WO2022060878A1

WO2022060878A1 - Methods for treating prostate cancer

Info

Publication number: WO2022060878A1
Application number: PCT/US2021/050520
Authority: WO
Inventors: Hosein KOUROS-MEHR; Mukul MINOCHA; Vijay Upreti
Original assignee: Amgen Inc.
Priority date: 2020-09-16
Filing date: 2021-09-15
Publication date: 2022-03-24

Abstract

The present invention relates to treatment methods for prostate cancer using bispecific T-cell engaging molecules that specifically bind to human prostate-specific membrane antigen (PSMA) and human CD3. In particular, the present invention relates to methods for treating prostate cancer, including metastatic castration-resistant prostate cancer, in a patient in need thereof comprising administering to the patient an initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein each initiation cycle and maintenance cycle comprises administering the bispecific T-cell engaging molecule according to specific dosage regimens. Pharmaceutical compositions comprising the bispecific T-cell engaging molecule for use in the methods are also disclosed.

Description

METHODS FOR TREATING PROSTATE CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/079,407, filed September 16, 2020, which is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The present application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The computer readable format copy of the Sequence Listing, which was created on September 7, 2021, is named A-2674-WO-PCT_ST25 and is 358 kilobytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to the fields of immuno-oncology and biopharmaceuticals. In particular, the invention relates to methods of treating prostate cancer by administering a bispecific T-cell engaging molecule that specifically binds to prostate-specific membrane antigen (PSMA) and cluster of differentiation 3 (CD3) in an initiation cycle and one or more maintenance cycles, wherein the initiation cycle and maintenance cycle each comprises administering the bispecific T-cell engaging molecule according to specific dosing regimens.

BACKGROUND OF THE INVENTION

[0004] Prostate cancer is the most frequently diagnosed non-cutaneous cancer in men with an estimated 164,690 new cases in the United States (US), accounting for 19% of new cancer cases in men (American Cancer Society, Cancer Facts & Figures, 2018). Prostate cancer deaths (26,730 in 2017 and an estimated 29,430 in 2018) account for 9% of all male cancer deaths in the US (National Comprehensive Cancer Network, Prostate Cancer, Version 3, 2018). In the European Union, there were an estimated 365,000 new cases of prostate cancer in 2015, with 72,000 and 77, 000 deaths estimated in 2012 and 2015, respectively (10% of total cancer deaths) (Crocetti, Epidemilogy of Prostate Cancer in Europe, 2015).

[0005] Metastasis is a primary cause of morbidity and mortality for prostate cancer. Since 1941, patients diagnosed with metastatic prostate cancer have received continuous androgen- deprivation therapy (ADT) in the form of surgical castration, chemical castration involving luteinizing hormone-releasing hormone agonist (LHRH)-modulating compounds, and/or antiandrogen therapy. In 2015, 2 trials (CHAARTED and STAMPEDE) demonstrated that in patients diagnosed with advanced prostate cancer, docetaxel combined with ADT conferred a 10 to 13.6 month improvement in median overall survival (OS) compared with ADT alone (James etal., Lancet, Vol. 387: 1163-1177, 2016; Sweeney et al., N Engl J Med., Vol. 373:737-746, 2015). In 2017, 2 trials (LATITUDE and STAMPEDE) demonstrated that the combination of ADT with abiraterone plus prednisone also conferred improvement in OS when compared to ADT alone (Fizazi et al., N Engl J Med., Vol. 377:352-360, 2017; James et al., N Engl J Med., Vol. 377:338-351, 2017). While abiraterone and docetaxel appear to have similar duration of survival benefit, the therapies differ significantly in their side effect profile, cost, and duration of treatment.

[0006] Metastatic prostate cancer often develops resistance to ADT (“castration-resistance”) due to increased intratumoral steroidogenesis, 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 demonstrated significant survival benefits in mCRPC patients. In first line mCRPC, abiraterone plus prednisone improved median OS from 30.3 to 34.7 months compared to 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 also demonstrated survival benefit (14.9 vs 11.3 months) in patients with bone metastases when combined with best standard care, which included older hormonal therapies, radiation, and bisphosphonates. Sipuleucel-T, an autologous cellular immunotherapy, increased median survival by 4.1 months compared with placebo, though PSA and radiographic responses were not observed. Cabazitaxel, a tubulin- binding taxane, increased median survival by 2.4 months compared with mitoxantrone, though many trial participants did not complete treatment due to toxicity. Other therapies include bone- targeted agents such as zoledronic acid and denosumab, which reduced the rate of skeletal adverse events, including pathologic fractures and spinal cord compression (Litwin and Tan, JAMA, Vol. 317:2532-2542, 2017). Recently, pembrolizumab demonstrated limited activity (5% overall response rate (ORR); n = 133) in patients with mCRPC and a number of combination trials with pembrolizumab are ongoing (Antonarakis et al., Journal of Clinical Oncology, Vol. 38(5): 395-405, 2020).

[0007] Recently, PSMA-targeted therapies have shown activity in mCRPC patients (Hofman et al., Lancet Oncol., Vol. 19(6):825-833, 2018). PSMA is a 100 kDa type-II integral membrane glycoprotein that is mainly expressed 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 in a subset of renal proximal tubules, some cells of the intestinal brush-border membrane, liver and rare cells in the 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 higher-grade lesions. There is a strong correlation between a 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 intense and homogeneous expression of PSMA within metastatic lesions localized to lymph nodes, bone, soft tissue, and lungs compared with benign prostatic tissues (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 carcinoma and metastatic disease has made PSMA an attractive target for the development of therapies for prostate cancer.

[0008] While recently approved therapies have demonstrated survival benefits for patients with mCRPC, drug resistance often complicates the disease course and contributes to relapse and mortality. There is an urgent need for therapies with novel mechanisms of action, such as PSMA-targeted immunotherapies, to treat prostate cancers resistant to chemohormonal therapies. SUMMARY OF THE INVENTION

[0009] The present invention is based, in part, on the identification of therapeutic regimens of a bispecific T-cell engaging molecule that specifically binds human PSMA and human CD3 for effectively treating prostate cancer, particularly mCRPC. Accordingly, in one embodiment, the present invention provides a method for treating prostate cancer in a patient in need thereof comprising administering to the patient an initiation cycle and at least one maintenance cycle of a bispecific T-cell engaging molecule that specifically binds to human PSMA and human CD3. [0010] In certain embodiments, the initiation cycle comprises administering one or more priming doses and a target dose of a PSMA x CD3 bispecific T-cell engaging molecule at a dosing interval of at least 7 days for a first period of time, such as from about 14 days to about 56 days or from about 21 days to about 28 days. In one embodiment, the duration of the initiation cycle (e.g. first period of time) is 28 days. In some embodiments, the initiation cycle comprises administering the PSMA x CD3 bispecific T-cell engaging molecule at a priming dose and a target dose, wherein the target dose is greater than the priming dose and is administered about 7 days after the first priming dose. In some such embodiments, the target dose is administered a second time during the initiation cycle at least 14 days after the first administration of the target dose.

[0011] In other embodiments of the methods of the invention, the initiation cycle comprises administering two or more priming doses and a target dose of a PSMA x CD3 bispecific T-cell engaging molecule. In one such embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, a second priming dose, and a target dose, wherein the second priming dose is greater than the first priming dose and the target dose is greater than the second priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose, and the target dose is administered about 7 days after the second priming dose. In another such embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, a second priming dose, a third priming dose, and a target dose, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose, the third priming dose is administered about 7 days after the second priming dose, and the target dose is administered about 7 days after the third priming dose.

[0012] The priming doses of the PSMA x CD3 bispecific T-cell engaging molecule may, in some embodiments, be lower than therapeutic doses but sufficient to induce T-cell activation in a patient to prime or prepare the patient to receive higher doses of the PSMA x CD3 bispecific T- cell engaging molecule such that administration of the higher doses results in a reduced number or severity of adverse events, like cytokine release syndrome. The priming doses can vary depending on the number of priming doses administered and the amount of the target dose. In certain embodiments, the priming doses of the PSMA x CD3 bispecific T-cell engaging molecule administered during the initiation cycle increase at one or more subsequent dosing intervals as a series of increasing dose steps. In some such embodiments, the priming doses of the PSMA x CD3 bispecific T-cell engaging molecule can increase in dose steps from about 10 pg to about 300 pg. For example, a first priming dose of the bispecific T-cell engaging molecule may be from about 10 pg to about 60 pg, a second priming dose of the bispecific T-cell engaging molecule may be from about 30 pg to about 180 pg, and a third priming dose of the bispecific T- cell engaging molecule may be from about 60 pg to about 300 pg.

