopes et al., Cancer Res., Vol. 50:6423-6429, 1990; Horoszewicz et al., Anticancer Res., Vol. 7:927-935, 1987). In prostate cancer, expression of PSMA increases with disease progression and is highest in metastatic disease, hormone refractory cases, and more aggressive lesions. There is a strong correlation between negative prognosis and cell surface expression of PSMA. Consistent with the correlation between PSMA expression and tumor stage, increased levels of PSMA are associated with androgen-independent prostate cancer (Wright et al., Urology, Vol. 48:326-334, 1996; Israeli et al., 1994, supra).Immunohistochemical analysis revealed relatively strong and uniform expression of PSMA in metastatic lesions located in lymph nodes, bone, soft tissue, and lungs compared with benign prostate tissue (Chang et al., Urology, Vol. 57: 1179-1183, 2001; Sweat et al., Urology, Vol. 52: 637-640, 1998; Murphy et al., Cancer, Vol. 78: 809-818, 1996). The restricted expression of PSMA and its upregulation in advanced cancer and metastatic disease make PSMA an attractive target for the development of therapeutics for prostate cancer. Advanced prostate cancer is a heterogeneous disease that frequently develops resistance to monotherapy (Carm et al., Sci Rep., Vol. 9: 13579, 2019). Therefore, combination therapy is necessary to overcome disease heterogeneity and resistance to therapy. Antiandrogen therapy such as enzalutamide and abiraterone has been reported to upregulate the expression of PSMA on prostate cancer cells (see, e.g., Deegen et al., Clin. Cancer Res., Vol. 27: 2928-2937, 2021; Aggarwal et al., Eur. Urol. Oncol., Vol. 1: 78-82, 2018; Emmett et al., J Nucl. Med., Vol. 60: 950-954, 2019). SMA-targeted T cell engagers are a new class of immunotherapy that bind to a patient's own T cells and redirect them to kill PSMA-expressing cancer cells. Combination therapy of PSMA-targeted T cell engagers with antiandrogen compounds that upregulate the expression of PSMA on cancer cells may provide synergistic antitumor effects. Indeed, in vitro experiments have shown that enzalutamide enhances the cytotoxicity of prostate cancer cells induced by PSMA-targeted T cell engagers (Deegen et al., 2021, supra). However, the side effect profile associated with such combination therapy in human patients is not well understood. Thus, there is a need in the art for a safe and effective approach to administer antiandrogen compounds in combination with PSMA-targeted immunotherapy to treat prostate cancer, particularly mCRPC. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Rawla,World J Oncol.,Vol.10:63-89,2019 [Non-Patent Document 2] Galletti et al.,Cancer Treat Rev.,Vol.57:16-27,2017

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Summary of the Invention

Means for Solving the Problems

[0008] The present invention is based, in part, on the identification of an administration schedule of an antiandrogen compound in combination with a PSMA-targeted T cell engager that improves the safety and tolerability of the combination therapy while maintaining optimal antitumor efficacy.Thus, in one embodiment, the present invention provides a method for treating prostate cancer in a patient in need thereof, comprising administering to the patient one or more cycles of an antiandrogen compound in combination with a T cell engaging molecule that specifically binds to human PSMA and human CD3, the first cycle comprising administering a first dose of the antiandrogen compound at least 3 days after administering a first therapeutic dose of the T cell engaging molecule.

[0009] In certain embodiments, the first cycle (also referred to herein as the initiation cycle) comprises administering a first dose of an antiandrogen compound about 4 to about 10 days, or about 5 to about 9 days, after administering a first therapeutic dose of a PSMA-targeted T-cell engaging molecule. For example, in some embodiments, the first cycle comprises administering a first dose of an antiandrogen compound about 5 days, about 6 days, or about 7 days after administering a first therapeutic dose of a PSMA-targeted T-cell engaging molecule.

[0010] In some embodiments of the methods of the invention, the first cycle further comprises administering one or more priming doses of the T cell engaging molecule to the patient prior to administering the first therapeutic dose of the T cell engaging molecule. Such priming doses, in some embodiments, are lower than the therapeutic dose of the T cell engaging molecule, but may be sufficient to induce T cell activation in the patient to prime or prepare the patient to receive a higher dose of the T cell engaging molecule, such that administration of a higher dose results in a reduction in the number or severity of adverse events, such as cytokine release syndrome. In some embodiments in which one or more priming doses of the PSMA-targeted T cell engaging molecule are administered to the patient in the first cycle, the first therapeutic dose of the T cell engaging molecule may be administered about 1 day to about 21 days, about 1 day to about 14 days, or about 1 day to about 7 days after administration of the first priming dose of the PSMA-targeted T cell engaging molecule to the patient. The first therapeutic dose of the PSMA-targeted T cell engaging molecule is administered about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the first priming dose of the PSMA-targeted T cell engaging molecule.

[0011] In various embodiments of the methods of the invention, the PSMA-targeted T cell engaging molecule is administered to the patient by intravenous infusion or subcutaneous injection. In some embodiments, one or more doses of the PSMA-targeted T cell engaging molecule are administered to the patient by continuous intravenous infusion over an extended period of time, such as a period of 1 day to 7 days. In some embodiments, one or more doses of the PSMA-targeted T cell engaging molecule are administered to the patient by bolus intravenous infusion (also referred to herein as short-term intravenous infusion) over a period of about 30 minutes to about 90 minutes. In certain embodiments, the first dose of the PSMA-targeted T cell engaging molecule in the first cycle is administered by continuous intravenous infusion over a period of 1 day to 7 days, such as a period of 3 days (i.e., a 72-hour infusion period). In some such embodiments, the first dose is a priming dose and may be less than the amount of the therapeutic dose. In related embodiments, one or more therapeutic doses of the PSMA-targeted T cell engaging molecule may then be administered to the patient by bolus intravenous infusion in the first cycle.

[0012] In some embodiments, the antiandrogen compound is orally administered to the patient. Depending on the antiandrogen compound used, the antiandrogen compound is administered to the patient in an oral dosage form, such as a tablet or capsule, once a day, twice a day, or three times a day. The antiandrogen compound can be abiraterone, abiraterone acetate, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide. In certain embodiments, the antiandrogen compound administered to the patient according to the method of the present invention is enzalutamide, abiraterone, abiraterone acetate, apalutamide, or darolutamide. In one embodiment, the antiandrogen compound is enzalutamide. Enzalutamide can be orally administered to the patient at a dose of about 160 mg once a day. In another embodiment, the antiandrogen compound is abiraterone or abiraterone acetate. In some such embodiments, abiraterone or abiraterone acetate is orally administered to a patient at a dose of about 1,000 mg once daily. In yet another embodiment, the antiandrogen compound is apalutamide, which may be orally administered to a patient at a dose of about 240 mg once daily. In yet another embodiment, the antiandrogen compound is darolutamide. In some embodiments, darolutamide is orally administered to a patient at a dose of about 600 mg twice daily.

[0013] The first cycle may have a duration of about 14 days to about 56 days, or about 21 days to about 28 days. In certain embodiments of the methods of the present invention, the first cycle has a duration of about 28 days. In such embodiments, the first cycle may include administering a PSMA-targeted T-cell engaging molecule on days 1, 8, 15, and 22 of the cycle, and administering an antiandrogen compound once daily on each of days 15 to 28 of the cycle. In other embodiments, the first cycle may include administering a PSMA-targeted T-cell engaging molecule on days 1 and 15 of the cycle, and administering an antiandrogen compound once daily on each of days 15 to 28 of the cycle. In certain other embodiments in which the first cycle is about 28 days, the first cycle may include administering a first dose (e.g., a priming dose) of the PSMA-targeted T-cell engaging molecule by continuous intravenous infusion over days 1-3 of the cycle, administering a therapeutic dose of the PSMA-targeted T-cell engaging molecule by bolus intravenous infusion on days 8 and 22 of the cycle, and orally administering an antiandrogen compound once daily on each of days 15-28 of the cycle. In such embodiments, the first dose (e.g., a priming dose) of the PSMA-targeted T-cell engaging molecule may be about 30 μg to about 300 μg or about 30 μg to about 150 μg, and the therapeutic dose of the PSMA-targeted T-cell engaging molecule may be about 90 μg to about 1.8 mg or about 100 μg to about 600 μg. In one specific embodiment, the first dose (e.g., the priming dose) of the PSMA-targeted T cell engaging molecule is about 90 μg and the therapeutic dose is about 150 μg or 300 μg.

[0014] In some embodiments, the method further comprises administering to the patient one or more maintenance cycles of an antiandrogen compound in combination with the PSMA-targeted T-cell engaging molecule. In one embodiment, the maintenance cycle comprises administering the antiandrogen compound orally once a day on each day of the cycle and administering a therapeutic dose of the PSMA-targeted T-cell engaging molecule by bolus intravenous infusion once every 7 days or once every 14 days. The duration of the maintenance cycle may be about 21 days to about 42 days, about 28 days to about 56 days, or about 21 days to about 28 days. In certain embodiments, the maintenance cycle has a duration of about 28 days. The maintenance cycle may be administered the day after completing the first cycle, for example, without a treatment-free period between the first cycle and the maintenance cycle. In another embodiment, the maintenance cycle is administered about 7 days after the completion of the first cycle, i.e., there is a 7-day treatment-free period between the first cycle and the maintenance cycle. A patient may receive multiple maintenance cycles, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more maintenance cycles. In some embodiments, maintenance cycles are administered to a patient until the patient responds to the treatment, for example, until the patient achieves a complete response.

[0015] In certain embodiments, the prostate cancer treated according to the method of the present invention is metastatic prostate cancer.Therefore, the patient treated according to the method of the present invention has metastatic prostate cancer or has been diagnosed with metastatic prostate cancer.Metastatic prostate cancer can be hormone-sensitive or resistant to hormone therapy.Therefore, in one embodiment, the patient treated according to the method of the present invention has metastatic castration-resistant prostate cancer or has been diagnosed with metastatic castration-resistant prostate cancer.

[0016] Prostate cancer patients treated according to the method of the present invention may have undergone one or more previous treatments for prostate cancer and may have failed or become intolerant, refractory, or resistant to one or more of these previous treatments. For example, in some embodiments, the patient has failed or become intolerant, refractory, or resistant to one or more chemotherapy regimens, such as taxane-containing chemotherapy regimens. Additionally or alternatively, the patient has failed or become intolerant, refractory, or resistant to one or more antiandrogen compounds, such as abiraterone, enzalutamide, apalutamide, or darolutamide. In such embodiments, the antiandrogen compound administered to the patient in combination with the PSMA-targeted T cell engagement molecule according to the method of the present invention is preferably an antiandrogen compound different from the antiandrogen compound the patient has previously received. In certain embodiments, the patient treated according to the method of the present invention may be: 177 are intolerant, refractory, or resistant to radioligand therapy such as Lu-PSMA-617.

[0017] In any embodiment of the methods disclosed herein, the PSMA-targeted T cell engaging molecule administered to the patient specifically binds to PSMA and CD3, preferably human PSMA and human CD3. Thus, the PSMA-targeted T cell engaging molecule comprises a first domain that specifically binds to human PSMA and a second domain that specifically binds to human CD3. The binding domain can comprise structural elements from an antibody or an antigen-binding fragment thereof, such as heavy and light chain variable regions. In one embodiment, either or both of the binding domains of the PSMA-targeted T cell engaging molecule used in the methods of the invention are single chain variable fragments (scFv). In some embodiments of the methods described herein, the PSMA-targeted T cell engaging molecule further comprises an Fc domain having one or more immunoglobulin Fc monomers. In such embodiments, the Fc domain can be a single chain Fc domain.

[0018] In certain embodiments, the PSMA-targeted T cell engaging molecule administered to a patient according to the methods of the invention comprises, in amino to carboxyl order, (i) a first domain that specifically binds human PSMA, (ii) a second domain that specifically binds human CD3, and (iii) an Fc domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, said two Fc monomers being fused to each other via a peptide linker. In one embodiment, the first domain comprises a first immunoglobulin heavy chain variable region (VH1) comprising a CDRH1 having the sequence of SEQ ID NO: 14, a CDRH2 having the sequence of SEQ ID NO: 16, and a CDRH3 having the sequence of SEQ ID NO: 20, and a first immunoglobulin light chain variable region (VL1) comprising a CDRL1 having the sequence of SEQ ID NO: 5, a CDRL2 having the sequence of SEQ ID NO: 8, and a CDRL3 having the sequence of SEQ ID NO: 9. In related embodiments, the second domain comprises a second immunoglobulin heavy chain variable region (VH2) comprising a CDRH1 having the sequence of SEQ ID NO: 49, a CDRH2 having the sequence of SEQ ID NO: 55, and a CDRH3 having the sequence of SEQ ID NO: 60, and a second immunoglobulin light chain variable region (VL2) comprising a CDRL1 having the sequence of SEQ ID NO: 43, a CDRL2 having the sequence of SEQ ID NO: 44, and a CDRL3 having the sequence of SEQ ID NO: 47. In some embodiments, the first domain of the PSMA-targeted T cell engaging molecule according to the invention (e.g., an anti-PSMA domain) comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 33, and a light chain variable region comprising the sequence of SEQ ID NO: 30. In these and other embodiments, the second domain of the PSMA-targeted T cell engaging molecule (e.g., an anti-CD3 domain) comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 72, and a light chain variable region comprising the sequence of SEQ ID NO: 70.

[0019] The PSMA-targeted T cell engaging molecule administered to a patient by the methods of the invention can comprise an Fc domain comprising (i) a first domain that specifically binds human PSMA and has the amino acid sequence of SEQ ID NO: 104, (ii) a second domain that specifically binds human CD3 and has the amino acid sequence of SEQ ID NO: 116, and (iii) two Fc monomers, each having the amino acid sequence of SEQ ID NO: 124, said two Fc monomers being fused to each other via a peptide linker. In a related embodiment, the Fc domain of the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 132. In a specific embodiment, the PSMA-targeted T cell engaging molecule used in the methods of the invention is a single chain polypeptide or a single chain fusion protein. Thus, any of the single chain polypeptides described in Table 6 herein are suitable for use in the methods of the invention. In a preferred embodiment, the PSMA-targeted T cell engaging molecule administered to a patient by the methods of the invention is a single chain polypeptide (e.g., acpatamab) comprising the amino acid sequence of SEQ ID NO: 140.

[0020] The present invention also provides pharmaceutical compositions of PSMA-targeted T-cell engaging molecules for use in combination with antiandrogen compounds in the methods described herein. The pharmaceutical compositions can include one or more pharma- ceutically acceptable diluents, carriers, or excipients, such as buffers, surfactants, and stabilizers. In certain embodiments, the pharmaceutical compositions include PSMA-targeted T-cell engaging molecules, buffers, surfactants, and stabilizers. In one embodiment, the pharmaceutical compositions include PSMA-targeted T-cell engaging molecules (e.g., single-chain polypeptides comprising the amino acid sequence of SEQ ID NO: 140), glutamate buffer, polysorbate 20 or polysorbate 80, and sucrose, at a pH of about 4.0 to about 4.4. In some embodiments, the pharmaceutical compositions can be lyophilized and reconstituted prior to administration to a patient.

[0021] The use of PSMA-targeted T cell engaging molecules in any of the methods described herein or in the preparation of a medicament for administration by any of the methods disclosed herein is specifically contemplated. For example, the present invention includes a PSMA-targeted T cell engaging molecule for use in combination with an antiandrogen compound in a method for the treatment of prostate cancer in a patient in need thereof, the method comprising administering a first cycle of a PSMA-targeted T cell engaging molecule and an antiandrogen compound, the first cycle comprising administering a first dose of the antiandrogen compound at least 3 days after administering a first therapeutic dose of the T cell engaging molecule. The present invention also includes the use of a PSMA-targeted T cell engaging molecule for the manufacture of a medicament for the treatment of prostate cancer in a patient in need thereof, the treatment comprising administering a first cycle of a PSMA-targeted T cell engaging molecule and an antiandrogen compound to the patient, the first cycle comprising administering a first dose of the antiandrogen compound at least 3 days after administering a first therapeutic dose of the T cell engaging molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention is based, in part, on the discovery that delaying the initiation of treatment with an antiandrogen compound for an extended period of time after administration of a PSMA-targeted T cell engaging molecule significantly reduces the number of serious adverse events and dose-limiting toxicities observed with the combination therapy compared to a regimen in which the PSMA-targeted T cell engaging molecule and the antiandrogen compound were initially administered simultaneously to the patient. See Example 1. In addition, the antitumor efficacy of the combination was comparable between the two regimens. These results are somewhat surprising for at least two reasons. Based on the mechanism of action of the antiandrogen compound and the PSMA-targeted T cell engaging molecule, overlapping toxicities were not expected because the potential adverse events of each molecule are different. For example, the most frequent adverse events of antiandrogen compounds such as enzalutamide are hot flashes and fatigue, while the most frequent adverse event of PSMA-targeted T cell engaging molecules is cytokine release syndrome (CRS). Second, antiandrogen compounds have been reported to upregulate the expression of PSMA on prostate cancer cells, suggesting that antiandrogen compounds should be administered before or at least simultaneously with PSMA-targeted T cell engaging molecules to observe synergistic effects on tumor cell cytotoxicity. However, delaying the initiation of treatment with antiandrogen compounds did not significantly affect the efficacy of the combination therapy (see Example 1). Thus, the present invention provides a method for administering antiandrogen compounds in combination with PSMA-targeted T cell engagers that improves the safety and tolerability of the combination therapy while maintaining optimal antitumor efficacy in patients with prostate cancer. Thus, in certain embodiments, the present invention provides a method for treating prostate cancer in a patient in need thereof, comprising administering to the patient one or more cycles of antiandrogen compounds in combination with human PSMA and T cell engaging molecules that specifically bind to human CD3, the first cycle comprising administering a first dose of antiandrogen compounds at least 3 days after administering a first therapeutic dose of T cell engaging molecules.

[0023] Prostate cancer is one of the most common types of cancer in men and occurs when cells within the prostate begin to grow uncontrollably. Most forms of prostate cancer are adenocarcinomas, which are tumors that form from glandular cells. Other forms of prostate cancer include small cell carcinoma, neuroendocrine tumors, transitional cell carcinoma, and sarcoma. Prostate cancer is initially confined to the prostate but can metastasize and spread to other tissues. Metastatic prostate cancer can be divided into two primary types: the first type in which the cancer has not been treated with androgen deprivation therapy ("metastatic hormone-sensitive prostate cancer" or mHSPC) and the second type in which the cancer is resistant to androgen deprivation therapy ("metastatic castration-resistant prostate cancer" or mCRPC). In prostate cancer, expression of PSMA increases with disease progression and is highest in metastatic disease, hormone-refractory cases, and higher-grade lesions.

[0024] Prostate cancer may not cause any signs or symptoms in its early stages. As the disease progresses, signs and symptoms of prostate cancer may include incontinence, dysuria, blood in semen or urine, erectile dysfunction, pain in the pelvic area or bones, or weakness in the legs or feet. Prostate cancer is typically diagnosed and monitored by one or more tests performed on a sample (e.g., blood, serum, plasma, semen, tissue) from a subject or patient suspected of having or developing prostate cancer. The sample may be any biological sample obtained from a human patient and may include bodily fluids such as blood, serum, plasma, semen, and urine, as well as tissues such as prostate tissue, lymph nodes, or tumor biopsies. A common test used to screen and / or monitor prostate cancer is the prostate-specific antigen (PSA) blood test. An elevation of PSA in the blood (e.g., serum or plasma) may be an indicator of the presence or progression of prostate cancer. Another test commonly used to detect or monitor prostate cancer is a prostate tissue biopsy. Prostate tissue biopsy samples are evaluated for the presence of abnormal or cancerous cells. If cancerous cells are present, prostate cancer may be assigned a grade based on the Gleason score grading system or other grading systems, which assign a grade based on how abnormal the cells look compared to normal cells. Gleason scores can range from 2 (non-invasive cancer) to 10 (highly invasive cancer). Prostate cancer may also be diagnosed using a variety of techniques, including, but not limited to, magnetic resonance imaging (MRI), computed tomography (CT), and other methods. 68 Gallium-PSMA-11, Pifluforastat F-18 (also known as 18 F-DCFPyL), or 18 Positron emission tomography (PET) using a radioactive tracer such as F-flurodeoxyglucose, or a bone scan (e.g., 98m It can be diagnosed or monitored using imaging tests, including bone scintigraphy using technetium-labeled radioactive tracers.

[0025] In certain embodiments, the patient treated by the methods of the invention has or has been diagnosed with prostate cancer. In such embodiments, the prostate cancer is PSMA positive, i.e., the tumor or cancer cells express PSMA as determined by standard immunohistochemical testing of biopsy samples or PSMA imaging methods. In some embodiments, the patient treated by the methods of the invention has a blood PSA level of 4 ng / mL or higher. In other embodiments, the patient treated by the methods of the invention has a blood PSA level of 10 ng / mL or higher. In yet other embodiments, the patient treated by the methods of the invention has a blood PSA level of 1 ng / mL or higher, and the PSA level has increased at least twice consecutively at least one week apart. In some embodiments, the patient treated by the methods of the invention has a prostate cancer with a Gleason score of 7. In other embodiments, the patient treated by the methods of the invention has a prostate cancer with a Gleason score of 8. In yet other embodiments, the patient treated by the methods of the invention has a Gleason score of 9 or 10. In some embodiments, the patient treated by the methods of the invention may be newly diagnosed with prostate cancer. Some patients in need of treatment may have been diagnosed with hormone-sensitive or castration-resistant prostate cancer.