[0013] In certain embodiments, the target dose of the PSMA x CD3 bispecific T-cell engaging molecule is a therapeutic dose and is generally greater than any of the priming doses administered to the patient. The target dose of the PSMA x CD3 bispecific T-cell engaging molecule can be from about 30 pg to about 1800 pg, for example from about 90 pg to about 1800 pg, from about 300 pg to about 900 pg, or from about 300 pg to about 600 pg. In some embodiments, the initiation cycle comprises administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose from about 10 pg to about 30 pg, a second priming dose from about 90 pg to about 180 pg, and a target dose from about 300 pg to about 900 pg. In other embodiments, the initiation cycle comprises administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose from about 10 pg to about 45 pg, a second priming dose from about 30 pg to about 110 pg, a third priming dose from about 90 pg to about 180 pg, and a target dose from about 300 pg to about 900 pg, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose. In one embodiment, the initiation cycle comprises administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose of about 10 pg, a second priming dose of about 30 pg, a third priming dose of about 90 pg, and a target dose of about 900 pg.

[0014] In some embodiments of the methods of the invention, the maintenance cycle comprises administering to the patient the target dose of the PSMA x CD3 bispecific T-cell engaging molecule once every 14 days (e.g. once every other week) for a second period time, such as from about 28 days to about 56 days. In one embodiment, the duration of the maintenance cycle (e.g. second period of time) is 28 days. In certain embodiments, the target dose of the bispecific T-cell engaging molecule administered during the maintenance cycle is the same at each biweekly dosing interval (e.g. a fixed dose for the entire maintenance cycle). In these and other embodiments, the target dose and dosing frequency (e.g. biweekly) of the bispecific T-cell engaging molecule administered during the maintenance cycle is the same from one maintenance cycle to the next maintenance cycle.

[0015] According to the methods of the invention, the maintenance cycle is administered after the initiation cycle. In one embodiment, the maintenance cycle is administered the following day after completing the initiation cycle, for example with no treatment-free periods between the initiation cycle and the maintenance cycle. In another embodiment, the maintenance cycle is administered about 7 days following the completion of the initiation cycle - i.e. there is a 7-day treatment-free period between the initiation cycle and the maintenance cycle. A patient may receive multiple maintenance cycles, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more maintenance cycles. In some embodiments, maintenance cycles are administered to the patient until the patient responds to treatment, for example achieves a complete response.

[0016] In some embodiments, the prostate cancer to be treated according to the methods of the invention is metastatic prostate cancer. Accordingly, the patient to be treated according to the methods of the invention has or is diagnosed with metastatic prostate cancer. The metastatic prostate cancer may be hormone-sensitive, or it may be resistant to hormone therapy. Thus, in one embodiment, the patient to be treated according to the methods of the invention has or is diagnosed with metastatic castration-resistant prostate cancer.

[0017] Prostate cancer patients to be treated according to the methods of the invention may have received one or more prior therapies for prostate cancer and have failed or become intolerant, refractory, or resistant to one or more of these prior therapies. For example, in some embodiments, the patients have failed or are intolerant, refractory, or resistant to one or more chemotherapy regimens, such as taxane-containing chemotherapy regimens. Additionally or alternatively, the patients have failed or are intolerant, refractory, or resistant to one or more antiandrogen therapies, such as abiraterone, enzalutamide, apalutamide, or darolutamide. In certain embodiments, the patients to be treated according to the methods of the invention have failed or are intolerant, refractory, or resistant to a radioligand therapy, such as ¹⁷⁷Lu-PSMA-617.

[0018] In certain embodiments of the methods described herein, the PSMA x CD3 bispecific T- cell engaging molecule is administered to the patient parenterally, preferably intravenously. The intravenous administration can be an intravenous infusion, such as intravenous infusion of about 30 min to about 3 hours or more preferably of about 30 min to about 90 min. In some embodiments, each of the doses of the bispecific T-cell engaging molecule administered during the initiation cycle and/or maintenance cycle is administered as an intravenous infusion.

[0019] In some embodiments, the methods of the invention may further comprise administering to the patient one or more premedications prior to administration of one or more (or all) doses of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle and/or maintenance cycle. In certain embodiments, the one or premedications are administered to the patient prior to each dose of the bispecific T-cell engaging molecule during the initiation cycle. Premedications can include antihistamines (e.g. diphenhydramine), glucocorticoids (e.g. dexamethasone), IL6 receptor antagonists (e.g. tocilizumab), and TNF-alpha antagonists (e.g. etanercept).

[0020] In other embodiments, the methods of the invention may further comprise administering to the patient one or more standard prostate cancer therapies, such as chemotherapy, radiation therapy, androgen deprivation therapy, or radioligand therapy, in combination with a PSMA x CD3 bispecific T-cell engaging molecule. In certain embodiments, the methods of the invention further comprise administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody during the initiation cycle and/or one or more maintenance cycles of the PSMA x CD3 bispecific T-cell engaging molecule. In some such embodiments, the PD-1 antagonist antibody or a PD-L1 antagonist antibody is administered to the patient once per cycle (initiation cycle and/or maintenance cycle). In one particular embodiment, the PD-1 antagonist antibody or a PD- L1 antagonist antibody is administered to the patient on the same day of the initiation cycle and/or maintenance cycle that the patient receives the target dose of the PSMA x CD3 bispecific T-cell engaging molecule. In some embodiments, the PD-1 antagonist antibody administered in combination with the PSMA x CD3 bispecific T-cell engaging molecule according to the methods of the invention is pembrolizumab, nivolumab, or cemiplimab. In other embodiments, the PD-1 antagonist antibody administered in combination with the PSMA x CD3 bispecific T- cell engaging molecule according to the methods of the invention is any one of the PD-1 antagonist antibodies listed in Table 7, such as antibody 20C1.9. In certain other embodiments, the PD-L1 antagonist antibody administered in combination with the PSMA x CD3 bispecific T- cell engaging molecule according to the methods of the invention is atezolizumab, avelumab, or durvalumab.

[0021] In any embodiments of the methods disclosed herein, the bispecific T-cell engaging molecule administered to the patient specifically binds to PSMA and CD3, preferably human PSMA and human CD3. Thus, the bispecific T-cell engaging molecule comprises a first binding domain that specifically binds to PSMA and a second binding domain that specifically binds to CD3. In certain embodiments, the first binding domain specifically binds to human PSMA and the second binding domain specifically binds to human CD3 epsilon. The binding domains can comprise structural elements from antibodies or antigen-binding fragments thereof, such as heavy and light chain variable regions. In one embodiment, either or both of the binding domains of the bispecific T-cell engaging molecule used in the methods of the invention is a single-chain variable fragment (scFv). In some embodiments of the methods described herein, the bispecific T-cell engaging molecules further comprise a third domain having one or more immunoglobulin Fc regions. In such embodiments, the third domain can be a single-chain Fc domain.

[0022] In certain embodiments, the bispecific T-cell engaging molecule administered to the patient according to the methods of the invention comprises, in an amino to carboxyl order: (i) a first domain that specifically binds to human PSMA, (ii) a second domain that specifically binds to human CD3, and (iii) a third domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, wherein said two Fc monomers are 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 a related embodiment, 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 bispecific T-cell engaging molecule 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 bispecific T-cell engaging molecule 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.

[0023] The bispecific T-cell engaging molecule administered to patients according to the methods of the invention may comprise (i) a first domain that specifically binds to human PSMA and has the amino acid sequence of SEQ ID NO: 104, (ii) a second domain that specifically binds to human CD3 and has the amino acid sequence of SEQ ID NO: 116, and (iii) a third domain comprising two Fc monomers each having the amino acid sequence of SEQ ID NO: 124, wherein said two Fc monomers are fused to each other via a peptide linker. In related embodiments, the third domain of the bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 132. In certain embodiments, the bispecific T-cell engaging molecule used in the methods of the invention is a single chain polypeptide or 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 bispecific T-cell engaging molecule administered to a patient according to the methods of the invention is a single chain polypeptide comprising the amino acid sequence of SEQ ID NO: 140.

[0024] The present invention also provides pharmaceutical compositions of PSMA x CD3 bispecific T-cell engaging molecules for use in the methods described herein. The pharmaceutical compositions can comprise one or more pharmaceutically acceptable diluents, carriers, or excipients, including buffers, surfactants, and stabilizing agents. In certain embodiments, the pharmaceutical compositions comprise a PSMA x CD3 bispecific T-cell engaging molecule, a buffer, a surfactant, and a stabilizing agent. In one embodiment, the pharmaceutical composition comprises a PSMA x CD3 bispecific T-cell engaging molecule (e.g. single chain polypeptide comprising the amino acid sequence of SEQ ID NO: 140), a 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 may be lyophilized and reconstituted prior to administration to a patient.

[0025] In some embodiments, the present invention also provides kits comprising a pharmaceutical composition disclosed herein and instructions for using the pharmaceutical composition to prepare and deliver, for example, by intravenous infusion, priming doses and target doses of the PSMA x CD3 bispecific T-cell engaging molecule for treating prostate cancer in a patient in need thereof. In embodiments in which the pharmaceutical composition is provided in a lyophilized or dry powder form, the kit may comprise a diluent and instructions for reconstituting the pharmaceutical composition prior to administration. In certain embodiments, the kits may further comprise one or more vials of intravenous solution stabilizer (IVSS) and instructions for using the IVSS for pre-treatment of IV bags prior to dilution of the pharmaceutical composition for delivery to the patient.

[0026] The use of PSMA x CD3 bispecific T-cell engaging molecules in any of the methods disclosed herein or for preparation of medicaments for administration according to any of the methods disclosed herein is specifically contemplated. For instance, the present invention includes a bispecific T-cell engaging molecule that specifically binds to human PSMA and human CD3 for use in a method for treating prostate cancer in a patient in need thereof, wherein the method comprises administering to the patient an initiation cycle and at least one maintenance cycle of the bispecific T-cell engaging molecule, wherein the initiation cycle comprises administering one or more priming doses and a target dose of the bispecific T-cell engaging molecule at a dosing interval of at least 7 days for a first period of time, wherein the maintenance cycle comprises administering the target dose of the bispecific T-cell engaging molecule once every 14 days for a second period of time, wherein the target dose is greater than said one or more priming doses and is about 30 pg to about 1800 pg, and wherein the maintenance cycle is administered after the initiation cycle. The present invention also includes the use of a bispecific T-cell engaging molecule that specifically binds to human PSMA and human CD3 for the manufacture of a medicament for the treatment of prostate cancer in a patient in need thereof, wherein the treatment comprises administering to the patient an initiation cycle and at least one maintenance cycle of the bispecific T-cell engaging molecule, wherein the initiation cycle comprises administering one or more priming doses and a target dose of the bispecific T-cell engaging molecule at a dosing interval of at least 7 days for a first period of time, wherein the maintenance cycle comprises administering the target dose of the bispecific T- cell engaging molecule once every 14 days for a second period of time, wherein the target dose is greater than said one or more priming doses and is about 30 pg to about 1800 pg, and wherein the maintenance cycle is administered after the initiation cycle.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the number of days on treatment with AMG 160 monotherapy for each of the thirty-three patients in six different target dose cohorts in the study. Observed responses (RECIST, PSA reductions, or CTC0) to treatment with AMG 160 are annotated to the right of the bars for each patient. CTC = circulating tumor cell; DLT = dose-limiting toxicities; NE = not evaluable; PSA = prostate-specific antigen; PR = partial response; SD = stable disease; (u) = unconfirmed. PR* occurred before but reported after data cutoff.

[0028] Figure 2 is a waterfall plot showing percentage change in PSA serum levels from baseline to best response in evaluable patients with mCRPC. Evaluable patients included those who had received > 1 dose of AMG 160 in one of six different target dose cohorts and had measurable PSA levels at baseline. PSA50 = PSA decrease of > 50%. The solid triangles denote patients who had failed prior treatment with ¹⁷⁷Lu-PSMA-617 radioligand therapy.

DETAILED DESCRIPTION

[0029] Bispecific T-cell engaging molecules are new immunotherapies being developed for the treatment of various cancers. These molecules typically have at least one binding domain that is specific for a cell-surface antigen expressed on cancer cells and at least another binding domain that is specific for cluster of differentiation 3 (CD3), a subunit of the T cell receptor complex expressed on T cells. Bispecific T cell engaging molecules are designed to connect T cells with target cancer cells and potently activate the inherent cytolytic potential of T cells against the target cancer cells. The first generation of bispecific T cell engaging molecules (see, e.g., WO 99/54440, WO 2005/040220, and WO 2008/119567) are typically administered by continuous intravenous infusion due to half-lives of less than a day. A second generation of bispecific T cell engaging molecules see, e.g., WO 2013/128027, WO 2014140358, WO 2014/144722, WO 2014/151910, WO 2017/134140) have been designed, at least in part, to increase the serum halflife of the molecules to enable dosing paradigms that do not involve continuous administration. [0030] Because the mechanism of action of bispecific T cell engaging molecules involves T cell activation, a potential side effect of these molecules is cytokine release syndrome (CRS). CRS can occur when large numbers of T cells are activated and release inflammatory cytokines. To minimize the effects of cytokine elevation and the development of CRS, bispecific T cell engaging molecules can be administered at lower doses or by employing anti-histamines or corticosteroid pre-treatments. In the case of second-generation bispecific T cell engaging molecules where the effects of the molecules, including the undesired side effects, may be prolonged due to the longer serum half-life, it is important to develop a dosing strategy that allows the patients to be exposed to efficacious doses as quickly as possible while at the same time limiting or avoiding the side effects associated with rapid cytokine elevation, such as CRS. The present invention addresses this need by providing novel dosing regimens for a bispecific T- cell engaging molecule that specifically binds to PSMA and CD3 (i.e. a PSMA x CD3 bispecific T-cell engaging molecule) for the treatment of prostate cancer. Accordingly, in one aspect, the present invention provides a method for treating prostate cancer in a patient in need thereof comprising administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule in an initiation cycle and at least one maintenance cycle as described further herein.

[0031] Prostate cancer is one of the most common types of cancer in men and occurs when cells in the prostate gland begin to grow out of control. Most forms of prostate cancer are adenocarcinomas, which are tumors formed from glandular cells. Other forms of prostate cancer include small cell carcinomas, neuroendocrine tumors, transitional cell carcinomas, and sarcomas. Prostate cancer is initially confined to the prostate gland but can metastasize and spread to other tissues. Metastatic prostate cancer can be divided into two primary types: a first type where the cancer has not been treated with androgen deprivation therapy (“metastatic hormone-sensitive prostate cancer” or mHSPC) and a second type where 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. [0032] In its early stages, prostate cancer may not cause any signs or symptoms. As the disease progresses, signs and symptoms of prostate cancer can include incontinence, trouble urinating, blood in semen or urine, erectile dysfunction, pain in pelvic area or bones, or weakness in legs or feet. Prostate cancers are typically diagnosed and monitored by one or more tests conducted on a sample (e.g. blood, serum, plasma, semen, tissue) from a subject or patient suspected of having or developing prostate cancer. A sample can be any biological sample obtained from a human patient and can include body fluids, such as blood, serum, plasma, semen, and urine, and tissues, such as prostate tissue, lymph nodes, or tumor biopsies. A common test used to screen and/or monitor for prostate cancer is the prostate-specific antigen (PSA) blood test. Elevation of PSA in the blood (e.g. serum or plasma) can 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. The prostate tissue biopsy sample is evaluated for the presence of abnormal or cancerous cells. If cancerous cells are present, the prostate cancer may be assigned a grade based on the Gleason score grading system or other grading system, which assigns a grade based on how abnormal the cells appear as compared to normal cells. Gleason scores can range from 2 (nonaggressive cancer) to 10 (very aggressive cancer). Prostate cancer may also be diagnosed or monitored using imaging tests including, but not limited to, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET) with radioactive tracers, such as ⁶⁸Gallium-PSMA-l l or ¹⁸F-flurodeoxy glucose, or bone scans (e.g. bone scintigraphy with ^98mtechnetium-labeled radiotracers).