[0026] In some embodiments, the patient treated by the method of the present invention has metastatic prostate cancer or has been diagnosed with metastatic prostate cancer.Metastatic prostate cancer can be hormone-sensitive or resistant to hormone therapy.Patients diagnosed with metastatic prostate cancer have evidence of prostate cancer cells outside the prostate, such as lymph nodes, bone, or other organs, most commonly the liver, lung, or brain.Evidence of the spread of cancer cells is typically detected by the presence of tumors or lesions in other tissues by one or more of the imaging methods mentioned above, such as CT, MRI, PET, bone scan, etc. In some embodiments, patients treated with the methods of the invention have evidence of progressive prostate cancer as indicated by lymph node or visceral tumor progression as defined by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, optionally with the Prostate Cancer Working Group 3 (PCWG3) amendment (see Eisenhauer et al., European Journal of Cancer, Vol. 45:228-247, 2009; Scher et al., J. Clin. Oncol, Vol. 34:1402-1418, 2016). In other embodiments, patients treated with the methods of the invention have evidence of progressive prostate cancer as indicated by bone scans (e.g., 98m Have evidence of progressive prostate cancer as demonstrated by the appearance of two or more new bone lesions as determined by bone scintigraphy using a technetium-labeled radioactive tracer.

[0027] In certain embodiments, the patient treated by the methods of the present invention has or has been diagnosed with metastatic castration-resistant prostate cancer (mCRPC). mCRPC is diagnosed when cancer progresses in patients with metastatic disease despite the patient having testosterone levels below those achieved by androgen deprivation therapy. In some embodiments, patients diagnosed with mCRPC may have failed or become refractory to treatment with androgen deprivation therapy, or may have relapsed after treatment. Androgen deprivation therapy includes surgical castration (e.g., bilateral orchiectomy), chemical castration with an LHRH agonist or antagonist (e.g., leuprolide, goserelin, triptorelin, histrelin, relugolix, or degarelix), or treatment with an antiandrogen compound such as an androgen biosynthesis inhibitor (e.g., abiraterone, ketoconazole), or an androgen receptor antagonist (e.g., flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide). In some embodiments, the patient treated by the method of the invention has a total serum testosterone level of 50 ng / dL (1.7 nmol / L) or less. In other embodiments, the patient treated by the method of the invention has a total serum testosterone level of 20 ng / dL (0.7 nmol / L) or less. In certain embodiments, the patient treated by the method of the invention has undergone bilateral orchiectomy. In another embodiment, the patient treated by the methods of the invention is receiving a continuous treatment regimen of an LHRH agonist or antagonist, hi yet another embodiment, the patient treated by the methods of the invention has had a bilateral orchiectomy and is receiving a continuous treatment regimen of an LHRH agonist or antagonist.

[0028] In patients with castration-resistant prostate cancer (CRPC), androgen receptor (AR) signaling is altered such that the patient's tumors develop a variety of mutations in genes that code for proteins in the androgen receptor signaling pathway (see Sartor and de Bono, N Engl J Med., Vol. 378: 645-657, 2018). Such mutations include mutations in the AR, FOXA1, ZBTB16, and SPOP genes, as well as genes involved in AKT signaling, DNA repair, and tumor suppression, such as PTEN, ET5, BRCA2, ATM, and CHEK2. Thus, the diagnosis of CRPC or mCRPC can be supplemented by gene expression profiling or genotyping to confirm the initial diagnosis and / or identify the subtype of CRPC or mCRPC.

[0029] The administration of the PSMA-targeted T cell engaging molecule in combination with an antiandrogen compound according to the method of the present invention is for the treatment of prostate cancer or other PSMA-expressing cancer or tumor. As used herein, the term "treatment" or "treating" refers to the application or administration of the T cell engaging molecule in combination with an antiandrogen compound to a patient who has or has been diagnosed with prostate cancer or other PSMA-positive malignancies, has symptoms of prostate cancer or other PSMA-positive malignancies, is at risk of developing prostate cancer or other PSMA-positive malignancies, or has a predisposition to prostate cancer or other PSMA-positive malignancies, for the purpose of treating, curing, alleviating, mitigating, altering, ameliorating, or improving one or more symptoms of prostate cancer or other PSMA-positive malignancies, the risk of developing prostate cancer or other PSMA-positive malignancies, or the predisposition to prostate cancer or other PSMA-positive malignancies. The term "treatment" encompasses any improvement in disease in a patient, including slowing or stopping the progression of prostate cancer or other PSMA-positive malignancies in the patient, reducing the number or severity of symptoms of prostate cancer or other PSMA-positive malignancies, or increasing the frequency or length of time that the patient is free of symptoms of prostate cancer or other PSMA-positive malignancies. The term "patient" includes human patients.

[0030] In certain embodiments of the methods of the present invention, administration of the PSMA-targeted T cell engaging molecule in combination with an antiandrogen compound reduces the blood level of PSA (e.g., serum or plasma level) in the patient by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, or about 100%, compared to the blood level of PSA in the patient before the start of treatment (i.e., before administration of the PSMA-targeted T cell engaging molecule and the antiandrogen compound). In some embodiments, administration of the PSMA-targeted T cell engaging molecule in combination with an antiandrogen compound reduces the blood level of PSA in the patient by 30% or more compared to the blood level of PSA in the patient before the start of treatment (i.e., PSA30 response). In other embodiments, administration of a PSMA-targeted T cell engagement molecule in combination with an antiandrogen compound reduces the patient's PSA blood level by 50% or more compared to the patient's PSA blood level before the start of treatment (i.e., PSA50 response). In yet other embodiments, administration of a PSMA-targeted T cell engagement molecule in combination with an antiandrogen compound reduces the patient's PSA blood level by 70% or more compared to the patient's PSA blood level before the start of treatment (i.e., PSA70 response). In yet other embodiments, administration of a PSMA-targeted T cell engagement molecule in combination with an antiandrogen compound reduces the patient's PSA blood level by 90% or more compared to the patient's PSA blood level before the start of treatment (i.e., PSA90 response). In certain embodiments of the methods of the invention, administration of a PSMA-targeted T cell engaging molecule in combination with an anti-androgen compound results in a PSA50 response in at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of prostate cancer patients.

[0031] In some embodiments of the methods of the present invention, administration of a PSMA-targeted T cell engaging molecule in combination with an anti-androgen compound induces a complete response, partial response, or stable disease response in at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of prostate cancer patients with measurable tumors or lesions prior to the initiation of treatment as determined by RECIST 1.1 criteria, optionally with PCWG3 modification (see Eisenhauer et al., European Journal of Cancer, Vol. 45:228-247, 2009; Scher et al., J. Clin. Oncol, Vol. 34:1402-1418, 2016). Complete response (CR) in the context of the present invention refers to a state in which all target lesions have disappeared (i.e., are no longer detectable) and pathological lymph nodes have a reduction in short axis to less than 10 mm. Partial response (PR) refers to a state in which there is at least a 30% reduction in the sum of the diameters of the target lesions compared to the sum of the diameters before the start of treatment. Stable disease (SD) refers to a state in which the target lesions have not decreased sufficiently to qualify as PR, but have not increased sufficiently to qualify as progressive disease (PD). PD refers to a state in which there is the appearance of one or more new target lesions, or there is an increase of at least 20% in the sum of the diameters of the target lesions compared to the smallest sum of the diameters that previously occurred, and there is an absolute increase in the sum of the diameters of at least 5 mm.

[0032] The efficacy of the treatment regimens described herein also includes: 68 Ga-PSMA-11 PET / CT imaging, Piflufolastat F-18( 18F-DCFPyL) PET / CT imaging or imaging with another PSMA radiographic PET tracer, the percentage of patients who achieve a circulating tumor cell (CTC) response, duration of response to treatment, time to disease progression, progression-free survival (PFS), and overall survival (OS). In certain embodiments, administration of a PSMA-targeted T cell engaging molecule in combination with an antiandrogen compound according to the methods of the invention increases the duration of response to treatment, time to disease progression, PFS, and / or OS compared to the duration of response to treatment, time to disease progression, PFS, and / or OS observed for a standard chemotherapy regimen (e.g., a taxane chemotherapy regimen) or a standard androgen deprivation therapy regimen.

[0033] In general, the methods of the present invention include administering a PSMA-targeted T cell engaging molecule in combination with an antiandrogen compound to a patient in one or more treatment cycles. A "treatment cycle" or "cycle" refers to a period during which the T cell engaging molecule and the antiandrogen compound are administered at a particular dosage and interval. According to the methods of the present invention, a patient can undergo multiple treatment cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more cycles). Treatment cycles can be administered to a patient continuously without a break between cycles, i.e., a period during which the T cell engaging molecule or the antiandrogen compound is not administered. Alternatively, a period during which the T cell engaging molecule, the antiandrogen compound, or both are not administered (e.g., a "treatment-free period" or "interruption") can be used between treatment cycles. The length of the treatment-free period can be adjusted based on the patient's characteristics and / or response to treatment.

[0034] Each of the treatment cycles involves administering a PSMA-targeted T-cell engaging molecule in combination with an antiandrogenic compound. The term "in combination" or "combination therapy" as used herein refers to a method of administration of two compounds such that the therapeutic effect of the PSMA-targeted T-cell engaging molecule coincides with the therapeutic effect of the antiandrogenic compound for one or more periods of time. Except for the first dose of the antiandrogenic compound and the first therapeutic dose of the PSMA-targeted T-cell engaging molecule, in the first treatment cycle described in more detail herein, the administration of the PSMA-targeted T-cell engaging molecule and the administration of the antiandrogenic compound can be substantially simultaneous or sequential (i.e., each compound is administered on different days in any order). Substantially simultaneous administration includes concurrent administration and can be achieved by administering a single formulation containing both compounds (e.g., a single IV bag containing both compounds) or by concurrently administering separate formulations containing each compound (e.g., on the same day). The antiandrogenic compound does not need to be administered at the same dosing frequency or interval as the PSMA-targeted T-cell engaging molecule. In general, the antiandrogen compound will be administered at a dose and / or time schedule determined for that compound. The antiandrogen compound may be administered on the same or different days of a treatment cycle as the PSMA-targeted T-cell engaging molecule. Exemplary doses and administration schedules of the PSMA-targeted T-cell engaging molecule and the antiandrogen compound for administration according to the methods of the present invention are provided herein.

[0035] In certain embodiments, the efficacy of the combination therapy, as assessed by any of the above methods, is greater than the efficacy of PSMA-targeted T cell engagement molecule monotherapy and the efficacy of antiandrogen compound monotherapy. For example, in some embodiments, administration of PSMA-targeted T cell engagement molecule in combination with antiandrogen compound produces PSA50 responses in a greater proportion of prostate cancer patients than those produced by monotherapy with either compound alone. In other embodiments, administration of PSMA-targeted T cell engagement molecule in combination with antiandrogen compound produces a greater number of CRs, PRs, and / or SDs in prostate cancer patients with measurable tumors or lesions, as determined by RECIST 1.1 criteria, optionally with PCWG3 modification, compared to the number of such responses resulting from monotherapy with either compound alone. In yet other embodiments, administration of PSMA-targeted T cell engagement molecule in combination with antiandrogen compound increases the duration of response to treatment, time to disease progression, PFS, and / or OS compared to the duration of response to treatment, time to disease progression, PFS, and / or OS observed with a monotherapy with either compound alone. In some embodiments, administration of a PSMA-targeted T cell engaging molecule in combination with an anti-androgen compound results in a synergistic increase in anti-tumor efficacy as assessed by any of the methods described above (e.g., PSA response, RECIST 1.1 response, PSMA tumor burden by PET / CT, CTC response, etc.).

[0036] In a preferred embodiment of the method of the present invention, the first treatment cycle (also called the initiation cycle) comprises administering a first therapeutic dose of PSMA-targeted T-cell engaging molecule, followed by a certain delay period, followed by administering a first dose of an antiandrogen compound. As described in the examples herein, when the initiation of treatment with an antiandrogen compound is delayed until about 7 days after the administration of the first therapeutic dose of a PSMA-targeted T-cell engaging molecule, the number and severity of adverse events are significantly reduced compared to when antiandrogen treatment is initiated simultaneously with the administration of the first dose of a PSMA-targeted T-cell engaging molecule. Without wishing to be bound by theory, it is believed that since CRS events are most severe and occur most frequently with the administration of the first therapeutic dose of a PSMA-targeted T-cell engaging molecule, delaying the initiation of treatment with an antiandrogen compound until CRS symptoms disappear or are remitted after the administration of the therapeutic dose (e.g., at least 3 days) improves the safety and tolerability profile of the combination therapy. Thus, in certain embodiments of the methods of the present invention, the first cycle comprises administering the first dose of the antiandrogen compound at least 3 days after administration of the first therapeutic dose of the PSMA-targeted T-cell engaging molecule. In some embodiments, the first cycle comprises administering the first dose of the antiandrogen compound about 3 to about 14 days, about 4 to about 10 days, or about 5 to about 9 days after administration of the first therapeutic dose of the PSMA-targeted T-cell engaging molecule. The delay period between administration of the first therapeutic dose of the PSMA-targeted T-cell engaging molecule and administration of the first dose of the antiandrogen compound in the first cycle can be about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In one embodiment, the first cycle comprises administering a first dose of an antiandrogen compound about 5 days after administration of a first therapeutic dose of a PSMA-targeted T-cell engaging molecule. In another embodiment, the first cycle comprises administering a first dose of an antiandrogen compound about 7 days after administration of a first therapeutic dose of a PSMA-targeted T-cell engaging molecule.

[0037] The first cycle or initiation cycle is typically administered to a patient when the patient begins a course of treatment with a PSMA-targeted T-cell engaging molecule. The first cycle described herein may also be administered to a patient when the patient resumes a course of treatment with a PSMA-targeted T-cell engaging molecule, such as after a treatment-free period, an interruption in administration (e.g., if the patient did not complete a previous treatment cycle), or after recurrence or progression of cancer in the patient. The first cycle regimen may also be administered when the patient switches to a course of treatment with a different PSMA-targeted T-cell engaging molecule or a different antiandrogen compound. In certain embodiments of the methods of the present invention, the duration of the first cycle or initiation cycle is about 14 days to about 56 days, such as about 14 days to about 28 days, about 21 days to about 42 days, about 28 days to about 49 days, or about 21 days to about 28 days. In certain embodiments, the duration of the first cycle or initiation cycle is about 28 days.

[0038] In one embodiment, the method of the present invention comprises administering to a patient a therapeutically effective dose of a PSMA-targeted T-cell engaging molecule and a therapeutically effective dose of an anti-androgen compound. A "therapeutically effective dose" or "therapeutic dose" refers to an amount of a compound sufficient to treat or ameliorate prostate cancer or one or more symptoms thereof, particularly a condition or symptom associated with cancer, or otherwise prevent, hinder, delay or reverse in any way the progression of prostate cancer or any other undesirable symptoms associated with prostate cancer. The amount of a therapeutic dose may vary depending on the characteristics of the patient being treated, the route of administration, the type, grade or stage of cancer diagnosed in the patient, and the particular PSMA-targeted T-cell engaging molecule administered to the patient. The therapeutic dose of a PSMA-targeted T-cell engaging molecule and / or an anti-androgen compound administered to a patient in a combination therapy of the present invention may differ from the therapeutic dose of the same compound used in a monotherapy. For example, the therapeutic dose of one or both compounds may be less than the therapeutic dose of the compound administered as part of a monotherapy, for example due to a synergistic anti-tumor effect. Specific therapeutic doses for PSMA-targeted T-cell engaging molecules and antiandrogen compounds can be determined from dose-finding human clinical trials, such as those described in the Examples, and in some cases can be extrapolated from relevant animal models of the particular cancer to be treated. Appropriate dosages of particular PSMA-targeted T-cell engaging molecules and antiandrogen compounds are described in more detail herein.

[0039] In some embodiments of the method of the present invention, the first dose of PSMA-targeted T cell engaging molecule and / or antiandrogen compound administered to the patient, for example in the first cycle, is a therapeutic dose. In other embodiments, the first dose of PSMA-targeted T cell engaging molecule and / or antiandrogen compound administered to the patient, for example in the first cycle, is not a therapeutic dose. For example, the first dose of PSMA-targeted T cell engaging molecule and / or antiandrogen compound administered to the patient can be a lower dose than a therapeutic dose (e.g., a titration or priming dose). Such a priming dose, particularly for PSMA-targeted T cell engaging molecule, can be used to reduce the number or severity of adverse events, such as CRS events, when the patient is first exposed to PSMA-targeted T cell engaging molecule. As used herein, the term "priming dose" refers to a dose or amount of a PSMA-targeted T cell engaging molecule that primes a patient for subsequent administration of a therapeutic dose of the PSMA-targeted T cell engaging molecule such that administration of a therapeutic dose results in fewer or less severe adverse events in the patient, e.g., fewer or less severe CRS events. In some embodiments, the priming dose may be lower than the therapeutic dose, but is sufficient to prime the patient's T cells, e.g., release cytokines such that subsequent administration of a higher or therapeutic dose of the T cell engaging molecule results in less increased cytokine secretion. In certain embodiments, the priming dose is sufficient to increase the percentage of activated peripheral T cells in the patient (e.g., increase the percentage of CD69+CD8+ peripheral T cells) compared to the percentage of activated T cells in the patient before receiving the dose of the T cell engaging molecule. The priming dose can be a fraction of the therapeutic dose, for example, about 10% to about 80% of the therapeutic dose, about 20% to about 75%, about 15% to about 50%, about 25% to about 70%, or about 30% to about 60% of the therapeutic dose. In one embodiment, the priming dose of the PSMA-targeted T cell engaging molecule is about 30% of the therapeutic dose. In another embodiment, the priming dose of the PSMA-targeted T cell engaging molecule is about 60% of the therapeutic dose.One or more priming doses of PSMA-targeted T cell engagement molecule can be administered to patient before administering therapeutic dose.In certain embodiments of the method of the present invention, the first dose of antiandrogen compound administered to patient in the first cycle is a therapeutic dose, and the first dose of PSMA-targeted T cell engagement molecule administered to patient in the first cycle is a priming dose.In certain other embodiments of the method of the present invention, the first dose of antiandrogen compound administered to patient in the first cycle is a therapeutic dose, and the first dose of PSMA-targeted T cell engagement molecule administered to patient in the first cycle is a therapeutic dose.

[0040] In certain embodiments of the methods of the invention, the first cycle further comprises administering one or more priming doses of a PSMA-targeted T cell engaging molecule to the patient prior to administration of the first therapeutic dose of the T cell engaging molecule. In such embodiments, the first therapeutic dose of the T cell engaging molecule may be administered about 1 day to about 21 days, about 1 day to about 14 days, or about 1 day to about 7 days after administration of the first priming dose of the PSMA-targeted T cell engaging molecule to the patient. In certain embodiments, the first therapeutic dose of the PSMA-targeted T cell engaging molecule is administered to the patient about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after the first priming dose of the PSMA-targeted T cell engaging molecule. In embodiments in which two or more priming doses of the PSMA-targeted T cell engaging molecule are administered to the patient, the priming doses may be the same or different. For example, in some such embodiments, the priming dose of the PSMA-targeted T cell engaging molecule may be increased in one or more subsequent dosing intervals as a series of increasing dosing steps. Such a stepwise dosing regimen may be used in embodiments in which two or more priming doses are administered prior to administration of a therapeutic dose during a first cycle, and may include one or more dosing steps (e.g., one or more dose escalations).

[0041] The antiandrogen compound administered by the method of the present invention refers to a compound that inhibits or blocks the biological activity of androgens in one or more cells or tissues of the body. In some embodiments, the antiandrogen compound inhibits or blocks the synthesis of androgens, for example, by inhibiting the function of the CYP17A1 enzyme. Exemplary androgen biosynthesis inhibitors include abiraterone and ketoconazole. In other embodiments, the antiandrogen compound is an androgen receptor antagonist, for example, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide. In certain embodiments, the antiandrogen compound used in the method of the present invention is abiraterone, abiraterone acetate, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide. In other embodiments, the antiandrogen compound is enzalutamide, abiraterone, abiraterone acetate, apalutamide, or darolutamide. In one particular embodiment, the antiandrogen compound used in the methods of the invention is enzalutamide. In another particular embodiment, the antiandrogen compound used in the methods of the invention is abiraterone or abiraterone acetate.

[0042] In certain embodiments of the method of the present invention, the antiandrogen compound is administered orally. A suitable therapeutic oral dose of the antiandrogen compound may be about 50 mg to about 1,000, about 150 mg to about 800 mg, or about 200 mg to about 600 mg, depending on the particular antiandrogen compound used. Such doses may be administered once a day, twice a day, or three times a day for the duration of the treatment cycle. In certain embodiments, the antiandrogen compound is orally administered to the patient once a day (QD). In some embodiments, the oral dose is in the form of a tablet or capsule. In some such embodiments, each tablet or capsule may contain a unit dose less than the total therapeutic dose administered, such that more than one tablet or capsule is administered to the patient at each particular dosing interval.

[0043] In some embodiments, the antiandrogen compound is enzalutamide and the therapeutic oral dose is about 40 mg to about 160 mg administered once daily (QD). In one particular embodiment of the method of the present invention, enzalutamide is orally administered to the patient once daily at a dose of 160 mg (e.g., four 40 mg tablets or capsules or two 80 mg tablets). In other embodiments, the antiandrogen compound is abiraterone or abiraterone acetate and the therapeutic oral dose is about 250 mg to about 1,000 mg or about 500 mg to about 1,000 mg administered once daily. In one such embodiment, abiraterone or abiraterone acetate is orally administered to the patient once daily at a dose of 1,000 mg (e.g., four 250 mg tablets or two 500 mg tablets). In embodiments in which the patient is administered abiraterone or abiraterone acetate, a 5 mg dose of prednisone may be orally administered twice daily (BID). In other embodiments in which the patient is administered abiraterone or abiraterone acetate, a 10 mg dose of prednisolone may be orally administered once daily. In certain other embodiments of the methods of the invention, the antiandrogen compound is darolutamide and the therapeutic oral dose is about 300 mg to about 600 mg administered twice daily (BID). In one such embodiment, darolutamide is orally administered to the patient twice daily in a 600 mg dose (e.g., two 300 mg tablets). The antiandrogen compound is apalutamide and the therapeutic oral dose is about 120 mg to about 240 mg administered once daily (QD). In one such embodiment, apalutamide is administered orally to a patient once daily in a dose of 240 mg (eg, four 60 mg tablets).

[0044] The methods described herein include administering to a patient a PSMA-targeted T cell engaging molecule, which refers to a T cell engaging molecule that specifically binds to PSMA and CD3. The term "T cell engaging molecule" refers to a molecule that includes at least one domain whose structure is derived from or includes the minimal structural features of an antibody, e.g., a full-length immunoglobulin molecule, that allows specific binding to an antigen on the surface of a T cell, such as CD3. Thus, a T cell engaging molecule according to the invention generally includes one or more binding domains, each of which typically includes the minimal structural requirements of an antibody that allows specific target binding. This minimal requirement may be defined, for example, by the presence of at least three light chain "complementarity determining regions" or CDRs (i.e., CDRL1, CDRL2, and CDRL3 of the VL region) and / or three heavy chain CDRs (i.e., CDRH1, CDRH2, and CDRH3 of the VH region), preferably all six CDRs of both the light chain variable region and the heavy chain variable region. T cell engaging molecules according to the present invention may comprise domains or regions (e.g., CDRs or variable regions) derived from monoclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies.

[0045] Preferably, the T cell engaging molecules used in the methods of the invention are proteins and comprise one or more polypeptide chains. Polypeptide, as used herein, refers to a polymer of amino acids comprising at least 50 amino acids, preferably at least 100 amino acids. In some embodiments, the T cell engaging molecules administered in accordance with the methods of the invention are single chain polypeptides. In other embodiments, the T cell engaging molecules administered in accordance with the methods of the invention comprise two or more polypeptide chains - e.g., polypeptide dimers or multimers. In certain embodiments, the T cell engaging molecules administered in accordance with the methods of the invention comprise four polypeptide chains, and may have, for example, the format of an antibody or immunoglobulin protein. In certain other embodiments, the T cell engaging molecules administered in accordance with the methods of the invention comprise three polypeptide chains.

[0046] As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each about 25 kDa) and two heavy chain polypeptides (each about 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from amino-terminus to carboxyl-terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino-terminus to carboxyl-terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε) and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG class and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA and IgD antibodies have three constant domains (CH1, CH2 and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3 and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e. between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0047] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions (more often called "complementarity determining regions" or CDRs). The CDRs from the two chains of each heavy / light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein (e.g., PSMA or CD3). From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically have the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDRs and FRs of a given antibody can be identified using this system. Other numbering systems for the amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0048] The T cell engaging molecule used in the method of the present invention is preferably at least a bispecific T cell engaging molecule. The term "bispecific T cell engaging molecule" refers to a molecule that can specifically bind to two different antigens. In the context of the present invention, such a bispecific T cell engaging molecule specifically binds to PSMA (e.g., human PSMA) on the cell surface of a target cell and CD3 (e.g., human CD3) on the cell surface of a T cell. The term "PSMA-targeted T cell engaging molecule" is used herein to refer to a T cell engaging molecule that specifically binds to PSMA and CD3. A T cell engaging molecule or a binding domain thereof "specifically binds" to a target antigen if it has a significantly higher binding affinity for the target antigen compared to its affinity for other unrelated proteins under similar binding assay conditions, such that the antigens can be distinguished. A T cell engaging molecule or a binding domain thereof that specifically binds to an antigen has a binding affinity for the target antigen that is significantly higher than 1×10 -6 The equilibrium dissociation constant (K D The T cell engaging molecule or its binding domain may bind to K D is 1×10 -8 In one embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the invention specifically binds to an antigen with a binding affinity of 5×10 -7 K below M D In another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the invention binds to human PSMA and / or human CD3 at 1×10 -7 K below M D In yet another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the invention binds to human PSMA and / or human CD3 at 5×10 -8 K below M D In another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the invention binds to human PSMA and / or human CD3 at 2×10 -8 K below M DIn a specific embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the invention binds to human PSMA and / or human CD3 at 1×10 -8 K below M D In another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the invention binds to human PSMA and / or human CD3 at 1×10 -9 K below M D and binds to human PSMA and / or human CD3.

[0049] Affinity is determined using a variety of techniques, one example of which is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the binding rate constant (k a Unit:M -1 s -1 ) and dissociation rate constant (k d Unit: s -1 ) can then be measured. D The ratio of kinetic rate constants (k d / k a In some embodiments, affinity is determined by a kinetic method, such as the equilibrium exclusion method (KExA) as described in Rathanaswami et al., Analytical Biochemistry, Vol. 373:52-60, 2008. The KinExA assay is used to determine the equilibrium dissociation constant (K D , M) and the binding rate constant (k a , M -1 s -1 From these values, the dissociation rate constant (k d , s -1 ) can be calculated (K D ×k aIn another embodiment, the affinity is determined by biolayer interferometry as described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015 and used in the Octet® system (Pall ForteBio). The rate constant (k a and k d ) and affinity constant (K D ) can be calculated in real time using biolayer interferometry. In some embodiments, the antibodies or binding domains thereof described herein have a resolution of about 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 or less for human PSMA and / or human CD3 d (dissociation rate constant) (lower values ​​indicate higher binding affinity) and / or approximately 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 K for human PSMA and / or human CD3 at or below M D The binding affinity of the agonist is preferably at least 100% and more preferably at least 150%.

[0050] In some embodiments, the PSMA-targeted T cell engaging molecule used in the methods of the present invention is an antibody and may have the general structure of a full-length immunoglobulin. For example, the PSMA-targeted T cell engaging molecule may comprise two full-length antibody heavy chains and two full-length antibody light chains. In certain embodiments, the T cell engaging molecule of the present invention is a heterodimeric antibody (used interchangeably herein as "heteroimmunoglobulin" or "heteroIg"), which refers to an antibody that comprises two different light chains and two different heavy chains. For example, in some embodiments, the heterodimeric antibody comprises a light chain and a heavy chain derived from an anti-PSMA antibody and a light chain and a heavy chain derived from an anti-CD3 antibody.

[0051] The PSMA-targeted T cell engaging molecules used in the methods of the invention may also include fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "rIgG" (a "half antibody" consisting of a heavy and light chain). The PSMA-targeted T cell engaging molecules according to the invention may also include modified fragments of antibodies. Examples of such modified fragments include, but are not limited to, single chain variable fragments (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, single chain Fab (scFab), Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab), tandem di-scFv, tandem tri-scFv, "minibodies" exemplified by structures as follows: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies (e.g. triabodies or tetrabodies) and single domain antibodies (e.g. nanobodies or single variable domain antibodies comprising only one variable region which may be VHH, VH or VL, which specifically binds to an antigen or target independent of other variable regions or domains). In some embodiments, the PSMA-targeted T cell engaging molecules used in the methods of the invention comprise a binding domain derived from a single variable domain antibody that comprises only a heavy chain variable region. In some such embodiments, the binding domain comprising the heavy chain variable region may be fused to one or more constant regions from an immunoglobulin heavy chain, such as a CH2, CH3, or Fc domain.

[0052] In certain embodiments, the PSMA-targeted T cell engaging molecules used in the methods of the invention are multivalent. The valency of a T cell engaging molecule refers to the number of individual antigen binding domains in the T cell engaging molecule. In the context of the present invention, for example, the terms "monovalent", "bivalent" and "tetravalent" with respect to a T cell engaging molecule refer to a T cell engaging molecule having one, two and four antigen binding domains, respectively. Thus, a multivalent T cell engaging molecule comprises two or more antigen binding domains. A T cell engaging molecule can have more antigen binding domains (e.g., higher valency) than specificity. For example, a T cell engaging molecule having two antigen binding domains for a first target (e.g., PSMA) and one antigen binding domain for a second target (CD3) - or vice versa - is considered to be trivalent (three antigen binding domains) and bispecific (binds to two antigens). In certain embodiments, the PSMA-targeted T cell engaging molecules used in the methods of the invention are bivalent. Thus, such a bispecific, bivalent T cell engaging molecule contains two antigen-binding domains: one antigen-binding domain for PSMA (e.g., human PSMA) and one antigen-binding domain for CD3 (e.g., human CD3).

[0053] Exemplary PSMA-targeted T cell engagement molecules used in the methods of the present invention are described in more detail herein.Other suitable PSMA-targeted T cell engagement molecules that can be administered in the combination therapy methods of the present invention are known in the art and are described in, for example, WO2010 / 037836, WO2011 / 121110, WO2012 / 145714, WO2017 / 023761, WO2017 / 121905, WO2017 / 134158, WO2018 / 098356, WO2019 / 224718, WO2020 / 206330, and WO2021 / 231976, all of which are incorporated herein by reference in their entirety. Exemplary ranges of therapeutic doses of PSMA-targeted T-cell engaging molecules that may be administered according to the methods of the present invention include, but are not limited to, doses of about 50 μg to about 200 mg, about 200 μg to about 80 mg, about 90 μg to about 30 mg, about 300 μg to about 15 mg, about 150 μg to about 2 mg, about 6 mg to about 25 mg, about 1 mg to about 20 mg, about 10 mg to about 100 mg, or about 50 mg to about 150 mg. Depending on the particular PSMA-targeted T-cell engaging molecule used and its half-life, the therapeutic dose of the PSMA-targeted T-cell engaging molecule may be administered at a dosing interval of once a week (QW), once every two weeks (Q2W), once every three weeks (Q3W), or once a month (QM). In certain embodiments, the therapeutic dose of the PSMA-targeted T-cell engaging molecule is administered once every 7 days or once a week. In other specific embodiments, a therapeutic dose of the PSMA-targeted T cell engaging molecule is administered once every 14 days or once every two weeks.

[0054] In some embodiments of the methods described herein, the PSMA-targeted T cell engaging molecule is administered parenterally to the patient. Parenteral administration refers to administration of a molecule by a route other than via the gastrointestinal tract, and may include intraperitoneal, intramuscular, intravenous, intraarterial, intradermal, subcutaneous, intracerebral, intraventricular, and intrathecal administration. In certain embodiments of the methods of the present invention, the PSMA-targeted T cell engaging molecule is administered intravenously to the patient. In other embodiments of the methods of the present invention, the PSMA-targeted T cell engaging molecule is administered subcutaneously to the patient.

[0055] Parenteral or intravenous administration may be performed by injection (e.g., using a needle and syringe) or infusion (e.g., via a catheter and pump system). It is envisioned that administration of the PSMA-targeted T cell engaging molecules according to some embodiments of the methods of the present invention is by intravenous injection or infusion. Typically, intravenous (IV) infusion is administered through a line, port, or catheter (small, flexible tubing), such as a central venous access, or central venous catheter (CVC), which is a catheter placed in a large vein, or a peripheral venous catheter (PVC), which is a catheter placed in a peripheral vein. Generally, a catheter or line may be placed through a vein in the neck (internal jugular vein), a vein in the chest (subclavian vein or axillary vein), a vein in the groin (femoral vein), or a vein in the arm (also known as a PICC line or peripherally inserted central catheter). A central IV line has a catheter that is advanced through a vein and drains into a large central vein (usually the superior vena cava, inferior vena cava) or even into the right atrium of the heart. Peripheral intravenous (PIV) lines are used in peripheral veins (veins in the arms, hands, legs and feet). A port is a central venous line that has no external connector; instead, it is covered with silicone rubber and has a small reservoir implanted under the skin. Medication is administered intermittently by inserting a small needle into the skin, piercing the silicone and entering the reservoir. When the needle is withdrawn, the reservoir cover naturally reseals. The cover can tolerate hundreds of needle sticks during its life.

[0056] In certain embodiments of the methods of the invention, one or more doses of PSMA-targeted T-cell engaging molecules are administered to a patient by continuous intravenous infusion over an extended period of time. As used herein, continuous intravenous infusion refers to a controlled method of intravenous administration of PSMA-targeted T-cell engaging molecules given without or substantially without interruption for a period of more than about 3 hours, more typically more than about 6 hours. Continuous intravenous infusion may be administered by a fluid delivery device or miniature pump system, including a fluid delivery mechanism for pumping fluid from a reservoir and a drive mechanism for driving the delivery mechanism. A pump system for such administration may include a needle or cannula for penetrating the patient's skin and delivering the infusion solution into the patient's body. The pump system may be connected to the patient for 24 hours to several days. Pump systems for delivering intravenous infusions are known in the art. Depending on the duration of the continuous infusion, it may be necessary to exchange or replace the bag or reservoir that contains the infusion solution within the pump system. During replacement of the bag or reservoir of the pump system, a temporary interruption of the otherwise uninterrupted flow of infusion solution may occur. Such a temporary interruption resulting from replacement of a bag or reservoir does not constitute an interruption or substantial interruption of intravenous administration, and the time during which the bag or reservoir is replaced is still considered to be within the time of continuous intravenous infusion, as that term is used herein.

[0057] In other embodiments of the methods of the present invention, one or more doses of the PSMA-targeted T cell engaging molecule are administered by bolus intravenous infusion. As used herein, bolus intravenous infusion, used interchangeably herein with short intravenous infusion, refers to a small intravenous infusion (e.g., 20 mL to 100 mL) administered over a period of at most 3 hours, more typically over a period of about 30 minutes to about 90 minutes. In some embodiments of the methods of the present invention, the bolus intravenous infusion is an intravenous infusion administered over a period of about 30 minutes to about 60 minutes. In certain embodiments of the methods of the present invention, the bolus intravenous infusion is an intravenous infusion administered over a period of about 60 minutes (e.g., 55 minutes to 65 minutes).

[0058] A combination of routes of administration can be used for administration of PSMA-targeted T cell engaging molecules according to the methods of the invention. For example, in some embodiments, the first dose of PSMA-targeted T cell engaging molecule (e.g., a priming dose), particularly in the first cycle, can be administered by continuous intravenous infusion over an extended period of time, such as 1-7 days, followed by subsequent doses (e.g., therapeutic doses) by bolus intravenous infusion or subcutaneous injection. Thus, in some embodiments of the methods of the invention, the first dose of PSMA-targeted T cell engaging molecule in the first cycle (i.e., the first priming dose) is administered by continuous intravenous infusion over a period of at least 24 hours, such as 1-14 days, 1-7 days, or 1-5 days. In certain embodiments, the first dose of PSMA-targeted T cell engaging molecule in the first cycle (e.g., the first priming dose) is administered by continuous intravenous infusion over a period of about 3 days (i.e., a 72-hour infusion). In these and other embodiments, the continuous intravenous infusion is given at a constant flow rate. That is, the continuous intravenous infusion delivers the dose of PSMA-targeted T cell engaging molecule at a constant rate over the infusion period. For example, for a dose of 3 mg, the continuous intravenous infusion given at a constant flow rate over 3 days delivers the dose at a rate of 1 mg per day, so that a total dose of 3 mg is delivered at the completion of the infusion period of 3 days. Alternatively, in some embodiments, the continuous intravenous infusion can be given at a variable flow rate, so that the dose is delivered at different doses per day over the infusion period. For example, in one such embodiment, the flow rate of the continuous infusion can be adjusted so that an increasing dose is given every day over the infusion period to deliver the total dose at the completion of the infusion period.

[0059] In some embodiments of the methods of the invention, the first cycle comprises administering a dose of the PSMA-targeted T-cell engaging molecule at a dosing interval of once every seven days (or once a week) and administering a dose of the antiandrogen compound at a dosing interval of once a day, the first dose of the antiandrogen compound being administered at least three days after the administration of the first therapeutic dose of the PSMA-targeted T-cell engaging molecule. An exemplary dosing schedule according to such an embodiment, in which the duration of the first cycle is 28 days, comprises administering a dose of the PSMA-targeted T-cell engaging molecule (e.g., by bolus intravenous infusion) on each of days 1, 8, 15, and 22 of the first cycle, and administering a dose of the antiandrogen compound (e.g., by oral administration) once per day on each of days 15-28 of the first cycle. In other embodiments of the methods of the invention, the first cycle comprises administering a dose of the PSMA-targeted T-cell engaging molecule at a dosing interval of once every 14 days (or once every two weeks) and administering a dose of the antiandrogen compound at a dosing interval of once a day, the first dose of the antiandrogen compound being administered at least 3 days after the administration of the first therapeutic dose of the PSMA-targeted T-cell engaging molecule. An exemplary dosing schedule according to these embodiments, in which the duration of the first cycle is 28 days, comprises administering a dose of the PSMA-targeted T-cell engaging molecule (e.g., by bolus intravenous infusion) on each of days 1 and 15 of the first cycle, and administering a dose of the antiandrogen compound (e.g., by oral administration) once per day on each of days 15-28 of the first cycle. In any of the foregoing embodiments, all of the doses of the PSMA-targeted T-cell engaging molecule administered during the first cycle may be the same and may be therapeutic doses. In alternative embodiments, the first dose of the PSMA-targeted T cell engaging molecule (e.g., the dose administered on day 1 of the cycle) may be different from all subsequent doses of the T cell engaging molecule administered during the first cycle. For example, the first dose of the PSMA-targeted T cell engaging molecule may be a priming dose as described herein, and all subsequent doses of the PSMA-targeted T cell engaging molecule may be therapeutic doses.

[0060] In certain embodiments of the methods of the invention, the first cycle comprises administering a priming dose of the PSMA-targeted T-cell engaging molecule by continuous intravenous infusion over about three days, followed by administering a therapeutic dose of the PSMA-targeted T-cell engaging molecule by bolus intravenous infusion at a dosing interval of seven or fourteen days, and administering a dose of an antiandrogen compound at a dosing interval of once daily, wherein the first dose of the antiandrogen compound is administered at least three days after the administration of the first therapeutic dose of the PSMA-targeted T-cell engaging molecule. In embodiments in which the first cycle has a duration of 28 days, an exemplary dosing schedule may include administration of a priming dose of the PSMA-targeted T-cell engaging molecule (which may be any of the priming doses described herein) by continuous intravenous infusion over days 1-3 of the first cycle, administration of a therapeutic dose of the PSMA-targeted T-cell engaging molecule by bolus intravenous infusion on days 8 and 22 of the first cycle, and oral administration of a dose of the antiandrogen compound once daily on each of days 15-28 of the first cycle.

[0061] In some embodiments, the method of the present invention further comprises administering to the patient at least one maintenance cycle of an antiandrogen compound in combination with a PSMA-targeted T cell engaging molecule after administration of the first cycle or the initiation cycle. As used herein, a "maintenance cycle" is a treatment cycle in which the PSMA-targeted T cell engaging molecule and the antiandrogen compound are administered at a dose and dosing frequency designed to maintain a threshold level of exposure of both compounds at therapeutic levels in the patient. In certain embodiments, the dosing frequency of the PSMA-targeted T cell engaging molecule, the antiandrogen compound, or both compounds used in the maintenance cycle is less than that used in the first cycle (i.e., the interval between dosing of the maintenance cycle is longer than the interval between dosing of the first cycle). In some embodiments, the maintenance cycle is administered immediately after the completion of the first initiation cycle. Thus, in such embodiments, there is no treatment-free period or interruption between the end of the first cycle and the start of the maintenance cycle. In one such embodiment, the maintenance cycle is administered the day after completing the first cycle or the initiation cycle. In other embodiments, there is a treatment-free period or break between the completion of the first or initiation cycle and the administration of the maintenance cycle.Preferably, the treatment-free period between the first or initiation cycle and the maintenance cycle is equal to or less than the administration interval of the PSMA-targeted T cell engaging molecule used in the maintenance cycle.In one embodiment, the maintenance cycle is administered about 7 days after the completion of the first or initiation cycle.In another embodiment, the maintenance cycle is administered about 14 days after the completion of the first or initiation cycle.

[0062] Multiple maintenance cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles) can be administered to a patient depending on the desired duration of treatment for that patient. For example, a patient can receive maintenance cycles of a PSMA-targeted T-cell engaging molecule in combination with an antiandrogen compound until the patient achieves a desired level of response, such as a complete or partial response. In some embodiments, two or more maintenance cycles are administered to the patient. In other embodiments, four or more maintenance cycles are administered to the patient. In still other embodiments, six to twelve maintenance cycles are administered to the patient. In certain embodiments, the maintenance cycles are administered consecutively with no treatment-free period between the maintenance cycles. If an interruption in treatment is required, the duration of the treatment-free period is ideally no more than twice the interval between administrations of the PSMA-targeted T-cell engaging molecule used in the maintenance cycle. For example, if the administration interval of the PSMA-targeted T cell engaging molecule used in the maintenance cycle is once every 14 days (e.g., once every two weeks), the treatment-free period between maintenance cycles is preferably about 28 days or less.

[0063] In certain embodiments of the methods of the present invention, the maintenance cycle comprises administering a therapeutic dose of a PSMA-targeted T-cell engaging molecule (such as any of the therapeutic doses described herein) with a dosing interval of at least 7 days, and orally administering an antiandrogen compound once a day on each day of the cycle. For example, in some embodiments of the methods of the present invention, the maintenance cycle comprises administering a therapeutic dose of a PSMA-targeted T-cell engaging molecule by bolus intravenous infusion or subcutaneous injection once every 7 days (e.g., once a week, QW administration) and orally administering an antiandrogen compound once a day on each day of the cycle. In other embodiments of the methods of the present invention, the maintenance cycle comprises administering a therapeutic dose of a PSMA-targeted T-cell engaging molecule by bolus intravenous infusion or subcutaneous injection once every 14 days (e.g., once every 2 weeks, Q2W administration) and orally administering an antiandrogen compound once a day on each day of the cycle. In yet other embodiments, the therapeutic dose of the PSMA-targeted T-cell engaging molecule may be administered by bolus intravenous infusion or subcutaneous injection at longer dosing intervals during the maintenance cycle, such as once every 3 weeks or once every 4 weeks. In such embodiments, the antiandrogen compound may continue to be administered orally once a day on each day of the cycle. Preferably, the therapeutic dose of the PSMA-targeted T-cell engaging molecule administered during the maintenance cycle is the same at each dosing interval, for example, once a week or once every two weeks (e.g., a fixed dose throughout the maintenance cycle). In these and other embodiments, the therapeutic dose and dosing frequency of the PSMA-targeted T-cell engaging molecule administered during the maintenance cycle is the same from one maintenance cycle to the next.