[0033] In certain embodiments, the patients to be treated according to the methods of the invention have or are diagnosed with prostate cancer. In such embodiments, the prostate cancer is PSMA positive - that is the tumors express PSMA as determined by standard immunohistochemical tests of biopsy samples or PSMA imaging methods. In some embodiments, the patients to be treated according to the methods of the invention have blood PSA levels of 4 ng/mL or greater. In other embodiments, the patients to be treated according to the methods of the invention have blood PSA levels of 10 ng/mL or greater. In still other embodiments, the patients to be treated according to the methods of the invention have blood PSA levels of 1 ng/mL or greater, wherein the PSA levels have increased on at least two successive occasions at least a week apart. In some embodiments, the patients to be treated according to the methods of the invention have prostate cancer with a Gleason score of 7. In other embodiments, the patients to be treated according to the methods of the invention have prostate cancer with a Gleason score of 8. In yet other embodiments, the patients to be treated according to the methods of the invention have prostate cancer with a Gleason score of 9 or 10. [0034] In some embodiments, the patients to be treated according to the methods of the invention have or are diagnosed with metastatic prostate cancer. The metastatic prostate cancer can be hormone sensitive or resistant to hormone therapy. Patients diagnosed with metastatic prostate cancer have evidence of cancerous prostate cells outside the prostate gland, such as in lymph nodes, bones, or other organs, most commonly liver, lung, or brain. Evidence of spread of the cancer cells is typically detected by the presence of tumors or lesions in other tissues by one or more of the imaging methods described above, such as CT, MRI, PET, or bone scans. In some embodiments, the patients to be treated according to the methods of the invention have evidence of progressive prostate cancer as shown by progression of lymph node or visceral tumors as defined by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 with Prostate Cancer Working Group 3 (PCWG3) modifications (see Eisenhauer et al., European Journal of Cancer, Vol. 45: 228-247, 2009; Scher etal., J. Clin, Oncol, Vol. 34: 1402-1418, 2016). In other embodiments, the patients to be treated according to the methods of the invention have evidence of progressive prostate cancer as shown by the appearance of two or more new bone lesions as determined by a bone scan (e.g. bone scintigraphy with ^98mtechnetium-labeled radiotracers). [0035] In certain embodiments, the patients to be treated according to the methods of the invention have or are diagnosed with metastatic castration-resistant prostate cancer (mCRPC). mCRPC is diagnosed when the cancer progresses in patients with metastatic disease even though the patients have testosterone levels at or below the testosterone levels achieved by androgen deprivation therapy. In some embodiments, patients who have been diagnosed with mCRPC may have failed, become refractory to, or have relapsed following treatment with an androgen deprivation therapy. Androgen deprivation therapy includes surgical castration (e.g. bilateral orchiectomy), chemical castration with LHRH agonists or antagonists (e.g. leuprolide, goserelin, triptorelin, histrelin, or degarelix), or treatment with anti-androgen therapies, such as androgen biosynthesis inhibitors (e.g. abiraterone, ketoconazole), or androgen receptor antagonists (e.g. flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide). In some embodiments, patients to be treated according to the methods of the invention have total serum testosterone levels of 50 ng/dL (1.7 nmol/L) or less. In other embodiments, patients to be treated according to the methods of the invention have total serum testosterone levels of 20 ng/dL (0.7 nmol/L) or less.

[0036] Androgen receptor (AR) signaling is altered in patients with castration-resistant prostate cancer (CRPC) and thus the patients’ tumors develop various mutations in genes encoding 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 R, FOXA1, ZBTB16, and SPOP genes along with mutations in genes involved in AKT signaling, DNA repair, and tumor suppression, such as PTEN, ET5, BRCA2, ATM, and CHEK2. Accordingly, diagnosis of CRPC or mCRPC can be supplemented by gene-expression profiling or genotyping to confirm an initial diagnosis and/or identify a subtype of CRPC or mCRPC.

[0037] The methods described herein are also applicable to treatment of other types of PSMA- expressing cancers. Immunohistochemistry studies demonstrate that PSMA is expressed on the surface of endothelial cells within the tumor vasculature of many tumor types, and is not expressed on normal vasculature (see, e.g., Schmidt et al., PLoS One, Vol. 12:e0186280, 2017; Wang et al., PLoS One, Vol. 10:e0125924, 2015; Chang, et. al., Cancer Res., Vol. 59:3192- 3198, 1999). In vivo, angiogenesis has been shown to be significantly impaired in PSMA-null mice or PSMA wild-type mice treated with a PSMA inhibitor, suggesting that PSMA participates in tumor-specific neovasculature growth (Nguyen et al., Mol Cancer Res., Vol. 14: 1045-1053, 2016; Conway et al. Mol Cell Biol., Vol. 26:5310-5324, 2006). Previous studies have shown that angiogenesis inhibitors can inhibit tumor growth by blocking new blood vessel formation and depriving the tumor of critical nutrients. PSMA has been shown to be expressed on tumor vasculature cells (e.g. neovasculature endothelial cells) in tumors in non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma, urinary bladder cancer, testicular cancer, colon cancer, glioblastoma, breast cancer, ovarian cancer, endometrial cancer, and melanoma. Accordingly, the present invention also provides methods for treating a patient having a PSMA-expressing tumor (i.e. PSMA-positive tumor), including patients having any of the aforementioned malignancies, comprising administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule according to any of the dosage regimens described herein. [0038] Administration of the PSMA x CD3 bispecific T-cell engaging molecule according to the methods of the invention is for the treatment of prostate cancer or other PSMA-expressing cancers or tumors. The term “treatment” or “treat” as used herein refers to the application or administration of the bispecific T-cell engaging molecule to a patient who has or is diagnosed with prostate cancer or other PSMA positive malignancy, has a symptom of prostate cancer or other PSMA positive malignancy, is at risk of developing prostate cancer or other PSMA positive malignancy, or has a predisposition to prostate cancer or other PSMA positive malignancy for the purpose of curing, healing, alleviating, relieving, altering, ameliorating, or improving prostate cancer or other PSMA positive malignancy, one or more symptoms of prostate cancer or other PSMA positive malignancy, the risk of developing prostate cancer or other PSMA positive malignancy, or predisposition toward prostate cancer or other PSMA positive malignancy. The term “treatment” encompasses any improvement of the disease in the patient, including the slowing or stopping of the progression of prostate cancer or other PSMA positive malignancy in the patient, a decrease in the number or severity of the symptoms of prostate cancer or other PSMA positive malignancy, or an increase in frequency or duration of periods where the patient is free from the symptoms of prostate cancer or other PSMA positive malignancy. The term “patient” includes human patients.

[0039] In certain embodiments of the methods of the invention, administration of the PSMA x CD3 bispecific T-cell engaging molecule reduces blood levels (e.g. serum or plasma levels) of PSA 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% relative to the blood levels of PSA in the patient prior to the start of the treatment (i.e. prior to the administration of the PSMA x CD3 bispecific T-cell engaging molecule). In some embodiments, administration of the PSMA x CD3 bispecific T-cell engaging molecule reduces PSA blood levels in the patient by 30% or greater relative to the PSA blood levels in the patient prior to the start of treatment (i.e. PSA30 response). In other embodiments, administration of the PSMA x CD3 bispecific T-cell engaging molecule reduces PSA blood levels in the patient by 50% or greater relative to the PSA blood levels in the patient prior to the start of treatment (i.e. PSA50 response). In yet other embodiments, administration of the PSMA x CD3 bispecific T-cell engaging molecule reduces PSA blood levels in the patient by 70% or greater relative to the PSA blood levels in the patient prior to the start of treatment (i.e. PSA70 response). In still other embodiments, administration of the PSMA x CD3 bispecific T-cell engaging molecule reduces PSA blood levels in the patient by 90% or greater relative to the PSA blood levels in the patient prior to the start of treatment (i.e. PSA90 response). In certain embodiments of the methods of the invention, administration of the PSMA x CD3 bispecific T-cell engaging molecule produces 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.

[0040] In some embodiments of the methods of the invention, administration of the PSMA x CD3 bispecific T-cell engaging molecule induces a complete response, a partial response, or a 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 start of treatment as determined by RECIST 1.1 criteria with PCWG3 modifications (see Eisenhauer et al., European Journal of Cancer, Vol. 45: 228- 247, 2009; Scher et al., J. Clin, Oncol, Vol. 34: 1402-1418, 2016). A complete response (CR) in the context of the invention refers to the condition in which all target lesions have disappeared (i.e. no longer detectable) and any pathological lymph nodes have a reduction in the short axis to less than 10 mm. A partial response (PR) refers to the condition in which there is at least a 30% decrease in the sum of diameters of target lesions relative to the sum of the diameters prior to the start of treatment. Stable disease (SD) refers to the condition where the target lesions have not reduced sufficiently to qualify as a PR but have not increased sufficiently to qualify as progressive disease (PD). PD refers to the condition in which there is the appearance of one or more new target lesions or there is at least 20% increase in the sum of diameters of target lesions relative to the smallest sum of diameters occurring previously and an absolute increase of the sum of diameters of at least 5 mm.

[0041] Efficacy of the therapeutic regimens described herein can also be assessed in terms of reduction of PSMA-positive tumor burden as assessed by ⁶⁸Ga-PSMA-l 1 PET/CT imaging (or other PSMA radiographic PET tracer) relative to PSMA-positive tumor burden prior to the start of treatment, percentage of patients achieving a circulating tumor cell (CTC) response, duration of response to treatment, time to progression of disease, progression-free survival (PFS), and overall survival (OS). In certain embodiments, administration of the PSMA x CD3 bispecific T- cell engaging molecule according to the methods of the invention increases the duration of response to treatment, time to progression of disease, PFS, and/or OS as compared to the duration of response to treatment, time to progression of disease, PFS, and/or OS observed for a standard chemotherapy regimen (e.g. a taxane chemotherapy regimen) or standard androgen deprivation therapy regimen (e.g. with abiraterone, enzalutamide, apalutamide, or darolutamide). [0042] In one aspect, the methods of the invention comprise administering to a patient a therapeutically effective dose of a PSMA x CD3 bispecific T-cell engaging molecule. A “therapeutically effective dose” or “therapeutic dose” refers to an amount sufficient to treat or ameliorate prostate cancer or one or more of its symptoms, particularly a state or symptoms associated with prostate cancer, or otherwise prevent, hinder, retard or reverse the progression of prostate cancer or any other undesirable symptom associated with prostate cancer in any way whatsoever. Suitable dosages of the PSMA x CD3 bispecific T-cell engaging molecule for each of the initiation and maintenance cycles are described in more detail herein. In certain embodiments, a therapeutically effective dose of a PSMA x CD3 bispecific T-cell engaging molecule is an amount sufficient to induce remission of prostate cancer in the patient. In these and other embodiments, a therapeutically effective dose of a PSMA x CD3 bispecific T-cell engaging molecule is an amount sufficient to prevent or delay metastasis of prostate cancer in the patient. In yet other embodiments, a therapeutically effective dose of a PSMA x CD3 bispecific T-cell engaging molecule is an amount sufficient to prolong or increase the survival of a patient diagnosed with prostate cancer. In still other embodiments, a therapeutically effective dose of a PSMA x CD3 bispecific T-cell engaging molecule is an amount sufficient to prevent or delay the progression of prostate cancer in the patient. A therapeutically effective dose can be administered in one or more administrations.

[0043] Generally, the methods of the invention comprise administering a PSMA x CD3 bispecific T-cell engaging molecule to the patient in one or more treatment cycles. A “treatment cycle” or “cycle” refers to a period of administration of the bispecific T-cell engaging molecule at specific dosages and dosing intervals. According to the methods of the invention, a patient can receive multiple treatment cycles (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more cycles). The treatment cycles can be administered to the patient consecutively with no break or period without administration of the bispecific T-cell engaging molecule between the cycles. Alternatively, a period without administration of the bispecific T-cell engaging molecule (e.g. a “treatment-free period” or “break”) can be employed between the treatment cycles. The length of the treatment-free period can be adjusted based on the patient’s characteristics and/or response to treatment.

[0044] In a preferred embodiment, the methods of the invention comprise administering a PSMA x CD3 bispecific T-cell engaging molecule to the patient in at least one initiation cycle and at least one maintenance cycle. An initiation cycle is preferably administered to a patient as the first treatment cycle when the patient begins a course of treatment with the PSMA x CD3 bispecific T-cell engaging molecule. An initiation cycle may also be administered to a patient when the patient re-starts a course of treatment with the PSMA x CD3 bispecific T-cell engaging molecule, for example, following a treatment-free period or a relapse or progression of prostate cancer. Although administration of one initiation cycle will typically be sufficient, in some embodiments of the methods of the invention, administration of two or more initiation cycles is contemplated. In one particular embodiment, only one initiation cycle is administered to the patient.

[0045] As used herein, an “initiation cycle” is a treatment cycle in which the bispecific T-cell engaging molecule is administered at two or more different doses at a dosing frequency designed to minimize adverse events associated with CRS while enabling exposure of the patient to a therapeutically effective dose of the PSMA x CD3 bispecific T-cell engaging molecule in the shortest time possible. In some embodiments, at least one of the two or more different doses administered during the initiation cycle is lower than a therapeutically effective dose but is a dose that is sufficient to increase the proportion of activated peripheral T-cells in the patient (e.g. increases the proportion of CD69+CD8+ peripheral T-cells) relative to the proportion of activated T-cells in the patient prior to receiving the dose of the bispecific T-cell engaging molecule. Without being bound by theory, a subtherapeutic dose is believed to prime the patient’s T-cells (e.g. to release cytokines) such that administration of a subsequent greater dose or therapeutic dose of the bispecific T-cell engaging molecule produces an attenuated increase in cytokine secretion thereby reducing the occurrence or severity of CRS responses in the patient. Thus, in certain embodiments, the initiation cycle comprises administering the bispecific T-cell engaging molecule at one or more priming doses and a target dose. As used herein, the term “priming dose” refers to a dose or amount of a PSMA x CD3 bispecific T-cell engaging molecule that primes a patient for administration of a greater dose of the bispecific T-cell engaging molecule such that the greater dose produces no CRS response or a reduced CRS response in the patient. In some embodiments, the priming dose is sufficient to produce an increase of about 10% to about 50% of activated peripheral T cells (e.g. CD69+CD8+ T cells) in the patient’s blood, for example as assessed by standard fluorescence activated cell sorting methods.

[0046] In certain embodiments of the methods of the invention, the initiation cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at one or more priming doses and a target dose, wherein the target dose is greater than the one or more priming doses. The amounts of the one or more priming doses may vary depending on the specific PSMA x CD3 bispecific T-cell engaging molecule employed in the treatment method, the grade or stage of prostate cancer to be treated, and one or more patient characteristics, such as age, co-morbidities, and other concomitant medications. Suitable priming doses of the PSMA x CD3 bispecific T-cell engaging molecule include, but are not limited to, doses of about 3 pg to about 300 pg, about 10 pg to about 300 pg, about 10 pg to about 150 pg, about 15 pg to about 200 pg, about 30 pg to about 180 pg, about 60 pg to about 300 pg, about 10 pg to about 90 pg, or about 10 pg to about 60 pg. In some embodiments, the one or more priming doses are about 10 pg to about 300 pg. In other embodiments, the one or more priming doses are about 10 pg to about 90 pg.

[0047] The term “target dose” is a dose intended to be a therapeutically effective dose. Like the amounts of the priming doses, the amounts of the target dose or therapeutic dose may vary depending on the characteristics of the patient to be treated, grade or stage of prostate cancer diagnosed in the patient, and specific PSMA x CD3 bispecific T-cell engaging molecule administered to the patient. In some embodiments, the target dose or therapeutic dose of the PSMA x CD3 bispecific T-cell engaging molecule administered to the patient according to the methods of the invention will be greater than any priming dose of the bispecific T-cell engaging molecule previously administered to the patient. Exemplary ranges of target doses or therapeutic doses of the PSMA x CD3 bispecific T-cell engaging molecule include, but are not limited to, doses of about 30 pg to about 1800 pg, about 90 pg to about 1800 pg, about 300 pg to about 900 pg, about 300 pg to about 600 pg, about 800 pg to about 1600 pg, about 600 pg to about 1200 pg, or about 150 pg to about 400 pg. In one embodiment, the target dose or therapeutic dose is about 30 pg to about 1800 pg. In another embodiment, the target dose or therapeutic dose is about 90 pg to about 1800 pg. In certain embodiments, the target dose or therapeutic dose is less than about 1 mg. For instance, in some embodiments, the target dose or therapeutic dose is about 300 pg to about 900 pg. In other embodiments, the target dose or therapeutic dose is about 300 pg to about 600 pg.