[0064] According to some embodiments of the methods of the present invention, the duration of the maintenance cycle is about 14 days to about 60 days, such as about 14 days to about 28 days, about 21 days to about 42 days, about 28 days to about 49 days, about 28 days to about 56 days, or about 21 days to about 28 days. In certain embodiments, the duration of the maintenance cycle is about 28 days. In some such embodiments, the therapeutic dose of the PSMA-targeted T-cell engaging molecule is administered by bolus intravenous injection on days 1 and 15 of each maintenance cycle, and the antiandrogen compound is administered orally once daily on each day of the maintenance cycle (i.e., each of days 1 to 28). In other embodiments, in which the duration of the maintenance cycle is about 28 days, the therapeutic dose of the PSMA-targeted T-cell engaging molecule is administered by bolus intravenous injection on days 1, 8, 15, and 22 of each maintenance cycle, and the antiandrogen compound is administered orally once daily on each day of the maintenance cycle (i.e., each of days 1 to 28).

[0065] In some embodiments, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises a first binding domain that specifically binds PSMA (e.g., human PSMA) and a second binding domain that specifically binds CD3 (e.g., human CD3). As used herein, the term "antigen-binding domain", used interchangeably with "binding domain", refers to a region of a T cell engaging molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity to the T cell engaging molecule for that antigen. In certain embodiments, one or more binding domains of a T cell engaging molecule may be derived from an antibody or an antigen-binding fragment thereof. For example, the binding domain of a PSMA-targeted T cell engaging molecule used in the methods of the invention may comprise one or more CDRs derived from the light and heavy chain variable regions of an antibody that specifically binds human PSMA and / or human CD3. In some embodiments, the anti-PSMA binding domain of the T cell engaging molecule comprises all six CDRs of the heavy and light chain variable regions of an anti-PSMA antibody described herein or known in the art, and the anti-CD3 binding domain of the T cell engaging molecule comprises all six CDRs of the heavy and light chain variable regions of an anti-CD3 antibody described herein or known in the art. In some embodiments, the binding domains (anti-PSMA binding domain, anti-CD3 binding domain or both) of the PSMA-targeted T cell engaging molecule used in the methods of the invention comprise a Fab, Fab', F(ab')2, Fv, single chain variable fragment (scFv) or nanobody. In one embodiment, both binding domains of the PSMA-targeted T cell engaging molecule are Fab fragments. In another embodiment, one binding domain of the PSMA-targeted T cell engaging molecule is a Fab fragment and the other binding domain is an scFv. In yet another embodiment, both binding domains of the PSMA-targeted T cell engaging molecule are scFv. In yet another embodiment, one binding domain of the PSMA-targeted T cell engaging molecule is a nanobody and the other binding domain is a Fab fragment.

[0066] As used in the context of the present invention, an "antigen-binding fragment", which is used interchangeably herein with "binding fragment" or "fragment", is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chains, but is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a heavy-chain-only antibody (e.g., a camelid heavy-chain antibody); VHH fragments, see Cortez-Retamozo et al., Cancer Research, Vol. 64: 2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments and CDR fragments, and may be derived from any mammalian source, such as human, mouse, rat, rabbit or camel. An antigen-binding fragment may compete with an intact antibody for binding to a target antigen, and may be produced by modification of an intact antibody (e.g., enzymatic or chemical cleavage) or may be synthesized de novo using recombinant DNA technology or peptide synthesis. In some embodiments, an antigen-binding fragment comprises at least one CDR from an antibody that binds the antigen, such as the heavy chain CDR3 from an antibody that binds the antigen. In other embodiments, an antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds the antigen or all three CDRs from the light chain of an antibody that binds the antigen. In yet other embodiments, an antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) from an antibody that binds the antigen.

[0067] Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments, each of which has a single antigen-binding site, and a residual "Fc" fragment, which contains all but the first domain of the immunoglobulin heavy chain constant region. The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and one immunoglobulin heavy chain CH1 region and variable region (VH). The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH domain and the CH1 domain from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0068] An "Fc fragment" or "Fc domain" of an immunoglobulin generally comprises two constant domains, namely, a CH2 domain and a CH3 domain, and optionally a CH4 domain. In certain embodiments, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises an Fc domain of an immunoglobulin. The Fc domain may be an Fc domain derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc domain comprises a CH2 domain and a CH3 domain derived from a human IgG1 or human IgG2 immunoglobulin. The Fc domain may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc domain may be modified to reduce or eliminate effector functions.

[0069] A "Fab' fragment" is a Fab fragment that has, at the C-terminus of the CH1 domain, one or more cysteine ​​residues from the antibody hinge region.

[0070] An "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by inter-heavy chain disulfide bridges at the hinge region.

[0071] An "Fv" fragment is the minimum fragment that contains a complete antigen recognition and binding site from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable domain (VH) and one immunoglobulin light chain variable domain (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. A single light or heavy chain variable domain (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with lower affinity than the entire binding site containing both VH and VL.

[0072] A "single-chain variable antibody fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, which domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0073] "Nanobodies" are the heavy chain variable regions of heavy chain antibodies. Such variable domains are the smallest fully functional antigen-binding fragments of such heavy chain antibodies, with a molecular mass of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies, devoid of light chains, occur naturally in certain species of animals, such as the nurse shark, nurse shark, and Camelidae, such as camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region, i.e., these functional antibodies are heavy chain-only homodimers (also called "heavy chain antibodies" or "HCAbs"). Camelized VHHs are reportedly recombined with IgG2 and IgG3 constant regions, containing hinge, CH2 and CH3 domains, and lacking the CH1 domain. Camelized VHH domains have been shown to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276: 26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41: 3628-36, 2002). Methods for making antibodies with camelized heavy chains are described, for example, in US Patent Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold and can provide a framework for long transmembrane loop structures. Human heavy chain antibodies can be produced from transgenic animals expressing human immunoglobulin genes, such as, for example, the UniAb™ antibodies produced by UniRat™ transgenic rats.

[0074] In certain embodiments, the binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises the immunoglobulin heavy chain variable region (VH) and immunoglobulin light chain variable region (VL) of an antibody or antibody fragment that specifically binds to a desired antigen. For example, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule of the invention comprises the VH and VL regions derived from an anti-PSMA antibody, e.g., any of the anti-PSMA antibodies or fragments thereof known in the art or described herein, and the anti-CD3 binding domain comprises the VH and VL regions derived from an anti-CD3 antibody, e.g., any of the anti-CD3 antibodies or fragments thereof known in the art or described herein. The binding domain that specifically binds to human PSMA or human CD3 can be derived from known antibodies against these antigens or from novel antibodies or antibody fragments obtained by novel immunization methods using antigenic proteins or fragments thereof, phage display, or other methods known in the art and described herein below. The antibody from which the binding domain of the PSMA-targeted T-cell engaging molecule is derived may be a monoclonal antibody, a recombinant antibody, a chimeric antibody, a human antibody, or a humanized antibody. In certain embodiments, the antibody from which the binding domain is derived is a monoclonal antibody. In these or other embodiments, the antibody is a human antibody or a humanized antibody and may be of the IgG1, IgG2, IgG3, or IgG4 type.

[0075] The first binding domain of the PSMA-targeted T cell engaging molecule used in the method of the present invention specifically binds to PSMA, preferably human PSMA. This binding domain is referred to herein as anti-PSMA binding domain. PSMA (prostate-specific membrane antigen; also known as glutamic acid carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamic acid peptidase I, or NAAG peptidase) is a type II membrane glycoprotein that is expressed primarily on prostate epithelial cells. More preferably, the first binding domain binds to PSMA on the surface of the target cell. A "target cell" can be any prokaryotic or eukaryotic cell that expresses PSMA on its surface; preferably, the target cell is a cell that is part of a human or animal body, such as a specific PSMA-expressing cancer or tumor cell. It is further envisaged that the first binding domain of the PSMA-targeted T cell engaging molecule binds to human PSMA, preferably PSMA on the surface of the target cell. It is also envisioned that the first binding domain binds to macaque PSMA, preferably macaque PSMA on the surface of a target cell. Exemplary amino acid sequences of the mature polypeptide and extracellular domains of human and macaque PSMA are provided in Table 1 below.

[0076] [Table 1-1]

[0077] [Table 1-2]

[0078] Examples of anti-PSMA binding domains from which the first binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention may be constructed or derived are described in WO 2010 / 037836, WO 2011 / 121110, WO 2017 / 134158 and WO 2020 / 206330, all of which are incorporated by reference in their entireties. Exemplary anti-human PSMA antibody light and heavy chain variable regions and associated CDRs from which the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule may be derived or constructed are shown in Tables 2A and 2B, respectively.

[0079] [Table 2A-1]

[0080] [Table 2A-2]

[0081] [Table 2B-1]

[0082] [Table 2B-2]

[0083] [Table 2B-3]

[0084] [Table 2B-4]

[0085] A domain that specifically binds human PSMA (e.g., an anti-PSMA binding domain) of a PSMA-targeted T cell engaging molecule suitable for use in the methods of the invention can comprise one or more of the light chain CDRs (i.e., CDRLs) and / or heavy chain CDRs (i.e., CDRHs) presented in Tables 2A and 2B, respectively. For example, in some embodiments, an anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a CDRL1 comprising the sequence of SEQ ID NO:5 or SEQ ID NO:6; a CDRL2 comprising the sequence of SEQ ID NO:7 or SEQ ID NO:8; a CDRL3 comprising a sequence selected from SEQ ID NOs:9-13; a CDRH1 comprising the sequence of SEQ ID NO:14 or SEQ ID NO:15; a CDRH2 comprising a sequence selected from SEQ ID NOs:16-19; and a CDRH3 comprising the sequence of SEQ ID NO:20.

[0086] In some embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3: (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 7, and 9, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 7, and 10, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 7, and 11, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 7, and 12, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 7, and 13, respectively; (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 8, and 9, respectively; or (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 8, and 9, respectively. In these and other embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3: (a) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 14, 16, and 20, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 14, 17, and 20, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 15, 18, and 20, respectively; or (d) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 14, 19, and 20, respectively.

[0087] In certain embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule suitable for use in the methods of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 9, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:14, 16 and 20, respectively; (b) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 10, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:14, 16 and 20, respectively; (c) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 11, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:14, 17 and 20, respectively; (d) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 12, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:14, 16 and 20, respectively; (e) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 13, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:14, 16 and 20, respectively; (f) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 11, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:15, 18 and 20, respectively; (g) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs:5, 7 and 11, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs:14, 16 and 20, respectively; (h) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 5, 8 and 9, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 14, 16 and 20, respectively; or (i) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 6, 8 and 9, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 14, 19 and 20, respectively. In a preferred embodiment, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises (i) a light chain variable region comprising CDRL1 having the sequence of SEQ ID NO: 5, CDRL2 having the sequence of SEQ ID NO: 8 and CDRL3 having the sequence of SEQ ID NO: 9, and (ii) a heavy chain variable region comprising CDRH1 having the sequence of SEQ ID NO: 14, CDRH2 having the sequence of SEQ ID NO: 16 and CDRH3 having the sequence of SEQ ID NO: 20.

[0088] In some embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule used in the methods of the invention comprises a heavy chain-only antibody, such as a heavy chain variable region from an antibody designated as Antibodies 51-57 in Table 2B. In related embodiments, the anti-PSMA binding domain comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3: (a) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 158, 163, and 167, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 164, and 168, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 160, 163, and 167, respectively; (d) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 160, 165, and 167, respectively; (e) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 161, 166, and 169, respectively; or (f) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 162, 166, and 169, respectively.

[0089] In some embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) from an antibody that specifically binds to human PSMA, such as an antibody described herein. A "variable region", as used herein interchangeably with "variable domain" (variable region of the light chain (VL), variable region of the heavy chain (VH)), refers to the region in each of the immunoglobulin light chain and the immunoglobulin heavy chain that is directly involved in binding the antibody to the antigen. As described above, the regions of the variable light chain and the variable heavy chain have the same general structure, each region comprising four framework (FR) regions, the sequences of which are widely conserved and linked by three CDRs. The framework regions adopt a beta-sheet structure, and the CDRs may form loops connecting the beta-sheet structures. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form the antigen-binding site together with the CDRs of the other chain. Thus, in some embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule used in accordance with the methods of the invention may comprise a light chain variable region selected from LV-01 to LV-12 (SEQ ID NOs: 21 to 32) as set forth in Table 2A and / or a heavy chain variable region selected from HV-01 to HV-14 (SEQ ID NOs: 33 to 39 and 170 to 176) as set forth in Table 2B, as well as binding fragments, derivatives and variants of these light and heavy chain variable regions. In certain embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule may comprise only a heavy chain variable region selected from HV-08 to HV-14 (SEQ ID NOs: 170 to 176), which corresponds to the heavy chain variable region from heavy chain-only antibodies 51 to 57 in Table 2B.

[0090] Each of the light chain variable regions listed in Table 2A may be combined with any of the heavy chain variable regions listed in Table 2B to form the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention. Examples of such combinations include, but are not limited to, (i) HV-01 with any of LV-01, LV-02, LV-03, LV-04, LV-05, and LV-10; (ii) LV-03 with HV-02; (iii) LV-06 with HV-03; (iv) LV-07 with HV-04; (v) LV-08 with HV-05; (vi) LV-09 with HV-05; (vii) LV-11 with HV-06; and (viii) LV-12 with HV-07.

[0091] In certain embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:21 and a heavy chain variable region comprising the sequence of SEQ ID NO:33. In some embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:22 and a heavy chain variable region comprising the sequence of SEQ ID NO:33. In other embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:23 and a heavy chain variable region comprising the sequence of SEQ ID NO:33. In yet other embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:24 and a heavy chain variable region comprising the sequence of SEQ ID NO:33. In some embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:25 and a heavy chain variable region comprising the sequence of SEQ ID NO:33. In a particular embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the sequence of SEQ ID NO: 34. In one embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 26 and a heavy chain variable region comprising the sequence of SEQ ID NO: 35. In another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 27 and a heavy chain variable region comprising the sequence of SEQ ID NO: 36. In another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the sequence of SEQ ID NO: 38. In yet another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 32 and a heavy chain variable region comprising the sequence of SEQ ID NO: 39.In some embodiments, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and a heavy chain variable region comprising the sequence of SEQ ID NO: 37. In a preferred embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 30, and a heavy chain variable region comprising the sequence of SEQ ID NO: 33.

[0092] In certain other embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising the sequence of any one of SEQ ID NOs: 170-176. In some such embodiments, the anti-PSMA binding domain does not comprise a light chain variable region. In one embodiment, the anti-PSMA binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 170. In another specific embodiment, the anti-PSMA binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 171. In another embodiment, the anti-PSMA binding domain comprises a first heavy chain variable region comprising the sequence of SEQ ID NO: 170 and a second heavy chain variable region comprising the sequence of SEQ ID NO: 171, the two heavy chain variable region sequences optionally being connected by a linker (e.g., a glycine-serine linker).

[0093] In some embodiments, an anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule for use in the methods of the invention comprises a light chain variable region comprising a sequence of contiguous amino acids that differs from a sequence of a light chain variable region of Table 2A, i.e., a VL selected from LV-01 to LV-12, by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, each such sequence difference being, independently, a deletion, insertion or substitution of one amino acid, the deletion, insertion and / or substitution resulting in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The light chain variable region of some anti-PSMA binding domains comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:21-32 (i.e., the light chain variable region of Table 2A).

[0094] In one embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 21-32. In another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 21-32. In yet another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 21-32.

[0095] In these and other embodiments, the anti-PSMA binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence of contiguous amino acids that differs from a heavy chain variable region sequence of Table 2B, i.e., a VH selected from HV-01 to HV-14, by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, each such sequence difference being, independently, a deletion, insertion or substitution of one amino acid, the deletion, insertion and / or substitution resulting in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The heavy chain variable region of some anti-PSMA binding domains comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 33-39 or SEQ ID NOs: 170-176 (i.e., the heavy chain variable region of Table 2B).

[0096] In one embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 33-39 and 170-176. In another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 33-39 and 170-176. In yet another embodiment, the anti-PSMA binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 33-39 and 170-176.

[0097] The term "identity" as used herein refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" as used herein means the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the smallest size of the compared molecules. For these calculations, gaps in the alignment (if any) must be addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity in the positions of amino acids in two polypeptides.Using a computer program such as BLAST or FASTA, two polypeptide or two polynucleotide sequences are aligned so that their respective residues match optimally (along the entire length of one or both sequences or along a predetermined portion of one or both sequences). The program provides a default start penalty and a default gap penalty, and a scoring matrix such as PAM 250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3, 1978) or BLOSUM62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) can be used with the computer program. Then, for example, the percent identity can be calculated as follows: the total number of perfect matches is multiplied by 100, and then divided by the sum of the length of the longer sequence in the matched span and the number of gaps introduced in the longer sequence to align the two sequences. In calculating the percent identity, the sequences to be compared are aligned to maximize the match between these sequences.

[0098] The GCG program package is a computer program that can be used to determine percent identity, and includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides are best matched (the "match span" determined by the algorithm). With this algorithm, a gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap opening penalty) and a comparison matrix such as PAM 250 or BLOSUM 62 are used. In certain embodiments, the algorithm also uses standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0099] Recommended parameters for determining percent identity of a polypeptide or nucleotide sequence using the GAP program include the following: Algorithm: Needleman et al. 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992 (ibid.); Gap penalty: 12 (but no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0.

[0100] A particular alignment scheme for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this aligned small region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) can be adjusted as necessary to result in alignment over at least 50 consecutive amino acids of the target polypeptide.

[0101] The second binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention specifically binds to CD3, preferably human CD3. This binding domain is referred to herein as the anti-CD3 binding domain. "CD3" (cluster of differentiation 3) is a T cell coreceptor composed of four chains. In mammals, the CD3 protein complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain and two CD3ε (epsilon) chains. These four chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the "T cell receptor complex" and generate activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta) and CD3ε (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling capacity of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene, which is located on chromosome 11 in humans.

[0102] Lysis of target cells redirected by recruitment of T cells by T cell engaging molecules that bind to CD3 on T cells and target proteins (e.g., PSMA) on target cells (e.g., tumor cells) typically involves cytolytic synapse formation and delivery of perforin and granzymes. Engaged T cells are capable of continuous target cell lysis and are not subject to immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation (see, e.g., WO 2007 / 042261).

[0103] In certain embodiments, the second binding domain of the PSMA-targeted T cell engagement molecule used in the methods of the present invention specifically binds to CD3 on the surface of T cells, more preferably to human CD3 on the surface of T cells. In some embodiments, the second binding domain of the PSMA-targeted T cell engagement molecule specifically binds to epsilon of CD3, preferably to epsilon of human CD3, for example, human CD3 epsilon on the surface of T cells. An exemplary amino acid sequence of the extracellular domain of human CD3 epsilon is provided below as SEQ ID NO: 40. 1 QDGNEEMGGI TQTPYKVSIS GTTVILTCPQ YPGSEILWQH NDKNIGGDED DKNIGSDEDH 61 LSLKEFSELE QSGYYVCYPR GSKPEDANFY LYLRARVCEN CMEMD (SEQ ID NO: 40)

[0104] Examples of anti-CD3 binding domains from which the second binding domain of the T cell engaging molecules used in the methods of the invention may be constructed or derived are described in WO 2017 / 042261 and WO 2008 / 119567, both of which are incorporated by reference in their entireties. Exemplary anti-human CD3 antibody light and heavy chain variable regions and associated CDRs from which the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecules may be derived or constructed are shown in Tables 3A and 3B, respectively.

[0105] [Table 3A]

[0106] [Table 3B-1]

[0107] [Table 3B-2]

[0108] A domain that specifically binds human CD3 (e.g., an anti-CD3 binding domain) of a PSMA-targeted T cell engaging molecule suitable for use in the methods of the invention can comprise one or more of the light chain CDRs (i.e., CDRLs) and / or heavy chain CDRs (i.e., CDRHs) presented in Tables 3A and 3B, respectively. For example, in some embodiments, an anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a CDRL1 comprising a sequence selected from SEQ ID NOs: 41-43, and 177; a CDRL2 comprising a sequence of SEQ ID NO: 44, 45, or 178; a CDRL3 comprising a sequence of SEQ ID NO: 46, 47, or 179; a CDRH1 comprising a sequence selected from SEQ ID NOs: 48-53, and 181; a CDRH2 comprising a sequence selected from SEQ ID NOs: 54-58, and 182; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 59-67, and 183.

[0109] In some embodiments, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule comprises a light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein (a) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively; (b) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 42, 45 and 46, respectively; (c) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 43, 44 and 47, respectively; or (d) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 177, 178 and 179, respectively. In these and other embodiments, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3: (a) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 48, 54, and 59, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 49, 55, and 60, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 50, 56, and 61, respectively; (d) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 51, 56, and 62, respectively; (e) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 52, 53, and 54, respectively; (f) CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 49, 54 and 64, respectively; (g) CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 53, 58 and 65, respectively; (h) CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 52, 57 and 66, respectively; (i) CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 50, 56 and 67, respectively; or (j) CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 181, 182 and 183, respectively.