[0048] The one or more priming doses administered prior to the administration of the target dose during the initiation cycle can be the same or different. For example, in one embodiment, the same dose of the PSMA x CD3 bispecific T-cell engaging molecule can be administered as a priming dose at each dosing interval on one or more occasions prior to administration of the target dose during the initiation cycle. In alternative embodiments, the one or more priming doses administered prior to the administration of the target dose during the initiation cycle may change from one dosing interval to the next. For instance, in some embodiments, the priming dose of the PSMA x CD3 bispecific T-cell engaging molecule may increase at one or more subsequent dosing intervals as a series of increasing dose steps. Such a step dosing regimen can be employed in embodiments in which two or more priming doses are administered prior to administration of the target dose during the initiation cycle and may comprise one or more dosage steps (e.g. one or more dose increases). For example, in one embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, followed by administration of a second priming dose, followed by administration of the target dose, wherein the second priming dose is greater than the first priming dose and the target dose is greater than the second priming dose. In another embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, followed by administration of a second priming dose, followed by administration of a third priming dose, followed by administration of the target dose, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose. One or more dosage steps between the priming doses can be used, for example, 2, 3, 4, or more dosage steps. In some embodiments, the step dosing regimen employed during the initiation cycle may comprise two dosage steps (i.e. three different doses administered including two priming doses and the target dose). In other embodiments, the step dosing regimen employed during the initiation cycle may comprise three dosage steps (i.e. four different doses administered including three priming doses and the target dose). Administration of a single priming dose followed by administration of a greater target dose during the initiation cycle may also be considered a step dosing regimen with one dosage step (i.e. two different doses administered).

[0049] In embodiments in which a step dosing regimen is employed during the initiation cycle, the step doses may increase proportionally over the dosing range. In alternative embodiments, the step doses may increase in smaller or larger steps over the dosing range, e.g. small steps at the earlier step doses and larger steps at the later step doses. In certain embodiments in which a step dosing regimen is employed during the initiation cycle, the initiation cycle comprises administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule at a priming dose from about 10 pg to about 60 pg followed by administration of a target dose of the bispecific T-cell engaging molecule from about 30 pg to about 1800 pg, wherein the target dose is greater than the priming dose. In embodiments in which the step dosing regimen comprises two dosage steps (i.e. three different doses administered including two priming doses and the target dose), the initiation cycle comprises administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose from about 10 pg to about 60 pg, followed by administration of a second priming dose from about 30 pg to about 180 pg, followed by administration of a target dose from about 30 pg to about 1800 pg, wherein the second priming dose is greater than the first priming dose and the target dose is greater than the second priming dose. In other embodiments in which the step dosing regimen comprises three dosage steps (i.e. four different doses administered including three priming doses and the target dose), the initiation cycle comprises administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose from about 10 pg to about 60 pg, followed by administration of a second priming dose from about 30 pg to about 180 pg, followed by administration of a third priming dose from about 60 pg to about 300 pg, followed by administration of a target dose from about 30 pg to about 1800 pg, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose. In any of the embodiments described above, the target dose of the PSMA x CD3 bispecific T-cell engaging molecule may be from about 90 pg to about 1800 pg, from about 300 pg to about 900 pg, or from about 300 pg to about 600 pg.

[0050] Any of the doses of a PSMA x CD3 bispecific T-cell engaging molecule described herein for administration during the initiation cycle can be administered at a dosing interval of at least 7 days. For instance, in one embodiment, the initiation cycle comprises administering one or more priming doses and a target dose of a PSMA x CD3 bispecific T-cell engaging molecule once every 7 days (e.g. QW or weekly dosing). In another embodiment, the initiation cycle comprises administering one or more priming doses and a target dose of a PSMA x CD3 bispecific T-cell engaging molecule once every 14 days (e.g. Q2W or biweekly dosing). In certain embodiments, during the initiation cycle the one or more priming doses of the PSMA x CD3 bispecific T-cell engaging molecule are administered at a weekly interval and the target doses are subsequently administered at a longer dosing interval, e.g. biweekly. In one such embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at a priming dose and a target dose, where the target dose is first administered about 7 days after the priming dose and the target dose is administered a second time at least 14 days after the first administration of the target dose. By way of illustration, such a dosing regimen comprises administering the bispecific T-cell engaging molecule at a priming dose on day 1 (DI) of the cycle, administering a target dose of the bispecific T-cell engaging molecule for a first time on day 8 (D8) of the cycle, and administering the target dose of the bispecific T-cell engaging molecule for a second time on day 22 (D22) of the cycle. In some embodiments in which a step dosing regimen is employed during the initiation cycle, the dose of the bispecific T-cell engaging molecule can be increased at each dosing interval, such as a weekly dosing interval, until the target dose is reached. For example, in some such embodiments, the initiation cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose on DI of the cycle, administering a second priming dose, which is greater than the first priming dose, on D8 of the cycle, and administering a target dose, which is higher than the second priming dose, on day 15 (DI 5) of the cycle. In other such embodiments, the initiation cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a first priming dose on DI of the cycle, administering a second priming dose, which is greater than the first priming dose, on D8 of the cycle, administering a third priming dose, which is greater than the second priming dose, on DI 5 of the cycle, and administering a target dose, which is higher than the third priming dose, on D22 of the cycle. [0051] In certain embodiments of the methods of the invention, the duration of the initiation cycle (e.g. first period of time) is from about 14 days to about 56 days, for example, from about 14 days to about 28 days, from about 21 days to about 42 days, from about 28 days to about 49 days, or from about 21 days to about 28 days. Thus, in some embodiments, the initiation cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at one or more of the doses described herein at a dosing interval of at least 7 days for a first period of time, wherein the first period of time is about 21 days to about 28 days. In one embodiment of the methods of the invention, the initiation cycle comprises administering the bispecific T-cell engaging molecule at one or more of the doses described herein once per week (e.g. weekly) for 21 days. In another embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at one or more of the doses described herein once per week (e.g. weekly) for 28 days. In yet another embodiment, the initiation cycle comprises administering the bispecific T-cell engaging molecule at step doses described herein once per week (e.g. weekly) until the target dose is reached and subsequently administering a target dose of the bispecific T-cell engaging molecule once every 14 days (e.g. biweekly), wherein the duration of the initiation cycle is 42 to 49 days.

[0052] The methods of the invention comprise administering at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule to the patient after administration of one or more initiation cycles. As used herein, a “maintenance cycle” is a treatment cycle in which the bispecific T-cell engaging molecule is administered at a dosing frequency designed to maintain a threshold level of exposure of the PSMA x CD3 bispecific T-cell engaging molecule at therapeutic levels in the patient. In some embodiments, the dosing frequency employed in the maintenance cycle is lower than the dosing frequency employed in the initiation cycle (i.e. the dosing interval in the maintenance cycle is longer than the dosing interval in the initiation cycle). In certain embodiments, the maintenance cycle is administered immediately after the completion of one or more initiation cycles. Accordingly, in such embodiments, there are no treatment-free periods or breaks between the end of the initiation cycle and the start of the maintenance cycle. In one such embodiment, the maintenance cycle is administered the following day after completing the initiation cycle. In other embodiments, there is a treatment-free period or break between the completion of the initiation cycle and the administration of the maintenance cycle. Preferably, the treatment-free period between the initiation cycle and the maintenance cycle is no longer than the dosing interval employed in the maintenance cycle. In one embodiment, the maintenance cycle is administered about 7 days following completion of the initiation cycle. In another embodiment, the maintenance cycle is administered about 14 days following completion of the initiation cycle. [0053] Multiple maintenance cycles (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles) can be administered to the patient depending on the desired duration of treatment for that patient. For instance, the patient may receive maintenance cycles of the PSMA x CD3 bispecific T-cell engaging molecule until the patient achieves a desired level of response, such as a complete response 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 periods between the maintenance cycles. If a treatment interruption is necessary, ideally the duration of the treatment-free period will be no greater than twice the dosing interval employed in the maintenance cycle. By way of example, if the dosing interval employed in the maintenance cycle is once every 14 days (e.g. biweekly), the treatment- free period between maintenance cycles will preferably be about 28 days or less.

[0054] In certain embodiments of the methods of the invention, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at any of the target doses as described herein once every 7 days (e.g. weekly, QW dosing) or once every 14 days (e.g. biweekly, Q2W dosing). For instance, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose or therapeutic dose from about 30 pg to about 1800 pg, about 90 pg to about 1800 pg, about 300 pg to about 900 pg, about 300 pg to about 600 pg, about 800 pg to about 1600 pg, about 600 pg to about 1200 pg, or about 150 pg to about 400 pg. In one embodiment, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose or therapeutic dose from about 30 pg to about 1800 pg once every 14 days. In another embodiment, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose or therapeutic dose from about 90 pg to about 1800 pg once every 14 days. In certain embodiments, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose or therapeutic dose from about 300 pg to about 900 pg once every 14 days. In other embodiments, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose or therapeutic dose from about 300 pg to about 600 pg once every 14 days. [0055] Preferably, the target dose of the bispecific T-cell engaging molecule administered during the maintenance cycle is the same at each weekly or biweekly dosing interval (e.g. a fixed dose for the entire maintenance cycle). In these and other embodiments, the target dose and dosing frequency of the bispecific T-cell engaging molecule administered during the maintenance cycle is the same from one maintenance cycle to the next maintenance cycle. In one particular embodiment of the methods of the invention, the maintenance cycle comprises administering the target dose of a PSMA x CD3 bispecific T-cell engaging molecule once per week (e.g. once every 7 days, weekly, or QW dosing). In another particular embodiment, the maintenance cycle comprises administering the target dose of a PSMA x CD3 bispecific T-cell engaging molecule once every other week (e.g. once every 14 days, biweekly, or Q2W dosing).

[0056] According to some embodiments of the methods of the invention, the duration of the maintenance cycle (e.g. second period of time) is from about 14 days to about 60 days, for example, from about 14 days to about 28 days, from about 21 days to about 42 days, from about 28 days to about 49 days, from about 28 days to about 56 days, or from about 21 days to about 28 days. Thus, in some embodiments, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose described herein once every 14 days (e.g. biweekly) for a second period of time, wherein the second period of time is about 28 days. In other embodiments, the maintenance cycle comprises administering a PSMA x CD3 bispecific T-cell engaging molecule at a target dose described herein once every 14 days (e.g. biweekly) for a second period of time, wherein the second period of time is about 56 days.

[0057] In certain embodiments, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises:

(a) administering the bispecific T-cell engaging molecule at a priming dose of about 10 pg to about 30 pg and a target dose of about 30 pg to about 90 pg, wherein the target dose is greater than the priming dose and is administered about 7 days after the priming dose; and

(b) administering the target dose of the bispecific T-cell engaging molecule a second time at least 14 days after the first administration of the target dose in (a); and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of about 30 pg to about 90 pg once every 14 days (e.g. biweekly; Q2W) for 28 days, wherein the maintenance cycle is administered about 14 days after the administration of the last dose in the initiation cycle. An exemplary dosing schedule according to these embodiments comprises administration of a priming dose (e.g. 10 pg) of the bispecific T- cell engaging molecule on day 1 (DI) and administration of a target dose (e.g. 90 pg) on day 8 (D8) and day 22 (D22) of a 28-day initiation cycle, followed by a treatment-free period of 7 days, followed by administration of the target dose (e.g. 90 pg) of the bispecific T-cell engaging molecule on day 1 (DI) and day 15 (D15) of a 28-day maintenance cycle. Thus, according to this dosing regimen, for the 56-day period encompassing both the 28-day initiation cycle and the 28- day maintenance cycle and starting with the first dose of the initiation cycle, the patient would be administered the bispecific T-cell engaging molecule on each of DI, D8, D22, day 36 (D36), and day 50 (D50). In some such embodiments, the priming dose is about 10 pg and the target dose is about 30 pg. In other such embodiments, the priming dose is about 10 pg and the target dose is about 90 pg.

[0058] In some embodiments, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T- cell engaging molecule, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose of about 10 pg to about 30 pg, a second priming dose of about 90 pg to about 180 pg, and a target dose of about 300 pg to about 900 pg, wherein the second priming dose is administered about 7 days after the first priming dose and the target dose is administered about 7 days after the second priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of about 300 pg to about 900 pg once every 14 days (e.g. biweekly; Q2W) for 28 days, wherein the maintenance cycle is administered about 14 days after the administration of the last dose in the initiation cycle. An exemplary dosing schedule according to these embodiments, in which a two-step dosing regimen is employed during the initiation cycle, comprises administration of the bispecific T-cell engaging molecule at a first priming dose (e.g. 10 pg) on DI, a second priming dose (e.g. 90 pg) on D8, and a target dose (e.g. 300 pg) on D15 of a 28-day initiation cycle, followed by administration of the target dose (e.g. 300 pg) of the bispecific T-cell engaging molecule on DI and D15 of a 28-day maintenance cycle, wherein the maintenance cycle is administered the following day after completing the 28-day initiation cycle. Thus, according to this dosing regimen, for the 56-day period encompassing both the 28-day initiation cycle and the 28-day maintenance cycle and starting with the first dose of the initiation cycle, the patient would be administered the bispecific T-cell engaging molecule on each of DI, D8, DI 5, day 29 (D29), and day 43 (D43).

[0059] In one embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 10

M^g; administering the bispecific T-cell engaging molecule at a second priming dose of about 90 pg about 7 days (e.g. a week) after the administration of the first priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 300 pg about 7 days (e.g. a week) after the administration of the second priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 300 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle.

[0060] In another embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 30 ktg; administering the bispecific T-cell engaging molecule at a second priming dose of about 90 pg about 7 days (e.g. a week) after the administration of the first priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 300 pg about 7 days (e.g. a week) after the administration of the second priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 300 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle. [0061] In yet another embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 30 M^g; administering the bispecific T-cell engaging molecule at a second priming dose of about 90 pg about 7 days (e.g. a week) after the administration of the first priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 600 pg about 7 days (e.g. a week) after the administration of the second priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 600 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle.

[0062] In still another embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 10 ktg; administering the bispecific T-cell engaging molecule at a second priming dose of about 90 pg about 7 days (e.g. a week) after the administration of the first priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 900 pg about 7 days (e.g. a week) after the administration of the second priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 900 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle.

[0063] In another embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 30 ktg; administering the bispecific T-cell engaging molecule at a second priming dose of about 90 pg about 7 days (e.g. a week) after the administration of the first priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 900 pg about 7 days (e.g. a week) after the administration of the second priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 900 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle. [0064] In other embodiments, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T- cell engaging molecule, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose of about 10 pg to about 45 pg, a second priming dose of about 30 pg to about 110 pg, a third priming dose of about 90 pg to about 180 pg, and a target dose of about 300 pg to about 900 pg, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose, the third priming dose is administered about 7 days after the second priming dose, and the target dose and is administered about 7 days after the third priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of about 300 pg to about 900 pg once every 14 days (e.g. biweekly; Q2W) for 28 days, wherein the maintenance cycle is administered about 14 days after the administration of the last dose in the initiation cycle. An exemplary dosing schedule according to these embodiments, in which a three-step dosing regimen is employed during the initiation cycle, comprises administration of the bispecific T-cell engaging molecule at a first priming dose (e.g. 10 pg) on DI, a second priming dose (e.g. 30 pg) on D8, a third priming dose (e.g. 90 pg) on DI 5, and a target dose (e.g. 900 pg) on D22 of a 28-day initiation cycle, followed by a treatment-free period of 7 days, followed by administration of the target dose (e.g. 900 pg) of the bispecific T-cell engaging molecule on DI and DI 5 of a 28-day maintenance cycle. Thus, according to this dosing regimen, for the 56-day period encompassing both the 28-day initiation cycle and the 28-day maintenance cycle and starting with the first dose of the initiation cycle, the patient would be administered the bispecific T-cell engaging molecule on each of DI, D8, DI 5, D22, D36, and D50.