[0110] In certain embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule suitable for use in the methods of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 48, 54 and 59, respectively; (b) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 49, 55 and 60, respectively; (c) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 50, 56 and 61, respectively; (d) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 51, 56 and 62, respectively; (e) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 42, 45 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 52, 57 and 63, respectively; (f) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 49, 54 and 64, respectively; (g) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 42, 45 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 53, 58 and 65, respectively; (h) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 52, 57 and 66, respectively; (i) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 43, 44 and 47, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 50, 56 and 67, respectively; (j) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 43, 44 and 47, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 49, 55 and 60, respectively; or (k) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 177, 178 and 179, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 181, 182 and 183, respectively. In a preferred embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises (i) a light chain variable region comprising CDRL1 having the sequence of SEQ ID NO: 43, CDRL2 having the sequence of SEQ ID NO: 44, and CDRL3 having the sequence of SEQ ID NO: 47, and (ii) a heavy chain variable region comprising CDRH1 having the sequence of SEQ ID NO: 49, CDRH2 having the sequence of SEQ ID NO: 55, and CDRH3 having the sequence of SEQ ID NO: 60. In a preferred embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises (i) a light chain variable region comprising CDRL1 having the sequence of SEQ ID NO: 177, CDRL2 having the sequence of SEQ ID NO: 178, and CDRL3 having the sequence of SEQ ID NO: 179, and (ii) a heavy chain variable region comprising CDRH1 having the sequence of SEQ ID NO: 181, CDRH2 having the sequence of SEQ ID NO: 182, and CDRH3 having the sequence of SEQ ID NO: 183.

[0111] The anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the methods of the invention may comprise a light chain variable region selected from LV-101 to LV-104 (SEQ ID NOs: 68 to 70 and 180) as shown in Table 3A and / or a heavy chain variable region selected from HV-101 to HV-110 (SEQ ID NOs: 71 to 79 and 184) as shown in Table 3B, as well as binding fragments, derivatives and variants of these light and heavy chain variable regions. Each of the light chain variable regions listed in Table 3A may be combined with any of the heavy chain variable regions listed in Table 3B to form the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention. Examples of such combinations include, but are not limited to, (i) LV-101 and HV-101; (ii) LV-101 and HV-102; (iii) LV-101 and HV-103; (iv) LV-101 and HV-104; (v) LV-101 and HV-106; (vi) LV-101 and HV-108; (vii) LV-102 and HV-105; (viii) LV-102 and HV-107; (ix) LV-103 and HV-109; (x) LV-103 and HV-102; and (xi) LV-104 and HV-110.

[0112] In certain embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 68 and a heavy chain variable region comprising the sequence of SEQ ID NO: 71. In some embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 68 and a heavy chain variable region comprising the sequence of SEQ ID NO: 72. In other embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 68 and a heavy chain variable region comprising the sequence of SEQ ID NO: 73. In yet other embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 68 and a heavy chain variable region comprising the sequence of SEQ ID NO: 74. In some embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 69 and a heavy chain variable region comprising the sequence of SEQ ID NO: 75. In a particular embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 68 and a heavy chain variable region comprising the sequence of SEQ ID NO: 76. In one embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 69 and a heavy chain variable region comprising the sequence of SEQ ID NO: 77. In another embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 68 and a heavy chain variable region comprising the sequence of SEQ ID NO: 78. In a preferred embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 70 and a heavy chain variable region comprising the sequence of SEQ ID NO: 72. In another embodiment, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 70 and a heavy chain variable region comprising the sequence of SEQ ID NO: 79. In yet other embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:180 and a heavy chain variable region comprising the sequence of SEQ ID NO:184.

[0113] In some embodiments, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence of contiguous amino acids that differs by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues from a light chain variable region sequence of Table 3A, i.e., a VL selected from LV-101 to LV-104, each such sequence difference being, independently, a deletion, insertion or substitution of one amino acid, the deletion, insertion and / or substitution resulting in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The light chain variable region of some anti-CD3 antibodies comprises a sequence of amino acids having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 68-70 and 180 (i.e., the light chain variable region of Table 3A).

[0114] In one embodiment, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 68-70 and 180. In another embodiment, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 68-70 and 180. In yet another embodiment, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 68-70 and 180.

[0115] In these and other embodiments, the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence of contiguous amino acids that differs from a sequence of a heavy chain variable region of Table 3B, i.e., a VH selected from HV-101 to HV-110, by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, each such sequence difference being, independently, a deletion, insertion or substitution of one amino acid, the deletion, insertion and / or substitution resulting in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The heavy chain variable region of some anti-CD3 binding domains comprises a sequence of amino acids having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 71-79 and 184 (i.e., the heavy chain variable region of Table 3B).

[0116] In one embodiment, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 71-79 and 184. In another embodiment, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 71-79 and 184. In yet another embodiment, the anti-CD3 binding domain of a PSMA-targeted T cell engaging molecule according to the invention comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 71-79 and 184.

[0117] According to certain embodiments, one or more of the binding domains of the PSMA-targeted T-cell engaging molecule used in the method of the invention is in the format of an scFv. In the scFv, the VH and VL regions are arranged (N-terminal to C-terminal) in the order VH-VL or VL-VH. It is envisaged that the VH and VL regions of the first and / or second binding domain are linked via a linker, preferably a peptide linker. In one embodiment of the first and / or second domain, the VH region is located at the N-terminus of the linker and the VL region is located at the C-terminus of the linker. The linker is preferably a peptide linker, more preferably a short peptide linker. Examples of suitable linkers include: GGGG (SEQ ID NO:80) GGGGS (SEQ ID NO:81) GGGGSGGGGS (SEQ ID NO:82) GGGGSGGGGSGGGGS (SEQ ID NO:83) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:84) GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 85) GGGGSGGGGSGGGGSGGGGGSGGGGS (SEQ ID NO: 86) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87) GGGGSGGGSGGGSGGGGGSGGGGGSGGGGGSGGGGS (SEQ ID NO: 88) PGGGGS (SEQ ID NO:89) PGGDGS (SEQ ID NO: 90) SGGGGS (SEQ ID NO:91) GGGGSGGGS (SEQ ID NO: 92) GGGGQ (SEQ ID NO: 93).

[0118] In the present context, a "short" linker has 2 to 50 amino acids, preferably 3 to 35 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 6 to 20 amino acids or 6 to 17 amino acids. The linker between the two variable regions of one binding domain may have a different length (e.g. may be longer) than the linker between the two binding domains. For example, the linker between the two variable regions of one or both binding domains may have a length of 8 to 16 amino acids, preferably 10 to 15, and the linker between the two binding domains may have a length of 3 to 10 amino acids, preferably 5 to 8. It is further envisaged that the peptide linker is a glycine / serine linker such as those shown in SEQ ID NOs: 81 to 92. In one embodiment, the anti-PSMA binding domain and / or the anti-CD3 binding domain of the PSMA-targeted T cell engaging molecule according to the present invention is an scFv comprising, from the N-terminus to the C-terminus, VH region-peptide linker-VL region, wherein the peptide linker comprises a glycine-serine linker such as the linker set forth in SEQ ID NO: 83. In related embodiments, the peptide linker between the anti-PSMA binding domain and the anti-CD3 binding domain (e.g., scFv domain) is the linker set forth in SEQ ID NO: 81 or SEQ ID NO: 91. Exemplary scFv domains of the anti-PSMA and anti-CD3 binding domains of PSMA-targeted T cell engaging molecules suitable for use in the methods of the invention are shown in Table 4 below.

[0119] [Table 4-1]

[0120] [Table 4-2]

[0121] [Table 4-3]

[0122] In certain embodiments, a PSMA-targeted T cell engaging molecule suitable for use in the methods of the invention comprises a first binding domain that specifically binds human PSMA and has an amino acid sequence selected from any one of SEQ ID NOs: 94-106, and a second binding domain that specifically binds human CD3 and has an amino acid sequence selected from any one of SEQ ID NOs: 107-116. In a preferred embodiment, the first binding domain of the PSMA-targeted T cell engaging molecule (e.g., an anti-PSMA binding domain) has the amino acid sequence of SEQ ID NO: 104. In another preferred embodiment, the second binding domain of the PSMA-targeted T cell engaging molecule (e.g., an anti-CD3 binding domain) has the amino acid sequence of SEQ ID NO: 116.

[0123] A PSMA-targeted T cell engaging molecule according to the invention may comprise any of the anti-PSMA scFv binding domains listed in Table 4 in combination with any of the anti-CD3 scFv binding domains listed in Table 4. For example, in some embodiments, the PSMA-targeted T cell engaging molecule comprises an anti-PSMA scFv binding domain of Table 4 and an anti-CD3 scFv binding domain of Table 4, where the anti-PSMA scFv binding domain is connected to the anti-CD3 scFv binding domain via a peptide linker, such as a peptide linker described herein. In certain embodiments, the PSMA-targeted T cell engaging molecule comprises, in amino to carboxyl order, an anti-PSMA scFv binding domain, a peptide linker, and an anti-CD3 scFv binding domain. In some such embodiments, the peptide linker comprises the sequence of SEQ ID NO:81 or SEQ ID NO:91.

[0124] The PSMA-targeted T cell engaging molecules according to the present invention can also include additional domains that can, for example, modulate the pharmacokinetic profile of the molecule. For example, the PSMA-targeted T cell engaging molecules can further include an immunoglobulin Fc domain, a domain derived from serum albumin (e.g., human serum albumin), or an albumin binding domain (e.g., including a human albumin binding peptide), and / or be conjugated to a polyethylene glycol chain to increase the serum half-life of the T cell engaging molecule. In certain embodiments, the PSMA-targeted T cell engaging molecules used in the methods of the present invention further include one or more immunoglobulin Fc domains. Each immunoglobulin Fc domain can include one or more Fc monomers. Each "Fc monomer" typically includes at least a CH2 domain and a CH3 domain from an immunoglobulin molecule. The Fc monomer can include a CH2 and a CH3 domain from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. As an example, the CH2 domain includes amino acids 231-340 of an IgG1 immunoglobulin, and the CH3 domain includes amino acids 341-446 of an IgG1 immunoglobulin, where the amino acid numbering is according to the EU numbering system as described in Edelman et al., Proc. Natl. Acad. USA, Vol. 63: 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health Publication No. 91-3242, Bethesda, MD (1991). Although the boundaries between the CH2 and CH3 domains may vary slightly for different IgG isoforms, the CH2 and CH3 domains in IgG2, IgG3 and IgG4 can be confirmed by alignment with the CH2 and CH3 domains in IgG1.

[0125] In some embodiments, the Fc monomer may comprise an immunoglobulin hinge region or a portion thereof. An immunoglobulin hinge region is typically the region defined by amino acids 216-231 (according to the EU numbering system) of an IgG immunoglobulin. In certain embodiments, the Fc monomer comprises a hinge region from an IgG1 immunoglobulin or a portion thereof. In some embodiments, the IgG1 region comprises the amino acid sequence DKTHTCPPCP (SEQ ID NO: 117) or EPKSCDKTHTCPPCP (SEQ ID NO: 118). In other embodiments, the Fc monomer comprises an IgG2 hinge region having the sequence ERKCCVECPPCP (SEQ ID NO: 119), an IgG3 hinge region having the sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 120), EPKSCDTPPCPRCP (SEQ ID NO: 121) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 122), or an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 123). In a specific embodiment, an Fc monomer comprises, in order from amino to carboxyl, an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain.

[0126] In certain embodiments, the PSMA-targeted T cell engaging molecule comprises an Fc domain with one Fc monomer. In alternative embodiments, the PSMA-targeted T cell engaging molecule comprises an Fc domain with two or more Fc monomers. For example, in one embodiment, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises an Fc domain with two Fc monomers. The two Fc monomers are present on separate polypeptide chains and can associate to form a dimer, for example, via non-covalent interactions and / or disulfide bonds (e.g., between cysteine ​​residues in the hinge region of the Fc monomers). In a preferred embodiment, the two Fc monomers are fused to each other via a peptide linker, preferably a linker of sufficient length to allow the Fc monomers to associate to form an intrachain dimer. The fusion of two Fc monomers to form a single polypeptide chain is referred to herein as a single chain Fc domain (scFc domain) and is described in more detail below.

[0127] The peptide linker through which the Fc monomers are fused together to form the single chain Fc domain preferably comprises at least 25 amino acid residues (e.g., 25, 26, 27, 28, 29, 30 or more). More preferably, the peptide linker comprises at least 30 amino acid residues (30, 31, 32, 33, 34, 35 or more). In some embodiments, the linker comprises up to 40 amino acid residues, more preferably up to 35 amino acid residues, even more preferably 30 amino acid residues. In certain embodiments, the peptide linker comprises repeats of glycine-serine residues, e.g., the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 81). In such embodiments, the peptide linker comprises (Gly4Ser) x where x is an integer equal to or greater than 5 (e.g., 6, 7, or 8). Preferably, the integer is 6 or 7, more preferably the integer is 6. In one particular embodiment, the peptide linker used to link two Fc monomers to form a single-chain Fc domain comprises the sequence of SEQ ID NO:86.

[0128] The Fc monomer may contain one or more amino acid substitutions compared to the native CH2 or CH3 immunoglobulin amino acid sequence, for example to modulate effector function, alter glycosylation, or enhance stability. For example, in one embodiment, the glycosylation site in the CH2 domain at amino acid position 297 according to EU numbering is eliminated by substituting an asparagine residue at this position with a different amino acid. In some embodiments, an N297G substitution is preferred. Stability enhancing mutations include substituting one or more amino acids in the CH2 and / or CH3 domain with cysteine ​​residues to promote disulfide bond formation. Preferably, certain pairs of residues are substituted with cysteines to preferentially form disulfide bonds with each other, thus limiting or preventing scrambling of disulfide bonds. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C (these amino acid positions are numbered according to the EU numbering system). In one particular embodiment, the Fc monomer incorporated into the Fc domain of the PSMA-targeted T cell engaging molecule comprises N297G, R292C, and V302C substitutions (these amino acid positions are numbered according to the EU numbering system).

[0129] In certain preferred embodiments, the PSMA-targeted T cell engaging molecules used in the methods of the invention comprise an Fc domain that is a single chain Fc domain. Thus, in certain such embodiments, the Fc domain comprises two Fc monomers, each monomer comprising an immunoglobulin hinge region, an immunoglobulin CH2 domain and an immunoglobulin CH3 domain, and the two Fc monomers are fused to each other via a peptide linker as described herein. Exemplary amino acid sequences of Fc monomers and single chain Fc (scFc) domains are provided in Table 5 below. In some embodiments, each of the Fc monomers of the Fc domain has an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOs: 124-131. In other embodiments, each of the Fc monomers of the Fc domain has an amino acid sequence selected from SEQ ID NOs: 124-131. In a preferred embodiment, each of the Fc monomers of the Fc domain comprises the amino acid sequence of SEQ ID NO: 124. In another preferred embodiment, each of the Fc monomers of the Fc domain comprises the amino acid sequence of SEQ ID NO: 125.

[0130] [Table 5-1]

[0131] [Table 5-2]

[0132] [Table 5-3]

[0133] The Fc domain of the PSMA-targeted T cell engaging molecule used in the methods of the invention may be any of the scFc domains shown in Table 5 or variants of these scFc domains. In one embodiment, the PSMA-targeted T cell engaging molecule according to the invention comprises an Fc domain comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOs: 132-139. In another embodiment, the PSMA-targeted T cell engaging molecule according to the invention comprises an Fc domain comprising an amino acid sequence selected from SEQ ID NOs: 132-139. In a preferred embodiment, the PSMA-targeted T cell engaging molecule according to the invention comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 132. In another preferred embodiment, the PSMA-targeted T cell engaging molecule according to the invention comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 133.

[0134] In certain embodiments, the PSMA-targeted T cell engaging molecules used in the methods of the invention are comprised, in amino to carboxyl order, of: (i) a first domain that specifically binds human PSMA, the first domain comprising a first immunoglobulin heavy chain variable region (VH1) and a first immunoglobulin light chain variable region (VL1); (ii) a second domain that specifically binds human CD3 comprising a second immunoglobulin heavy chain variable region (VH2) and a second immunoglobulin light chain variable region (VL2); and (iii) an Fc domain comprising two Fc monomers; Includes. In such embodiments, each Fc monomer can comprise an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, and the two Fc monomers are fused to each other via a peptide linker, such as any of the peptide linkers described herein.

[0135] In some embodiments, the PSMA-targeted T cell engaging molecule is comprised, in amino to carboxyl order: (i) a first domain that specifically binds to human PSMA, comprising a VH1 comprising a CDRH1 having the sequence of SEQ ID NO: 14 or SEQ ID NO: 15, a CDRH2 having a sequence selected from SEQ ID NOs: 16 to 19, and a CDRH3 having the sequence of SEQ ID NO: 20, and a VL1 comprising a CDRL1 having the sequence of SEQ ID NO: 5 or SEQ ID NO: 6, a CDRL2 having the sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and a CDRL3 having a sequence selected from SEQ ID NOs: 9 to 13; and (ii) a second domain that specifically binds to human CD3, comprising a VH2 comprising a CDRH1 having a sequence selected from SEQ ID NOs: 48 to 53, a CDRH2 having a sequence selected from SEQ ID NOs: 54 to 58, and a CDRH3 having a sequence selected from SEQ ID NOs: 59 to 67, and a VL2 comprising a CDRL1 having a sequence selected from SEQ ID NOs: 41 to 43, a CDRL2 having the sequence of SEQ ID NO: 44 or SEQ ID NO: 45, and a CDRL3 having the sequence of SEQ ID NO: 46 or SEQ ID NO: 47; and (iii) an Fc domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain and a CH3 domain, the two Fc monomers being fused to each other via a peptide linker; In such embodiments, VH1 comprises a sequence selected from SEQ ID NOs: 33-39 and VL1 comprises a sequence selected from SEQ ID NOs: 21-32. In these and other embodiments, VH2 comprises a sequence selected from SEQ ID NOs: 71-79 and VL2 comprises a sequence selected from SEQ ID NOs: 68-70. In one embodiment, VH1 comprises the sequence of SEQ ID NO: 33 and VL1 comprises the sequence of SEQ ID NO: 30. In a related embodiment, VH2 comprises the sequence of SEQ ID NO: 72 and VL2 comprises the sequence of SEQ ID NO: 70.

[0136] In a preferred embodiment, the PSMA-targeted T cell engaging molecule is comprised, in amino to carboxyl order, of: (i) a first domain that specifically binds human PSMA, comprising a VH1 comprising a CDRH1 having the sequence of SEQ ID NO: 14, a CDRH2 having the sequence of SEQ ID NO: 16, and a CDRH3 having the sequence of SEQ ID NO: 20, and a VL1 comprising a CDRL1 having the sequence of SEQ ID NO: 5, a CDRL2 having the sequence of SEQ ID NO: 8, and a CDRL3 having the sequence of SEQ ID NO: 9; and (ii) a second domain that specifically binds human CD3, comprising a VH2 comprising a CDRH1 having the sequence of SEQ ID NO: 49, a CDRH2 having the sequence of SEQ ID NO: 55, and a CDRH3 having the sequence of SEQ ID NO: 60, and a VL2 comprising a CDRL1 having the sequence of SEQ ID NO: 43, a CDRL2 having the sequence of SEQ ID NO: 44, and a CDRL3 having the sequence of SEQ ID NO: 47; and (iii) an Fc domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain and a CH3 domain, the two Fc monomers being fused to each other via a peptide linker; Includes.

[0137] In certain embodiments, a peptide linker, such as those described herein, connects the first domain to the second domain and / or the second domain to the Fc domain. Thus, in some embodiments, PSMA-targeted T cell engaging molecules according to the invention comprise, in amino to carboxyl order: (i) a first domain that specifically binds human PSMA; (ii) a first peptide linker having an amino acid sequence selected from SEQ ID NOs: 81-83 and 91; (iii) a second domain that specifically binds human CD3; (iv) a second peptide linker having an amino acid sequence selected from SEQ ID NOs: 80 to 83 and 89 to 91; (v) a first Fc monomer; (vi) a third peptide linker having an amino acid sequence selected from SEQ ID NOs: 85 to 88; and (vii) a second Fc monomer Includes.

[0138] In certain embodiments, the PSMA-targeted T cell engaging molecules according to the invention are comprised, in amino to carboxyl order, of: (i) a first domain (e.g., an anti-PSMA binding domain) having an amino acid sequence selected from SEQ ID NOs: 94-106; (ii) a first peptide linker having an amino acid sequence selected from SEQ ID NOs: 81-83 and 91; (iii) a second domain (e.g., an anti-CD3 binding domain) having an amino acid sequence selected from SEQ ID NOs: 107-116; (iv) a second peptide linker having an amino acid sequence selected from SEQ ID NOs: 80 to 83 and 89 to 91; (v) a first Fc monomer having an amino acid sequence selected from SEQ ID NOs: 124 to 131; (vi) a third peptide linker having an amino acid sequence selected from SEQ ID NOs: 85 to 88; and (vii) a second Fc monomer having an amino acid sequence selected from SEQ ID NOs: 124 to 131; Includes.

[0139] In a preferred embodiment, the PSMA-targeted T cell engaging molecules according to the invention are comprised, in amino to carboxyl order, of: (i) a first domain (e.g., an anti-PSMA binding domain) having the amino acid sequence of SEQ ID NO: 104; (ii) a first peptide linker having the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:91; (iii) a second domain (e.g., an anti-CD3 binding domain) having the amino acid sequence of SEQ ID NO: 116; (iv) a second peptide linker having the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 81; (v) a first Fc monomer having the amino acid sequence of SEQ ID NO: 124; (vi) a third peptide linker having the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87; and (vii) a second Fc monomer having the amino acid sequence of SEQ ID NO: 124. Includes.

[0140] In certain embodiments, the PSMA-targeted T cell engaging molecule used in the methods of the invention is a single-chain polypeptide or single-chain fusion protein. As used herein, a "single-chain polypeptide" or "single-chain fusion protein" refers to a molecule consisting of only one polypeptide chain. That is, all of the domains in the T cell engaging molecule are optionally linked via a peptide linker to form a single polypeptide chain. One example of such a single-chain polypeptide or single-chain fusion protein in the context of the present invention is a single-chain polypeptide comprising, in amino to carboxyl order, a PSMA scFv domain, a first peptide linker, an anti-CD3 scFv domain, a second peptide linker, and an scFc domain. Exemplary PSMA-targeted T cell binding single-chain polypeptides or single-chain fusion proteins that can be used in the methods of the invention are shown in Table 6 below. Other PSMA-targeted T cell binding single-chain polypeptides or single-chain fusion proteins suitable for use in the methods of the invention are described in WO 2017 / 134158, which is incorporated herein by reference in its entirety.

[0141] [Table 6-1]

[0142] [Table 6-2]

[0143] [Table 6-3]

[0144] [Table 6-4]

[0145] [Table 6-5]

[0146] [Table 6-6]

[0147] [Table 6-7]

[0148] [Table 6-8]

[0149] In certain embodiments, the PSMA-targeted T cell engaging molecule administered to a patient by the methods of the invention comprises an amino acid sequence selected from SEQ ID NOs: 140-157. In one embodiment, the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 141. In another embodiment, the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 144. In yet another embodiment, the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 147. In yet another embodiment, the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 150. In a preferred embodiment, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises the amino acid sequence of SEQ ID NO: 140 (e.g., acpatamab).

[0150] The PSMA-targeted T cell engaging molecules used in the methods of the invention may be variants of the single chain polypeptides shown in Table 6, comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 140-157. In one embodiment, the PSMA-targeted T cell engaging molecules according to the invention comprise an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 140-157. In another embodiment, the PSMA-targeted T cell engaging molecules according to the invention comprise an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NO: 140-157. In certain embodiments, the sequence changes occur in the peptide linker region and / or the single chain Fc domain.

[0151] In other embodiments, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises two or more polypeptide chains. For example, in some embodiments, the PSMA-targeted T cell engaging molecule comprises three polypeptide chains, where the first polypeptide is a light chain from an anti-CD3 antibody, the second polypeptide is a heavy chain from an anti-CD3 antibody, and the third polypeptide is a modified heavy chain comprising a heavy chain variable region from an anti-PSMA heavy chain only antibody. Such three polypeptide chain T cell engaging molecules are described in WO 2020 / 206330, which is incorporated herein by reference in its entirety. Thus, in certain embodiments, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises: (a) a first polypeptide comprising or consisting of a light chain polypeptide derived from an anti-CD3 antibody; (b) a second polypeptide comprising or consisting of a heavy chain polypeptide from an anti-CD3 antibody; and (c) a third polypeptide comprising a heavy chain variable region from an anti-PSMA antibody fused to a heavy chain immunoglobulin constant region. Exemplary polypeptide sequences for the construction of multi-chain PSMA-targeted T cell engaging molecules are shown in Table 7. In some such embodiments, the first polypeptide comprises the sequence of SEQ ID NO: 185, the second polypeptide comprises the sequence of SEQ ID NO: 186, and the third polypeptide comprises the sequences of SEQ ID NOs: 187-192. In one particular embodiment, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises a first polypeptide comprising the sequence of SEQ ID NO: 185, a second polypeptide comprising the sequence of SEQ ID NO: 186, and a third polypeptide comprising the sequence of SEQ ID NO: 187. In another particular embodiment, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises a first polypeptide comprising the sequence of SEQ ID NO: 185, a second polypeptide comprising the sequence of SEQ ID NO: 186, and a third polypeptide comprising the sequence of SEQ ID NO: 191. In yet another particular embodiment, the PSMA-targeted T cell engaging molecule used in the methods of the invention comprises a first polypeptide comprising the sequence of SEQ ID NO: 185, a second polypeptide comprising the sequence of SEQ ID NO: 186, and a third polypeptide comprising the sequence of SEQ ID NO: 192.

[0152] [Table 7-1]

[0153] [Table 7-2]

[0154] In certain embodiments, the methods of the invention comprise administering to a patient in need of treatment for prostate cancer one or more cycles of an antiandrogen compound in combination with a PSMA-targeted T cell engaging molecule, wherein the first cycle is about 28 days: administering a first dose (e.g., a priming dose) of about 30 μg to about 300 μg of a T cell engaging molecule by continuous intravenous infusion over days 1-3 of the cycle; administering a therapeutic dose of about 90 μg to about 1.8 mg of a T cell engaging molecule by bolus intravenous infusion on days 8 and 22 of the cycle; and administering an antiandrogen compound orally once daily on each of days 15 through 28 of the cycle.

[0155] In some embodiments of the methods of the present invention, the first cycle is about 28 days and comprises: administering a first dose (e.g., a priming dose) of about 90 μg of a T cell engaging molecule by continuous intravenous infusion over days 1-3 of the cycle; administering a therapeutic dose of about 150 μg or about 300 μg of a T cell engaging molecule by bolus intravenous infusion on days 8 and 22 of the cycle; and administering an antiandrogen compound orally once daily on each of days 15 through 28 of the cycle.

[0156] In any of the foregoing embodiments, the PSMA-targeting T cell engaging molecule may be akapatamab, a single chain polypeptide comprising the sequence of SEQ ID NO: 140, and the antiandrogen compound may be enzalutamide, abiraterone, abiraterone acetate, darolutamide, or apalutamide. For example, in some embodiments, the methods of the invention comprise administering to a patient in need of treatment for prostate cancer one or more cycles of enzalutamide in combination with akapatamab, the first cycle being about 28 days: administering a first dose (e.g., a priming dose) of about 90 μg of acpatamab by continuous intravenous infusion on days 1-3 of the cycle; administering a therapeutic dose of about 150 μg or about 300 μg of acpatamab by bolus intravenous infusion on days 8 and 22 of the cycle; and administering enzalutamide orally at a dose of approximately 160 mg once daily on each of days 15 through 28 of the cycle.

[0157] In another embodiment, the methods of the invention comprise administering to a patient in need of treatment for prostate cancer one or more cycles of abiraterone or abiraterone acetate in combination with akapatamab, wherein the first cycle is about 28 days and comprises: administering a first dose (e.g., a priming dose) of about 90 μg of acpatamab by continuous intravenous infusion on days 1-3 of the cycle; administering a therapeutic dose of about 150 μg or about 300 μg of acpatamab by bolus intravenous infusion on days 8 and 22 of the cycle; and administering abiraterone or abiraterone acetate orally at a dose of approximately 1,000 mg once daily on each of days 15 through 28 of the cycle.

[0158] In certain embodiments, the methods of the invention comprise administering to a patient in need of treatment for prostate cancer one or more cycles of darolutamide in combination with akapatamab, wherein the first cycle is about 28 days and comprises: administering a first dose (e.g., a priming dose) of about 90 μg of acpatamab by continuous intravenous infusion on days 1-3 of the cycle; administering a therapeutic dose of about 150 μg or about 300 μg of acpatamab by bolus intravenous infusion on days 8 and 22 of the cycle; and administering darolutamide orally at a dose of approximately 600 mg twice daily on each of days 15 through 28 of the cycle.

[0159] In other specific embodiments, the methods of the invention comprise administering to a patient in need of treatment for prostate cancer one or more cycles of apalutamide in combination with akapatamab, wherein the first cycle is about 28 days and comprises: administering a first dose (e.g., a priming dose) of about 90 μg of acpatamab by continuous intravenous infusion on days 1-3 of the cycle; administering a therapeutic dose of about 150 μg or about 300 μg of acpatamab by bolus intravenous infusion on days 8 and 22 of the cycle; and administering apalutamide orally at a dose of approximately 240 mg once daily on each of days 15 through 28 of the cycle.

[0160] In any of the foregoing embodiments, the method can further include administering to the patient a maintenance cycle comprising administering a therapeutic dose of a PSMA-targeted T-cell engaging molecule (e.g., acapatamab) by bolus intravenous infusion once every 14 days (Q2W) and administering an antiandrogen compound orally once or twice daily on each day of the cycle.

[0161] In certain embodiments of the method of the present invention, one or more premedications may be administered to the patient prior to administration of the first dose of the PSMA-targeted T cell engaging molecule in the first cycle. In some embodiments, the premedication is administered to the patient prior to administration of each dose of the PSMA-targeted T cell engaging molecule in the first cycle. The premedication may also be administered to the patient prior to administration of one or more doses of the PSMA-targeted T cell engaging molecule in one or more maintenance cycles. In some embodiments, the premedication is administered to the patient only prior to administration of one or more doses in the first cycle, and is not administered to the patient prior to administration of a dose of the PSMA-targeted T cell engaging molecule in a subsequent treatment cycle (e.g., a maintenance cycle). In this particular context, "prior to" is intended to mean within 72 hours, 48 ​​hours, 36 hours, 24 hours, 18 hours, 16 hours, 12 hours, 6 hours, 5 hours, 4 hours, or 3 hours, preferably within 120 minutes, 90 minutes, 60 minutes, or 30 minutes, prior to the start of administration of the PSMA-targeted T cell engaging molecule. Depending on the type of premedication used and the route by which it is administered, the premedication may be administered, for example, 30-120 minutes or 30-60 minutes prior to the start of administration of the PSMA-targeted T cell engaging molecule. The premedication may be administered, for example, to prevent or reduce the severity of an infusion-related reaction and / or to prevent or reduce the severity of cytokine release syndrome or symptoms thereof.

[0162] In some embodiments, the premedication is an antihistamine. The antihistamine is administered orally or intravenously and is administered at a dose equivalent to 50 mg of diphenhydramine iv. Suitable antihistamines that can be administered as a premedication include azatadine (maximum dose, e.g., 4 mg / day), brompheniramine (maximum dose, e.g., 30 mg / day), cetirizine (maximum dose, e.g., 15 mg / day), chlorpheniramine (maximum dose, e.g., 30 mg / day), clemastine (maximum dose, e.g., 10 mg / day), cyproheptadine (maximum dose, e.g., 15 mg / day), desloratadine (maximum dose, e.g., 7 mg / day), and cyclosporine (maximum dose, e.g., 7 mg / day). ), dexchlorpheniramine (maximum dose, e.g., 15 mg / day), diphenhydramine (maximum dose, e.g., 350 / day), doxylamine (maximum dose, e.g., 180 mg / day), fexofenadine (maximum dose, e.g., 200 mg / day), loratadine (maximum dose, e.g., 15 mg / day), and phenindamine (maximum dose, e.g., 180 mg / day), administered orally, parenterally, or rectally.

[0163] In another embodiment, the premedication is a glucocorticoid. Glucocorticoids are a class of corticosteroids, which are a class of steroid hormones. Glucocorticoids are corticosteroids that bind to the glucocorticoid receptor. A less common synonym is glucocorticosteroid. Cortisol (known as hydrocortisone when used as a drug) is the most important human glucocorticoid. A variety of synthetic glucocorticoids are much more potent than cortisol and have been produced for therapeutic use. Cortisol is the standard of comparison for glucocorticoid potency. An example of a commonly prescribed alternative steroid equivalent may be prednisone (5 mg) = cortisone (25 mg) = dexamethasone (0.75 mg) = hydrocortisone (20 mg) = methylprednisolone (4 mg). These doses represent pharmacological doses equivalent to systemic administration of glucocorticoids. Glucocorticoids can be administered orally or intravenously, and can be administered at a dose equivalent to 4-20 mg dexamethasone iv (equivalence referring to glucocorticoid potency). The dose of glucocorticoid can be the same in each administration (i.e., each time glucocorticoid premedication is administered). Alternatively, the dose of glucocorticoid can be reduced in subsequent administrations, for example by 50% of the previous dose, if there is no or minimal evidence of infusion reaction and / or CRS symptoms after the previous administration of PSMA-targeted T cell engaging molecule. In certain embodiments, glucocorticoids are administered as premedication only during the first cycle, and not in subsequent treatment cycles (e.g., maintenance cycles).

[0164] Examples of glucocorticoids used as premedication include, but are not limited to, cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, budesonide, triamcinolone, cloprednol, deflazacort, fluocortolone, cortivazol, paramethasone, fluticasone, fluticasone propionate, triamcinolone acetonide, and combinations and / or pharmaceutically acceptable derivatives thereof. Various glucocorticoids can be used alone or in combination. Dexamethasone, prednisone and prednisolone are preferred glucocorticoids for use as premedication according to the method of the present invention. In certain embodiments of the method of the present invention, the glucocorticoid administered to the patient before administration of one or more (or all) doses of PSMA-targeted T cell engaging molecules during the first cycle and / or maintenance cycle is dexamethasone. Dexamethasone can be administered in a dose of about 4-20 mg, 6-18 mg, 8-16 mg, about 16 mg, or about 8 mg per administration. In some embodiments of the methods of the invention, dexamethasone is administered to the patient prior to administration of each dose of PSMA-targeted T cell engaging molecule during the first cycle. In these and other embodiments, dexamethasone is administered orally to the patient in a dose of about 8 mg about 6-16 hours prior to administration of each dose of PSMA-targeted T cell engaging molecule during the first cycle. In other embodiments, dexamethasone is administered intravenously to the patient in a dose of about 8 mg within about 1 hour prior to administration of each dose of PSMA-targeted T cell engaging molecule during the first cycle. In yet another embodiment, the methods of the invention further comprise orally administering to the patient an 8 mg dose of dexamethasone (or an equivalent dose of other glucocorticoid) about 6-16 hours prior to administration of each dose of the PSMA-targeted T-cell engaging molecule during the first cycle, and intravenously administering to the patient an 8 mg dose of dexamethasone (or an equivalent dose of other glucocorticoid) within 1 hour prior to administration of each dose of the PSMA-targeted T-cell engaging molecule.

[0165] A patient may be treated according to the method of the present invention for a set treatment period. A "treatment period" begins upon administration of a first dose of PSMA-targeted T-cell engaging molecule in a first or initiation cycle and ends upon administration of a final dose of PSMA-targeted T-cell engaging molecule and / or antiandrogen compound in a maintenance cycle. The treatment period may be about 3 months to about 36 months, about 12 months to about 24 months, or about 6 months to about 12 months. For example, the treatment period may be about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 18 months, about 21 months, about 24 months, about 27 months, about 30 months, about 33 months, or about 36 months. In some embodiments, the treatment period is about 6 months. In some embodiments, the treatment period is about 9 months. In yet other embodiments, the treatment period is about 12 months. The duration of treatment can be adjusted for each patient depending on the patient's response to treatment. In one particular embodiment, the patient is treated according to the methods of the present invention until the patient achieves a complete response or until no evidence of prostate cancer is otherwise detectable in the patient.

[0166] In some embodiments, the patient treated by the method of the present invention may have failed or be intolerant to one or more previous prostate cancer therapies, such as chemotherapy, radiation therapy, androgen deprivation therapy, or radioligand therapy. As used herein, a patient may be considered to have failed a treatment if the patient's cancer progresses after a standard treatment regimen (e.g., the size of the prostate tumor increases; the presence, number, or size of metastatic lesions increases; blood levels of PSA increase). A patient may also be considered to have failed a treatment if the patient cannot tolerate the treatment or if the treatment is contraindicated for the patient. As the term is used herein, a patient is considered to be refractory or resistant to a treatment if the patient's cancer does not respond after continued administration of the treatment or loses initial response. As used herein, a patient is considered to have relapsed after a treatment if the signs and symptoms of prostate cancer return after the patient has experienced remission from the disease (e.g., blood levels of PSA increase, cancerous cells in the prostate, appearance of metastatic lesions, etc.).

[0167] In certain embodiments, the patient treated by the method of the present invention has failed or is intolerant to one or more chemotherapy regimens. In a related embodiment, the patient treated by the method of the present invention is refractory or resistant to one or more chemotherapy regimens. Standard chemotherapy regimens for treating prostate cancer typically include regimens using mitoxantrone, estramustine, carboplatin, oxaliplatin, cisplatin, and taxane chemotherapy regimens, such as docetaxel, cabazitaxel, or paclitaxel. In one embodiment, the patient treated by the method of the present invention has failed or is resistant to one or more taxane chemotherapy regimens. In another embodiment, the patient treated by the method of the present invention has failed or is intolerant, refractory, or resistant to two or more taxane chemotherapy regimens. In such an embodiment, the patient may have failed or is intolerant, refractory, or resistant to docetaxel regimen and / or cabazitaxel regimen.

[0168] In other embodiments, the patient treated by the methods of the present invention has failed or is intolerant, refractory, or resistant to one or more androgen deprivation therapies, including but not limited to surgical castration (e.g., bilateral orchiectomy), chemical castration with LHRH agonists or antagonists (e.g., leuprolide, goserelin, triptorelin, histrelin, or degarelix), or treatment with antiandrogen compounds such as androgen biosynthesis inhibitors (e.g., abiraterone, ketoconazole), or androgen receptor antagonists (e.g., flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide). In certain embodiments, the patient has failed or is intolerant, resistant or refractory to at least one previous antiandrogen compound, such as abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.In related embodiments, the patient has failed or is intolerant, resistant or refractory to one or more antiandrogen compounds selected from abiraterone, enzalutamide, apalutamide, and darolutamide.In such embodiments where the patient has failed or is intolerant, resistant or refractory to previous antiandrogen compound treatment, the antiandrogen compound administered to the patient according to the combination therapy of the present invention is preferably a different antiandrogen compound from the one the patient has previously received.

[0169] In certain other embodiments, the patient treated by the methods of the invention has failed or is intolerant, refractory, or resistant to radioligand therapy. Radioligand therapy is an agent that includes a radionuclide or radioisotope covalently attached to a targeting ligand (e.g., an antibody, peptide, or small molecule) that specifically binds to a protein on the surface of a cancer cell. In some embodiments, the patient is refractory or resistant to radioligand therapy, which includes a radionuclide (e.g., lutetium-177 ( 177 Lu), Actinium-225 ( 225 Ac), Yttrium-90( 90 Y), or iodine-131 ( 131 I)) PSMA-targeted radioligand therapy. PSMA-targeted radiotherapy includes: 177 Lu-PSMA-617, 225 Ac-PSMA-617, 225 Ac-huJ591, 177 Lu-huJ591, 90 Y-huJ591, and 131 PSMA-targeted radiotherapy includes, but is not limited to, I-MIP-1095. PSMA-targeted radiotherapy is described in Czerwinksa et al., Molecules, Vol. 25:1743, 2020, which is incorporated herein by reference in its entirety. In one embodiment, the patient treated by the method of the present invention is 177 have failed or are intolerant, refractory or resistant to Lu-PSMA-617 radioligand therapy. In another embodiment, the patient treated by the method of the present invention is 225 have failed or are intolerant, refractory or resistant to Ac-PSMA-617 radioligand therapy.

[0170] The PSMA-targeted T cell engaging molecule used in the method of the present invention can be prepared by any number of conventional methods.For example, the PSMA-targeted T cell engaging molecule can be produced by recombinant expression system using any method known in the art.See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.) Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).

[0171] The PSMA-targeted T cell engaging molecule or its components (e.g., Fv fragment, Fc monomer) can be expressed in hybridoma cell lines or non-hybridoma cell lines. Expression vectors or expression constructs encoding the T cell engaging molecules can be used to transform mammalian, insect or microbial host cells. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. The term "expression vector" or "expression construct" as used herein refers to a recombinant nucleic acid molecule that includes a desired coding sequence and appropriate nucleic acid control sequences required for the expression of this operably linked coding sequence in a particular host cell. Expression vectors can include, but are not limited to, sequences that affect or control transcription, translation, and introns, if present, sequences that affect RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and optionally other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence, optionally encoded by the expression vector, may also be operably linked to the coding sequence of interest, so that the expressed polypeptide may be secreted by the recombinant host cell, making it easier to isolate the polypeptide of interest from the cell, if desired.

[0172] The recombinant expression vector or construct generally comprises a nucleic acid molecule encoding a polypeptide comprising one or more of the following: one or more CDRs provided herein, a light chain constant region, a light chain variable region, a heavy chain constant region (e.g., CH1, CH2 and / or CH3), a heavy chain variable region, a hinge region, an Fc domain and / or another scaffold portion of an anti-PSMA antibody or an anti-CD3 antibody. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In embodiments where the T cell engaging molecule is a single chain polypeptide or a single chain fusion protein, the nucleic acid contained in the recombinant expression vector typically encodes a full-length single chain polypeptide (e.g., a full-length single chain fusion protein). The vector is typically selected to be functional in the particular host cell to be utilized (i.e., the vector is compatible with the host's cellular machinery and may allow for gene amplification and / or expression). In some embodiments, vectors are used that utilize protein-fragment complementation assays using protein reporters such as dihydrofolate reductase (see, e.g., U.S. Pat. No. 6,270,964, which is incorporated herein by reference). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies or BD Biosciences (formerly "Clontech"). Other useful vectors for cloning and expressing T cell engaging molecules include those described in Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84:439-44, which is incorporated herein by reference. Additional suitable expression vectors are discussed, for example, in Methods Enzymol., vol. 185 (DV Goeddel, ed.), 1990, New York: Academic Press.

[0173] Expression vectors used in any of the host cells to produce PSMA-targeted T cell engaging molecules usually contain sequences for cloning and expression of exogenous nucleotide sequences encoding the T cell engaging molecule or components thereof. Such sequences, collectively referred to as "flanking sequences", will in certain embodiments typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selection marker element.

[0174] Optionally, the vector may contain a sequence encoding a "tag", i.e., an oligonucleotide molecule located at the 5' or 3' end of the sequence encoding the PSMA-targeted T cell engaging molecule, encoding poly-His (such as hexa-His) or another "tag" for which commercially available antibodies exist, such as the FLAG® tag, HA (influenza virus hemagglutinin) or myc. This tag is usually fused to the polypeptide upon expression and can serve as a means for affinity purification or detection of the PSMA-targeted T cell engaging molecule from the host cell. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified T cell engaging molecule by various means, such as using specific peptidases for cleavage.

[0175] Expression and cloning vectors typically contain a promoter that is recognized by a host cell and operably linked to a nucleic acid molecule encoding a PSMA-targeted T cell engaging molecule. As used herein, the term "operably linked" refers to two or more nucleic acid sequences being linked together to produce a nucleic acid molecule capable of inducing transcription of a given gene and / or synthesis of a desired protein molecule. For example, a control sequence in a vector "operably linked" to a protein coding sequence is ligated to the protein coding sequence such that expression of the protein coding sequence occurs under conditions compatible with the transcriptional activity of the control sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Numerous promoters recognized by a variety of potential host cells are well known to those skilled in the art. Suitable promoters for use with mammalian host cells include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). A suitable promoter is operably linked to a polynucleotide encoding, for example, a PSMA-targeted T cell engaging molecule or component thereof by removing the promoter from the source nucleic acid by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[0176] The expression vector for recombinant production of the PSMA-targeted T cell engaging molecule described herein can be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the desired flanking sequences are not originally present in the vector, they can be obtained individually and ligated into the vector. The method used to obtain each of the flanking sequences is known to those skilled in the art. The expression vector can be introduced into a host cell, thereby producing the PSMA-targeted T cell engaging molecule encoded by the nucleic acid present in the vector.

[0177] After the vector is constructed and one or more nucleic acid molecules encoding a PSMA-targeted T-cell engaging molecule or a component thereof are inserted into the appropriate site of the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. As used herein, the term "host cell" refers to a cell that has been transformed or is capable of being transformed with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the morphology or genetic make-up of the progeny is identical to the original parent cell, so long as the gene of interest is present. A host cell that contains an isolated polynucleotide or isolated nucleic acid encoding a PSMA-targeted T-cell engaging molecule, preferably operably linked to at least one expression control sequence (e.g., promoter or enhancer), is a "recombinant host cell."

[0178] Transformation of an expression vector for a polypeptide into a selected host cell can be carried out by well known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell to be used.

[0179] The host cells synthesize the T cell engaging molecule when cultured under appropriate conditions, and the T cell engaging molecule can then be harvested from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, the modification of the polypeptide that is desired or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. Suitable host cells include, but are not limited to, prokaryotic cells (e.g., E. coli, B. subtilis), yeast cells (Saccharomyces cerevisiae, Pichia pastoris), and mammalian cells (e.g., Chinese hamster ovary (CHO), human embryonic kidney (HEK)). In some embodiments, CHO cells are the preferred host cells for expressing PSMA-targeted T cell engaging molecules.

[0180] Host cells are transformed or transfected with the above expression vectors for the production of T cell engaging molecules and cultured in conventional nutrient media modified appropriately for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Host cells used to produce T cell engaging molecules can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; or WO 87 / 00195 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., the drug Gentamicin™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature and pH will be those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0181] When the host cells are cultured, the T cell engaging molecules may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the T cell engaging molecules are produced intracellularly, as a first step, the host cells are lysed (e.g., by mechanical shearing, osmotic shock, or enzymatic methods) and particulate debris (e.g., host cells and lysed fragments) are removed, for example, by centrifugation, microfiltration, or ultrafiltration. If the T cell engaging molecules are secreted into the culture medium, the T cell engaging molecules may be separated from the host cells by centrifugation or microfiltration, and optionally subsequently concentrated by ultrafiltration. The PSMA-targeted T cell engaging molecules may be further purified or partially purified using one or more chromatographic steps, such as, for example, affinity chromatography (e.g., protein A, protein L, or protein G affinity chromatography), cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, or mixed-format chromatography.

[0182] The PSMA-targeted T cell engaging molecules and antiandrogen compounds are generally administered to patients in pharmaceutical compositions that may include pharma- ceutically acceptable carriers, excipients, or diluents. "Pharmaceutically acceptable" refers to molecules, compounds, and compositions that are non-toxic to human recipients at the dosages and concentrations used and / or do not cause allergic or adverse reactions when administered to humans. In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition.In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; coatings; monosaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (sodium, potassium, magnesium , calcium, acetate, hydrochloride, phosphate, etc.); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapear, etc.); stability enhancers (sucrose or sorbitol); isotonicity enhancers (alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants. Methods and suitable materials for formulating molecules for therapeutic use are known in the pharmaceutical art and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.Pharmaceutical compositions comprising a PSMA-targeted T cell engaging molecule or an antiandrogenic compound administered according to the methods of the invention include, but are not limited to, solid compositions, liquid compositions, frozen compositions and lyophilized compositions.

[0183] If the pharmaceutical composition is lyophilized, the lyophilized material is reconstituted with an appropriate liquid prior to administration. The lyophilized material can be reconstituted, for example, in bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was present prior to lyophilization.

[0184] In some embodiments, the selection of carriers and excipients for incorporation into pharmaceutical compositions affects the physical state, stability, in vivo release rate and in vivo clearance rate of the T cell engaging molecule or antiandrogenic compound. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for the T cell engaging molecule can be water for injection, saline solution, optionally supplemented with other materials or excipients commonly found in compositions for parenteral administration.

[0185] In certain embodiments, the pharmaceutical composition comprises an effective amount of a PSMA-targeted T cell engaging molecule or an antiandrogen compound and one or more excipients. The effective amount can be a therapeutic dose or a smaller amount, such as a priming dose or a unit dose. Excipients can be used for a variety of purposes, such as adjusting the physical, chemical or biological properties of the formulation (such as adjusting the viscosity) and / or to stabilize such formulations against degradation and deterioration due to stresses that occur, for example, during manufacture, shipping, storage, preparation for use and administration.

[0186] In some embodiments, the pharmaceutical composition comprising an effective amount of a PSMA-targeted T cell engaging molecule administered to a patient according to the methods of the invention comprises a buffer. A buffer is used to maintain the composition at physiological pH or slightly lower pH, typically within a pH range of about 4.0 to about 6.5. Suitable buffers include, but are not limited to, glutamate, acetate, Tris, citrate, histidine, succinate and phosphate buffers. In certain embodiments, the pharmaceutical composition administered according to the methods described herein comprises a glutamate buffer, particularly an L-glutamate buffer. A pharmaceutical composition comprising a glutamate buffer can have a pH of about 4.0 to about 5.5, a pH of about 4.0 to about 4.4, or a pH of about 4.2 to about 4.8.

[0187] The pharmaceutical composition comprising an effective amount of PSMA-targeted T cell engaging molecules may further comprise a surfactant. The term "surfactant" as used herein refers to a substance that serves to reduce the surface tension of the dissolved liquid. Surfactants may be included in the pharmaceutical composition for a variety of purposes, including, for example, to prevent or control aggregation, particle formation and / or surface adsorption in liquid formulations, or to prevent or control these phenomena during lyophilization and / or the reconstitution process in lyophilized formulations. Surfactants include, for example, amphiphilic organic compounds that exhibit partial solubility in both organic solvents and aqueous solutions. Common characteristics of surfactants include their ability to reduce the surface tension of water, reduce the interfacial tension between oil and water, and also form micelles. Surfactants that may be incorporated into the pharmaceutical composition used in the methods of the present invention include both non-ionic and ionic surfactants. Suitable nonionic surfactants include, but are not limited to, alkyl poly(ethylene oxide), alkyl polyglucosides such as octyl glucoside and decyl maltoside, fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA and cocamide TEA.Specific examples of nonionic surfactants include, for example, polysorbates, including polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.; poloxamers and polyethylene glycols (PEGs), including poloxamer 188, poloxamer 407, or polyethylene-polypropylene glycol, also known as poloxalkol or poly(ethylene oxide)-poly(propylene oxide).Suitable ionic surfactants include, for example, anionic, cationic and zwitterionic surfactants. Anionic surfactants include, but are not limited to, soaps, fatty acid salts, sulfonate or carboxylate surfactants such as sodium dodecyl sulfate (SDS), ammonium lauryl sulfate and other alkyl sulfates.Cationic surfactants include, but are not limited to, quaternary ammonium surfactants such as cetyltrimethylammonium bromide (CTAB), other alkyltrimethylammonium salts, cetylpyridinium chloride, polyethoxylated tallow amine (POEA) and benzalkonium chloride. Zwitterionic or amphoteric surfactants include, for example, dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycinate. In certain embodiments, the pharmaceutical composition administered according to the methods described herein comprises a non-ionic surfactant. In one embodiment, the non-ionic surfactant is polysorbate 20. In another embodiment, the non-ionic surfactant is polysorbate 80.

[0188] In certain embodiments, the pharmaceutical composition comprising an effective amount of PSMA-targeted T cell engaging molecule further comprises a stabilizer. As used herein, the term "stabilizer" refers to an excipient that stabilizes the native conformation of a polypeptide or T cell engaging molecule and / or prevents or reduces physical or chemical degradation of the polypeptide or T cell engaging molecule. Suitable stabilizers include, but are not limited to, polyols (e.g., sorbitol, glycerol, mannitol, xylitol, maltitol, lactitol, erythritol and threitol), sugars (e.g., fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, rhamnose, galactose, maltose, sucrose, trehalose, sorbose, sucralose, melezitose and raffinose) and amino acids (e.g., glycine, methionine, proline, lysine, arginine, histidine or glutamic acid). In some embodiments, the pharmaceutical composition comprises a sugar as a stabilizer. In these and other embodiments, the sugar is sucrose.

[0189] Exemplary pharmaceutical compositions comprising PSMA-targeted T-cell engaging molecules are described in WO 2018 / 141910, which is incorporated herein by reference in its entirety. In certain embodiments, pharmaceutical compositions useful for treating prostate cancer according to the methods described herein comprise about 0.5 mg / ml to about 2 mg / ml of PSMA-targeted T-cell engaging molecules, about 5 mM to about 20 mM L-glutamic acid, about 0.005% to about 0.015% weight / volume (w / v) of polysorbate (e.g., polysorbate 20 or polysorbate 80), and about 7% (w / v) to about 12% (w / v) of sucrose. In other embodiments, the pharmaceutical compositions include about 0.5 mg / ml to about 1 mg / ml of PSMA-targeted T cell engaging molecule, about 8 mM to about 12 mM L-glutamic acid, about 0.008% (w / v) to about 0.012% (w / v) of polysorbate (e.g., polysorbate 20 or polysorbate 80), and about 8% (w / v) to about 10% (w / v) of sucrose. The pH of these formulations ranges from about 4.0 to about 4.4 (e.g., a pH of about 4.0, about 4.1, about 4.2, about 4.3, or about 4.4).

[0190] Any of the pharmaceutical compositions comprising the PSMA-targeted T cell engaging molecules described herein can be lyophilized and reconstituted, for example, with sterile water for injection, prior to administration to a patient. The reconstitution volume can be from about 0.5 ml to about 5 ml, depending on the protein content after lyophilization and the desired concentration of the T cell engaging molecule in the reconstituted solution. The reconstituted solution can be further diluted with a diluent (e.g., saline and / or intravenous solution stabilizer (IVSS)) prior to administration to a patient, as necessary, to administer the doses described herein according to the methods of the invention.

[0191] Any of the PSMA-targeted T cell engaging molecules described herein, including the single chain polypeptides described in Table 6, can be incorporated into any of the pharmaceutical compositions described above and administered to a patient according to the methods described herein. In a preferred embodiment, the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 140 (e.g., akapatamab). In another preferred embodiment, the PSMA-targeted T cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 141.

[0192] The antiandrogen compound that is administered according to the method of the present invention will typically be formulated in oral dosage form.Oral dosage forms include, but are not limited to, tablets, pills, pellets, capsules, powders, lozenges, granules, liquids, suspensions, emulsions, syrups, elixirs, and sprays.Some oral dosage forms, such as tablets or capsules, may contain enteric coatings or films.

[0193] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. EXAMPLES

[0194] Example 1. Phase 1b Study of AMG 160 in Combination with Enzalutamide in Patients with Metastatic Castration-Resistant Prostate Cancer AMG 160 (also known as acapatamab) is a half-life extended (HLE) BiTE® (bispecific T cell engager) molecule that binds both human PSMA and human CD3 and contains a single chain IgG Fc domain. The amino acid sequence of AMG 160 is set forth in SEQ ID NO: 140. AMG 160 is designed to bind to a patient's T cells and kill prostate cancer cells via binding of CD3 on the T cells and PSMA on the cancer cells. Enzalutamide is an androgen receptor inhibitor indicated for the treatment of patients with castration-resistant prostate cancer and, in some areas, metastatic hormone-sensitive prostate cancer. However, patients receiving enzalutamide may experience disease progression or recurrence due in part to mutations in the androgen receptor (e.g., AR-V7) and increased androgen receptor expression, among other mechanisms (Galletti et al., Cancer Treat Rev., Vol. 57:16-27, 2017). These resistance mechanisms do not necessarily confer resistance to immunotherapy such as AMG 160, so combination therapy with AMG 160 and enzalutamide may provide better efficacy. In addition, enzalutamide has been reported to rapidly upregulate the expression of PSMA in prostate cancer cells and enhance AMG 160-mediated cytotoxicity of prostate cancer cells in vitro (see, e.g., Deegen et al., Clin. Cancer Res., Vol. 27: 2928-2937, 2021; Aggarwal et al., Eur. Urol. Oncol., Vol. 1: 78-82, 2018; Emmett et al., J Nucl. Med., Vol. 60: 950-954, 2019). The objective of the study was to evaluate the safety, tolerability, and preliminary efficacy of AMG 160 in combination with enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC).

[0195] After signing the informed consent, patients entered a screening period (up to 28 days) during which their eligibility was assessed. Eligible patients had mCRPC with histologically or cytologically confirmed adenocarcinoma of the prostate, without pure neuroendocrine differentiation or small cell features. Patients who were to receive enzalutamide for the first time in the metastatic setting were eligible to participate in the study. Specifically, patients who met all of the following key inclusion criteria were enrolled in the study: Histologically or cytologically confirmed mCRPC without pure neuroendocrine differentiation or small cell features; · had undergone bilateral orchiectomy or received continuous androgen deprivation therapy (ADT) with gonadotropin-releasing hormone (GnRH) agonists or antagonists; Total serum testosterone concentration was 50 ng / dL or less or 1.7 nmol / L or less; and I was scheduled to receive enzalutamide for the first time for mCRPC.

[0196] Patients were excluded from the study if they: (i) had a confirmed history or current autoimmune or other disease requiring permanent immunosuppressive therapy; (ii) had previously received enzalutamide; (iii) had CNS metastases or meningeal disease; (iv) had previous treatment with a taxane for mCRPC; (v) had major surgery and / or radiation within 4 weeks; (vi) had used a strong CYP2C8 inhibitor (e.g., gemfibrozil) within 7 days prior to the first dose of AMG 160, or a strong CYP3A4 inducer (e.g., carbamazepine, phenobarbital, phenytoin, rifabutin, rifampin, rifapentine, St. John's wort) within 28 days prior to the first dose of AMG 160; or (vii) had used an AMG Prior to the first dose of 160, drugs with narrow therapeutic indices that are substrates of CYP3A4 (e.g., alfentanil, cyclosporine, dihydroergotamine, ergotamine, fentanyl, pimozide, quinidine, sirolimus, and tacrolimus), CYP2C9 (e.g., phenytoin, warfarin), or CYP2C19 (e.g., S-mephenytoin, clopidogrel) were used.

[0197] In the first patient cohort (Cohort 1), AMG 160 was administered by continuous intravenous infusion (also called extended IV (eIV) infusion) at a dose of 0.09 mg over 72 hours on days 1-3 of a 28-day cycle in cycle 1, followed by a therapeutic dose of 0.3 mg administered as a brief IV infusion (sIV; approximately 60 minutes) on days 8 and 22 of the 28-day cycle (see Table 8 below). The initial 0.09 mg dose was administered at a constant rate over the 3-day continuous infusion period, such that the total dose was delivered at a rate of 0.03 mg / day for 3 days. There was a 7-day infusion-free period between cycles 1 and 2 to maintain a 14-day dosing interval between the infusion on day 22 of cycle 1 and the infusion on day 1 of cycle 2. In cycle 2 and all subsequent cycles, AMG 160 was administered at a dose of 0.3 mg by brief IV infusion (approximately 60 minutes) every 2 weeks (Q2W) on days 1 and 15 of a 28-day cycle. The day of the first dose of AMG 160 was defined as day 1 in each of the 28-day cycles. In cohort 1, enzalutamide was administered orally (PO) at a dose of 160 mg once daily (QD) starting on day 1 of cycle 1 (i.e., the first day of treatment with AMG 160) or up to 3 days after day 1 of cycle 1.

[0198] All patients were pretreated with 8 mg PO dexamethasone (or equivalent dose of other corticosteroid) 6-16 hours prior to all doses of AMG 160 in cycle 1. In addition, dexamethasone 8 mg IV (or equivalent dose of other corticosteroid) was administered within 1 hour prior to all doses of AMG 160 in cycle 1. On cycle 2 day 1, if the subject experienced grade 2 or higher cytokine release syndrome (CRS) in cycle 1, they were given 8 mg dexamethasone IV premedication. Otherwise, dexamethasone premedication was reduced in cycle 2 if tolerated (e.g., 4-8 mg dexamethasone on cycle 2 day 1 and 0-4 mg dexamethasone on cycle 2 day 15). Corticosteroid eye drops (e.g., 1% prednisolone acetate eye drops, 2 drops per eye 4 times per day) were used with AMG 160 infusions: (1) within 6 hours prior to the start of the infusion, (2) during the entire infusion period, and (3) 48 hours after the end of infusions in cycles 1 and 2. From cycle 3 onwards, eye drop prophylaxis was administered at the investigator's discretion. Patients received treatment cycles of AMG 160 in combination with enzalutamide until disease progression or unacceptable toxicity.

[0199] The antitumor activity of AMG 160 in combination with enzalutamide was evaluated based on objective response by RECIST 1.1 criteria (assessed by CT or MRI scans), prostate specific antigen (PSA) response, circulating tumor cell (CTC) response, and 68 gallium( 68 Ga)-PSMA-11 (or Piflufolastat F-18 ( 18 F-DCFPyL)) Positron Emission Tomography (PET) / Computed Tomography (CT) and 18Radiologic response was assessed by several measures, including F-fluorodeoxyglucose (FDG) PET / CT scans, progression-free survival (radiography and PSA), and overall survival. CT / magnetic resonance imaging (MRI) scans were performed at baseline and every 8 weeks for the first 6 months of treatment, then every 12 weeks. Tumor burden assessment was based on RECIST 1.1. A second MRI / CT scan was performed at least 4 weeks after the first detection of radiologic progression to confirm disease progression (PD). Responses (partial response (PR) and complete response (CR)) were confirmed by repeated serial assessments at least 4 weeks after the first detection of radiologic response.

[0200] PSA30 / 50 / 70 / 90 responses were defined as a 30%, 50%, 70%, and 90% decrease, respectively, in serum PSA levels in at least two post-treatment measurements taken at least 3 weeks apart. CTC responses were defined as CTC0 (decrease in CTCs from >0 to 0) or CTC conversion (from ≥5 CTCs / 7.5 mL blood to ≤4 CTCs / 7.5 mL blood) measured in whole blood. 68 Ga-PSMA-11 or Pifluforastat F-18( 18 F-DCFPyL) PET / CT scans were performed at baseline to assess PSMA-positive tumor burden and at 12 and 24 weeks after treatment initiation for response assessment. 18 F-FDG PET / CT scan was performed at baseline.

[0201] Adverse events were assessed using the Common Terminology Criteria for Adverse Events (CTCAE), version 5, available at the following web address: ctep.cancer.gov / protocolDevelopment / electronic_applications / ctc.htm, except that CRS was graded according to the criteria described in Lee et al., Blood, Vol. 124: 188-195, 2014. Tumor lysis syndrome was graded according to the Cairo Bishop criteria referenced in Coiffier et al., J. Clin. Oncol., Vol. 26: 2767-2778, 2008, and immune effector cell-associated neurological syndrome (ICANS) was assessed using the criteria described in Lee et al., Biol. Blood Marrow Transplant., Vol. 25: 625-638, 2019.

[0202] Four patients were enrolled in cohort 1, and at the time of data analysis, all four patients were continuing treatment. Three of the four patients were evaluable for dose-limiting toxicity (DLT). Of these three patients, two developed DLTs (grade 3 CRS and grade 3 anemia requiring transfusion). Both DLTs developed during combination therapy (i.e., after the patient received both AMG 160 and enzalutamide). All four subjects experienced grade 2 CRS, which was associated with the first or second dose of AMG 160. There were 10 serious adverse events related to the AMG 160 study drug. All four patients had a PSA90 response, all of which were confirmed responses. All four patients were assessed to have stable disease according to on-treatment radiographic scans. None of the four patients were RECIST-evaluable, as none of the patients had measurable target lesions according to RECIST 1.1 criteria. Non-RECIST-evaluable patients can only be evaluated as having complete response (CR), stable disease (SD), or progressive disease (PD).

[0203] Preliminary results from cohort 1 showed that the combination of enzalutamide and AMG 160 at this dose level provided remarkable efficacy in all evaluable patients with PSA90 response and positive radiographic response (stable disease). However, the combination of enzalutamide treatment initiated simultaneously with AMG 160 at this dose level caused most patients to develop DLTs. These results are somewhat surprising as the overlapping toxicity of the two drugs was not expected given the differences in the biological mechanisms of each drug and the reported safety risks. Two new cohorts (cohort 2a and cohort 2b) were opened, and the treatment dose of AMG 160 was reduced from 0.3 mg to 0.15 mg (see Table 8 below). In addition, in cohort 2b, the start of enzalutamide treatment was delayed until day 15 of cycle 1. The specific dosing regimens of AMG 160 and enzalutamide in cohorts 2a and 2b were as follows: Cohort 2a o In cycle 1, AMG 160 was administered by eIV infusion at a dose of 0.09 mg over 72 hours on days 1-3 of a 28-day cycle, followed by a therapeutic dose of 0.15 mg as a short IV infusion (sIV; approximately 60 minutes) on days 8 and 22 of a 28-day cycle. For cycle 2 and all other subsequent cycles, AMG 160 was administered at a dose of 0.15 mg by sIV every 2 weeks on days 1 and 15 of a 28-day cycle. There was a 7-day infusion-free period between cycles 1 and 2. o 160 mg enzalutamide orally once daily starting on Cycle 1 Day 1 or continuing until 3 days after Cycle 1 Day 1. Cohort 2b o In cycle 1, 0.09 mg AMG 160 was administered by eIV infusion over 72 hours on days 1-3 of a 28-day cycle, followed by a therapeutic dose of 0.15 mg as an sIV infusion on days 8 and 22 of a 28-day cycle. For cycle 2 and all other subsequent cycles, AMG 160 was administered at a dose of 0.15 mg by sIV every 2 weeks on days 1 and 15 of a 28-day cycle. There was a 7-day infusion-free period between cycles 1 and 2. Enzalutamide 160 mg orally once daily starting on day 15 of cycle 1.

[0204] Three patients were enrolled in each of cohorts 2a and 2b. As of the data analysis date, two of the three patients continued treatment in cohort 2a, and one patient discontinued due to adverse events (grade 3 CRS and grade 3 myocardial infarction). Two of the three patients in cohort 2b continued treatment, while one patient discontinued due to progressive disease. In cohort 2a, where enzalutamide treatment was initiated on cycle 1 day 1 (i.e., simultaneously with AMG 160), all three patients were evaluable for DLTs, and one patient of these three patients developed a DLT (grade 3 hearing loss) during the combination therapy period. One patient experienced grade 3 CRS, while the other two patients had grade 2 CRS as their worst grade. There were three serious adverse events related to the AMG 160 study drug. All three patients had a decline in PSA levels with one PSA90 response (confirmed), one PSA50 response (confirmed), and one PSA30 response (unconfirmed). At the time of data analysis, two of the three patients had on-treatment radiographic scans and one of the two patients was RECIST evaluable (i.e., had measurable target lesions at baseline). Of these two patients, one patient had a confirmed partial response and one patient had stable disease. Two of the three patients enrolled in cohort 2a had previously been treated with another antiandrogen therapy: one patient had previously received abiraterone and the other had previously received abiraterone and darolutamide.

[0205] In cohort 2b, in which the initiation of enzalutamide treatment was delayed until day 15 of cycle 1 (i.e., after the patient had received the first two doses of AMG 160, including the first therapeutic dose of AMG 160 on day 8 of cycle 1), all three patients were evaluable for DLTs, but none were observed. Two of the patients experienced grade 2 CRS, while one patient had grade 1 CRS as the worst grade. There were no serious adverse events. Regarding antitumor activity, two patients had confirmed PSA90 responses and one patient did not show a PSA response. Only one patient who was not RECIST-evaluable (i.e., did not have a measurable target lesion) had an on-treatment scan and had stable disease at the time of data analysis. One of the three patients in cohort 2b had been pretreated with antiandrogen therapy (apalutamide).

[0206] The results of cohorts 2a and 2b showed that delaying the initiation of enzalutamide treatment until week 2 of cycle 1 (e.g., approximately 7 days after administration of the first therapeutic dose of AMG 160) significantly improved the safety profile of the combination therapy, as no patients in cohort 2b experienced DLTs or serious adverse events. The severity of CRS associated with AMG 160 administration was also reduced when the initiation of enzalutamide treatment was delayed. Antitumor efficacy was comparable between cohorts 2a and 2b, with two out of three patients in both cohorts demonstrating at least a PSA50 response. Given these results, four patients were enrolled in cohort 1a, where the therapeutic dose of AMG 160 was increased to 0.3 mg and enzalutamide treatment was initiated on day 15 of cycle 1 (see Table 8 below). Preliminary results from one patient in cohort 1a who previously received abiraterone treatment showed that this dosing regimen resulted in a PSA90 (unconfirmed) response. The antitumor efficacy of the combination with the dosing regimen is expected to be similar in the other two enrolled patients, and the tolerability profile is expected to be significantly improved compared to that observed in cohort 1, with few or no DLTs or serious adverse events observed.

[0207] [Table 8]

[0208] A second interim analysis of data from cohorts 1, 1a, 2a, and 2b was performed. The results of this second interim analysis are summarized in Table 9 below. The results of this second interim analysis are consistent with the results of the first interim analysis described above. Specifically, when the first dose of enzalutamide was delayed until after the first administration of a therapeutic dose of AMG 160 (cohorts 1a and 2b), patients had less severe serious adverse events, DLTs, and CRS events compared to when enzalutamide was initiated on the same day as the first dose of AMG 160. When AMG 160 was administered at a therapeutic dose of 0.15 mg (cohorts 2a and 2b), PSA responses were comparable between cohorts, indicating that delaying the initiation of enzalutamide treatment did not affect the antitumor efficacy of the combination therapy. At the 0.3 mg therapeutic dose level of AMG 160, only two of four patients in cohort 1a had a PSA90 response compared to all four patients in cohort 1 in which enzalutamide was initiated on day 1. However, all patients in cohort 1a were previously treated with at least one novel hormonal therapy (NHT), whereas three of four patients in cohort 1 were NHT-naive. This may explain the lower PSA response rate in cohort 1a. Only two of 14 patients in all four cohorts were RECIST 1.1 evaluable at the time of data analysis. Interestingly, the only objective RECIST response was observed in cohort 1a, where AMG 160 was administered at a therapeutic dose of 0.3 mg and the first dose of enzalutamide was delayed until day 15 of cycle 1.

[0209] Overall, the results of this study indicate that delaying the administration of the first dose of enzalutamide compared with the administration of the first therapeutic dose of AMG 160 improves the tolerability of the combination therapy, such that a higher therapeutic dose of AMG 160 can be administered to maximize the antitumor efficacy of the combination.

[0210] [Table 9]

[0211] Example 2. Phase 1b Study of AMG 160 in Combination with Abiraterone in Patients with Metastatic Castration-Resistant Prostate Cancer Abiraterone is a cytochrome P450 (CYP)17 inhibitor indicated in combination with prednisone (or in some areas prednisolone) for the treatment of patients with mCRPC and, in some areas, metastatic or high-risk castration-sensitive prostate cancer. Similar to enzalutamide and other NHTs, abiraterone has been reported to upregulate the expression of PSMA, the target of the AMG 160 bispecific T cell engager, on prostate cancer cells (see Aggarwal et al., Eur. Urol. Oncol., Vol. 1:78-82, 2018; Emmett et al., J Nucl. Med., Vol. 60:950-954, 2019). Thus, combination therapy with AMG 160, PSMA x CD3 T cell engager, and abiraterone may provide synergistic antitumor effects. The primary objective of this study was to evaluate the safety, tolerability, and preliminary efficacy of AMG 160 in combination with abiraterone in patients with mCRPC.

[0212] After signing informed consent, patients entered a screening period (up to 28 days) during which their eligibility was assessed. Eligible patients had mCRPC with histologically or cytologically confirmed adenocarcinoma of the prostate, without pure neuroendocrine differentiation or small cell features. Patients who were to receive abiraterone for the first time in the metastatic setting were eligible to participate in the study. Specifically, patients who met all of the following key inclusion criteria were enrolled in the study: Histologically or cytologically confirmed mCRPC without pure neuroendocrine differentiation or small cell features; · had undergone bilateral orchiectomy or received continuous androgen deprivation therapy (ADT) with gonadotropin-releasing hormone (GnRH) agonists or antagonists; Total serum testosterone concentration was 50 ng / dL or less or 1.7 nmol / L or less; and · I was scheduled to receive abiraterone for the first time for mCRPC.

[0213] Patients were excluded from the study if they: (i) had a confirmed history or current autoimmune or other disease requiring permanent immunosuppressive therapy; (ii) had previously received abiraterone; (iii) had CNS metastases or meningeal disease; (iv) had previous treatment with a taxane for mCRPC; (v) had major surgery and / or radiation within 4 weeks; (vi) had moderate or severe hepatic dysfunction (Child-Pugh classes B and C) at baseline; (vii) had uncontrolled hypertension, hypokalemia, or fluid retention; (viii) had a history of or current adrenal insufficiency; (ix) had used a strong CYP3A4 inducer (e.g., carbamazepine, phenobarbital, phenytoin, rifabutin, rifampin, rifapentine, St. John's wort) within 28 days prior to the first dose of AMG 160; or (x) had MG Concomitant use of drugs with a narrow therapeutic index that are sensitive substrates for CYP2D6 (e.g., thioridazine) within 7 days prior to the first dose of 160.

[0214] AMG 160 (amino acid sequence set forth in SEQ ID NO: 140) was administered as described in Example 1, with the first dose of cycle 1 administered as a 72-hour extended IV infusion on days 1-3, followed by therapeutic doses administered by short IV infusion (sIV; approximately 60 minutes) on days 8 and 22 of cycle 1. After a 7-day infusion-free period between cycles 1 and 2, AMG 160 was administered by short IV infusion Q2W on days 1 and 15 of cycle 2, as well as all other subsequent cycles. Abiraterone was administered orally at a dose of 1,000 mg once daily beginning on day 15±3 of cycle 1. The day of the first dose of AMG 160 was defined as day 1 in each of the 28-day cycles. A 5 mg dose of prednisone was administered orally twice daily (BID) on the same day that patients received abiraterone. In some areas, a 10 mg dose of prednisolone was administered once daily instead of prednisone. Table 10 below summarizes the dosing regimens in cycle 1 for the two patient cohorts.

[0215] [Table 10]

[0216] All patients were pretreated with 8 mg PO dexamethasone (or equivalent dose of other corticosteroid) 6-16 hours prior to all doses of AMG 160 in cycle 1. In addition, dexamethasone 8 mg IV (or equivalent dose of other corticosteroid) was administered within 1 hour prior to all doses of AMG 160 in cycle 1. On cycle 2 day 1, if the subject experienced grade 2 or higher CRS in cycle 1, they received 8 mg dexamethasone IV premedication. Otherwise, dexamethasone premedication was reduced in cycle 2 if tolerated (e.g., 4-8 mg dexamethasone on cycle 2 day 1 and 0-4 mg dexamethasone on cycle 2 day 15). Corticosteroid eye drops (e.g., 1% prednisolone acetate eye drops, 2 drops per eye 4 times per day) were used with AMG 160 infusions: (1) within 6 hours before the start of the infusion, (2) during the entire infusion period, and (3) 48 hours after the end of infusions in cycles 1 and 2. From cycle 3 onwards, eye drop prophylaxis was administered at the discretion of the investigator. Patients received treatment cycles of AMG 160 in combination with abiraterone until disease progression or unacceptable toxicity. Adverse events and antitumor efficacy were evaluated as described in Example 1.

[0217] Four patients were enrolled in cohort 1 and three patients were initially enrolled in cohort 2. At the time of data analysis, all four patients in cohort 1 continued treatment, two of the three patients in cohort 2 continued treatment, and one patient discontinued due to radiographic disease progression. All four patients in cohort 1 were DLT-evaluable, but no DLTs were observed. Two patients experienced one serious adverse event each, but both events were unrelated to the AMG 160 study drug. Two of the four patients experienced grade 2 CRS, and the other two patients experienced grade 1 CRS as the worst grade. No hepatotoxicity was observed. Regarding preliminary antitumor efficacy, all four patients in cohort 1 had post-baseline PSA measurements, one patient had a PSA90 response (confirmed), one patient had a PSA70 response (confirmed), and one patient had a PSA30 response (confirmed). Two of the four patients (one of whom had RECIST measurable target lesions) had on-treatment scans at the time of data analysis, with one patient showing a confirmed partial response and the other patient having stable disease.

[0218] In cohort 2, two of the three patients were DLT-evaluable, one of whom developed a DLT (grade 3 rash) on day 9 of cycle 1 before receiving abiraterone. Thus, no DLTs occurred during combination therapy. There were six adverse events related to the AMG 160 study drug, but no hepatotoxicity was observed. Two patients had grade 2 CRS and one patient had grade 1 CRS as the worst grade. One patient out of three patients in cohort 2 had a PSA70 response (confirmed) and one patient had a PSA30 response (confirmed). All three of the patients in cohort 2 had been previously treated with enzalutamide. One patient had also been pretreated with darolutamide. All three patients in cohort 2 did not have measurable target lesions by RECIST (i.e., RECIST-non-evaluable). Results of on-treatment scans for these patients revealed that two patients had stable disease and the other had undetermined progressive disease. Three additional patients will be enrolled in cohort 2 to further study the combination at this dose level.

[0219] An additional 3 patients were enrolled in cohort 2, and 4 patients were enrolled in the expansion cohort receiving the same dosing regimen as cohort 2 (Table 10). A second interim analysis was performed, and a summary of the results of this analysis is provided below in Table 11. Only 2 patients out of 10 patients enrolled in cohort 2 and the expansion cohort had a DLT, only 1 patient discontinued AMG 160 treatment due to a treatment-emergent adverse event, and only 1 patient had a grade 3 CRS event that occurred on day 15 of cycle 2. Of the 10 patients enrolled in cohort 2 and the expansion cohort, 4 had at least a PSA50 response, and 2 patients had a PSA90 response. Only 2 of the 10 patients had not received prior treatment with NHT (i.e., were NHT-naive). Only two patients enrolled in Cohort 2 and the expansion cohort were RECIST 1.1 evaluable (i.e., had measurable baseline disease according to RECIST 1.1 criteria), one of whom had a partial response and the other had stable disease.

[0220] The results of this study indicate that the combination of AMG 160 and abiraterone is tolerable and results in a reduction in PSA serum levels in the majority of patients and a 50% objective response rate when abiraterone treatment is initiated approximately 2 weeks after the initiation of AMG 160 treatment (and approximately 7 days after administration of the first therapeutic dose of AMG 160).

[0221] [Table 11]

[0222] All publications, patents, and patent applications described and cited herein are hereby incorporated by reference in their entirety. It is understood that the disclosed invention is not limited to the specific methods, procedures, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the appended claims.

[0223] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A combination for use in a method of treating prostate cancer in a patient requiring treatment for prostate cancer, the combination comprising a T cell engagement molecule and an antiandrogen compound, wherein the T cell engagement molecule specifically binds to human prostate-specific membrane antigen (PSMA) and human CD3, and the method comprises administering one or more cycles of the combination to the patient, the first cycle comprising administering a first dose of the antiandrogen compound about 4 to about 10 days after administering a first therapeutic dose of the T cell engagement molecule.

2. The combination for use according to claim 1, wherein the first cycle comprises administering the first dose of the anti-androgen compound about five days after administering the first therapeutic dose of the T-cell engagement molecule.

3. The combination for use according to claim 1, wherein the first cycle comprises administering the first dose of the anti-androgen compound about seven days after administering the first therapeutic dose of the T-cell engagement molecule.

4. The combination for use according to claim 1, characterized in that the T cell engagement molecule is administered by intravenous injection.

5. The combination for use according to claim 1, further comprising administering one or more priming doses of the T cell engagement molecule before administering the first therapeutic dose of the T cell engagement molecule.

6. The combination for use according to claim 5, characterized in that the first priming dose of the T cell engagement molecule is administered by continuous intravenous infusion over a period of 1 to 7 days.

7. The combination for use according to claim 1, characterized in that the anti-androgen compound is administered orally once daily.

8. The combination for use according to claim 1, wherein the anti-androgen compound is enzalutamide, abiraterone, abiraterone acetate, apalutamide, or darolutamide.

9. The combination for use according to claim 8, wherein the anti-androgen compound is enzalutamide.

10. The combination for use according to claim 9, characterized in that the enzalutamide is administered orally once daily at a dose of about 160 mg.

11. The combination according to claim 8, wherein the anti-androgen compound is abiraterone or abiraterone acetate.

12. The combination for use according to claim 11, characterized in that the abiraterone or abiraterone acetate is administered orally once daily at a dose of about 1,000 mg.

13. The method further comprises administering a maintenance cycle of the combination to the patient, the maintenance cycle comprising administering the anti-androgen compound orally once daily on each day of the cycle and administering a therapeutic dose of the T-cell engagement molecule by bolus intravenous infusion once every seven or fourteen days, the combination for use according to claim 1.

14. The T cell engagement molecule is composed of amino and carboxyl molecules in that order. (i) A first domain that specifically binds to human PSMA, comprising a first immunoglobulin heavy chain variable region (VH1) and a first immunoglobulin light chain variable region (VL1); (ii) A second domain that specifically binds to human CD3, comprising a second immunoglobulin heavy chain variable region (VH2) and a second immunoglobulin light chain variable region (VL2); and (iii) An Fc domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain and a CH3 domain, wherein the two Fc monomers are fused to each other via a peptide linker. A combination for use according to claim 1, including the following:

15. The first domain includes VH1, which contains CDRH1 having the sequence of SEQ ID NO: 14, CDRH2 having the sequence of SEQ ID NO: 16, and CDRH3 having the sequence of SEQ ID NO: 20; and VL1, which contains CDRL1 having the sequence of SEQ ID NO: 5, CDRL2 having the sequence of SEQ ID NO: 8, and CDRL3 having the sequence of SEQ ID NO: 9; The combination for use according to claim 14, wherein the second domain includes VH2 having the sequence of sequence number 49, CDRH2 having the sequence of sequence number 55, and CDRH3 having the sequence of sequence number 60, and VL2 having the sequence of sequence number 43, CDRL2 having the sequence of sequence number 44, and CDRL3 having the sequence of sequence number 47.

16. The combination for use according to claim 15, wherein VH1 comprises the sequence of sequence number 33, VL1 comprises the sequence of sequence number 30, VH2 comprises the sequence of sequence number 72, and VL2 comprises the sequence of sequence number 70.

17. The combination for use according to claim 14, wherein the T cell engagement molecule is a single-chain polypeptide comprising the sequence of Sequence ID No.

140.

18. The combination for use according to any one of claims 1 to 17, wherein the prostate cancer is metastatic prostate cancer.

19. The combination for use according to claim 18, wherein the prostate cancer is metastatic castration-resistant prostate cancer.

20. The combination for use according to any one of claims 1 to 17, wherein the patient has a total serum testosterone level of 50 ng / dL or less.

21. A composition for use in a method for treating prostate cancer in a patient requiring treatment for prostate cancer, wherein the composition comprises an antiandrogen compound, the composition is characterized by being administered in combination with a T-cell engagement molecule, the T-cell engagement molecule specifically binds to human prostate-specific membrane antigen (PSMA) and human CD3, and the method comprises administering one or more cycles of the composition to the patient, the first cycle comprising administering a first dose of the antiandrogen compound about 4 to about 10 days after administration of a first therapeutic dose of the T-cell engagement molecule.

22. A composition for use in a method for treating prostate cancer in a patient requiring treatment for prostate cancer, wherein the composition comprises a T cell engagement molecule, the T cell engagement molecule specifically binds to human prostate-specific membrane antigen (PSMA) and human CD3, the composition is administered in combination with an antiandrogen compound, the method comprising administering one or more cycles of the composition to the patient, the first cycle comprising administering a first dose of the antiandrogen compound about 4 to about 10 days after administration of a first therapeutic dose of the T cell engagement molecule.

23. The composition for use according to claim 21 or 22, wherein the anti-androgen compound is enzalutamide, abiraterone, abiraterone acetate, apalutamide, or darolutamide.

24. The composition for use according to claim 23, wherein the anti-androgen compound is enzalutamide.

25. The composition for use according to claim 23, wherein the antiandrogen compound is abiraterone or abiraterone acetate.

26. The T cell engagement molecule is composed of amino and carboxyl molecules in that order. (i) A first domain that specifically binds to human PSMA, comprising a first immunoglobulin heavy chain variable region (VH1) and a first immunoglobulin light chain variable region (VL1); (ii) A second domain that specifically binds to human CD3, comprising a second immunoglobulin heavy chain variable region (VH2) and a second immunoglobulin light chain variable region (VL2); and (iii) An Fc domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain and a CH3 domain, wherein the two Fc monomers are fused to each other via a peptide linker. A composition for use according to claim 21 or 22, comprising:

27. ​​The first domain includes VH1 having the sequence of SEQ ID NO: 14, CDRH2 having the sequence of SEQ ID NO: 16, and CDRH3 having the sequence of SEQ ID NO: 20, and VL1 having CDRL1 having the sequence of SEQ ID NO: 5, CDRL2 having the sequence of SEQ ID NO: 8, and CDRL3 having the sequence of SEQ ID NO: 9; The composition for use according to claim 26, wherein the second domain comprises VH2 including CDRH1 having the sequence of SEQ ID NO: 49, CDRH2 having the sequence of SEQ ID NO: 55, and CDRH3 having the sequence of SEQ ID NO: 60, and VL2 including CDRL1 having the sequence of SEQ ID NO: 43, CDRL2 having the sequence of SEQ ID NO: 44, and CDRL3 having the sequence of SEQ ID NO:

47.

28. The composition for use according to claim 27, wherein VH1 comprises the sequence of sequence number 33, VL1 comprises the sequence of sequence number 30, VH2 comprises the sequence of sequence number 72, and VL2 comprises the sequence of sequence number 70.

29. The composition for use according to claim 26, wherein the T cell engagement molecule is a single-chain polypeptide comprising the sequence of SEQ ID NO:

140.

30. The composition for use according to claim 21 or 22, wherein the prostate cancer is metastatic prostate cancer.

31. The composition for use according to claim 30, wherein the prostate cancer is metastatic castration-resistant prostate cancer.