[0065] In one embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 10

M^g; administering the bispecific T-cell engaging molecule at a second priming dose of about 30 pg about 7 days (e.g. a week) after the administration of the first priming dose; administering the bispecific T-cell engaging molecule at a third priming dose of about 90 pg about 7 days (e.g. a week) after the administration of the second priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 900 pg about 7 days (e.g. a week) after the administration of the third priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 900 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle.

[0066] In another embodiment, the methods of the invention comprise administering to the patient at least one initiation cycle and at least one maintenance cycle of a PSMA x CD3 bispecific T-cell engaging molecule, wherein the initiation cycle comprises: administering the bispecific T-cell engaging molecule at a first priming dose of about 10 ktg; administering the bispecific T-cell engaging molecule at a second priming dose of about 50 pg about 7 days (e.g. a week) after the administration of the first priming dose; administering the bispecific T-cell engaging molecule at a third priming dose of about 150 pg about 7 days (e.g. a week) after the administration of the second priming dose; and administering the bispecific T-cell engaging molecule at a target dose of about 300 pg about 7 days (e.g. a week) after the administration of the third priming dose; and wherein the maintenance cycle comprises administering the bispecific T-cell engaging molecule at the target dose of 300 pg once every 14 days (e.g. once every other week) for 28 days, wherein the maintenance cycle is administered about 14 days (e.g. two weeks) following administration of the target dose in the initiation cycle.

[0067] In certain embodiments of the methods of the invention, one or more premedications can be administered to the patient prior to the administration of a first dose of a PSMA x CD3 bispecific T-cell engaging molecule in the initiation cycle. In some embodiments, the premedication is administered to the patient prior to administration of each dose of the bispecific T-cell engaging molecule in the initiation cycle. The premedication may also be administered to the patient prior to administration of one or more doses of the bispecific T-cell engaging molecule in one or more maintenance cycles. In some embodiments, the premedication is only administered to the patient prior to administration of one or more doses during the initiation cycle and is not administered to the patient prior to administration of any dose of the bispecific T-cell engaging molecule in a subsequent treatment cycle (e.g. a maintenance cycle). It is envisaged that “prior to”, in this specific context, means within 72 hours, 48 hours, 36, hours, 24 hours, 18 hours, 16 hours, 12 hours, 6 hours, 5 hours, 4 hours, or 3 hours, and preferably within 120, 90, 60 or 30 minutes before the start of administration of the bispecific T-cell engaging molecule. Depending on the type of premedication used and the route by which it is administered, the premedication may e.g. be administered 30-120 or 30-60 minutes prior to start of administration of the bispecific T-cell engaging molecule. The premedication may be administered e.g. to prevent or reduce severity of infusion-related reactions and/or to prevent or reduce severity of cytokine release syndrome or its symptoms.

[0068] In some embodiments, the premedication is an antihistamine. The antihistamine can be administered orally or intravenously and can be administered at a dose equivalent to diphenhydramine 50 mg i.v. Suitable antihistamines that can be administered as a premedication include, but are not limited to, antihistamines of oral, parenteral or rectal route such as: 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), dexchlorpheniramine (maximum dose e.g. 15 mg/day), diphenhydramine (maximum dose e.g. 350 mg/per 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).

[0069] In other embodiments, 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 medication) is the most important human glucocorticoid. A variety of synthetic glucocorticoids, some far more potent than cortisol, have been created for therapeutic use. Cortisol is the standard of comparison for glucocorticoid potency. One example for commonly prescribed replacement steroid equivalents may be prednisone (5 mg) = cortisone (25 mg) = dexamethasone (0.75 mg) = hydrocortisone (20 mg) = methylprednisolone (4 mg). These doses indicate the equivalent pharmacologic dose of systemic glucocorticoids. The glucocorticoid can be administered orally or intravenously and can be administered at a dose equivalent to 4-20 mg dexamethasone i.v. (the equivalence referring to the glucocorticoid potency). The dose of glucocorticoid can be the same at each administration (i.e. at each time the glucocorticoid premedication is administered). Alternatively, the dose of glucocorticoid can be reduced in subsequent administrations, e.g. by 50% of the previous dose, if there are no or minimal signs of infusion reactions and/or CRS symptoms following the previous administration of the bispecific T-cell engaging molecule. In certain embodiments, glucocorticoids are only administered as premedications during the initiation cycle and are not administered in subsequent treatment cycles (e.g. maintenance cycles).

[0070] Examples of glucocorticoids to be used as a 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, as well as combinations and/or pharmaceutically acceptable derivatives thereof. The different glucocorticoids may be used alone or in combination. Dexamethasone, prednisone and prednisolone are preferred glucocorticoids for use as a premedication according to the methods of the invention. In certain embodiments of the methods of the invention, the glucocorticoid administered to the patient prior to administration of one or more (or all) doses of the bispecific T-cell engaging molecule during the initiation cycle and/or maintenance cycle is dexamethasone. Dexamethasone can be administered at a dose of about 4-20 mg, 6-18 mg, 8-16 mg, about 16 mg, or about 8 mg at each administration. In some embodiments of the methods of the invention, dexamethasone is administered to the patient prior to the administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In these and other embodiments, dexamethasone is orally administered to the patient at a dose of about 8 mg about 6-16 hours prior to administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In other embodiments, dexamethasone is intravenously administered to the patient at a dose of about 8 mg within one hour prior to administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In still other embodiments, the methods of the invention further comprise administering during the initiation cycle an 8 mg dose of dexamethasone orally (or equivalent dose of other glucocorticoid) to the patient about 6-16 hours prior to administration of each dose of the bispecific T-cell engaging molecule and administering an 8 mg dose of dexamethasone intravenously (or equivalent dose of other glucocorticoid) to the patient within one hour prior to administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule.

[0071] In certain embodiments, the premedication can be an IL-6 receptor antagonist, such as tocilizumab. Tocilizumab has been reported to effectively reduce or reverse symptoms of CRS induced by T cell-engaging therapies. See, e.g., Maude et al., Cancer J., Vol. 20: 119-122, 2014. Tocilizumab can be administered at a dose of about 1 mg/kg to about 20 mg/kg body weight, about 8 mg/kg to about 12 mg/kg body weight, or about 4 mg/kg to about 8 mg/kg body weight. Tocilizumab can be administered about 1 hour to about 2 hours prior to each dose of the PSMA x CD3 bispecific T-cell engaging molecule in the initiation cycle and/or one or more maintenance cycles. Additionally or alternatively, tocilizumab can be administered immediately after each dose of the PSMA x CD3 bispecific T-cell engaging molecule in the initiation cycle and/or one or more maintenance cycles. In some embodiments, tocilizumab is administered about 2 hours prior to each dose of the PSMA x CD3 bispecific T- cell engaging molecule in the initiation cycle. Other antagonists of IL-6/IL-6 receptor signaling, such as siltuximab, olokizumab, clazakizumab, sarilumab, and sirukumab, can be used as a premedication according to the methods of the invention to reduce the occurrence or severity of CRS.

[0072] In some embodiments, the premedication is a tumor necrosis factor alpha (TNF-alpha) antagonist. CRS symptoms have been previously reported to be mediated in part by release of TNF-alpha (Lee et al., Blood, Vol. 124: 188-195, 2014; Grupp et al., N Engl J Med., Vol. 368: 1509-1518, 2013). Recent studies have suggested that treatment with TNF-alpha antagonists prior to administration of immunotherapy agents may mitigate CRS symptoms (Li et al., Sci Transl Med., Vol. 11(508), 2019; Lee et al., 2014, supra, Grupp et al., 2013, supra). Accordingly, in certain embodiments, the methods of the invention further comprise administering to the patient a TNF-alpha antagonist prior to administration of each dose of the bispecific T-cell engaging molecule during the initiation cycle and/or one or more maintenance cycles. Examples of TNF-alpha antagonists that can be used as a premedication include, but are not limited to, etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab. In particular embodiments of the methods of the invention, the TNF-alpha antagonist administered to the patient prior to administration of one or more (or all) doses of the bispecific T-cell engaging molecule during the initiation cycle and/or maintenance cycle is etanercept. Etanercept can be administered at a dose of about 10 mg to 100 mg, about 25 mg to about 75 mg, about 40 mg to about 60 mg, or about 50 mg at each administration and can be administered subcutaneously or intravenously. In some embodiments of the methods of the invention, etanercept is administered to the patient prior to the administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In some such embodiments, etanercept is subcutaneously administered to the patient at a dose of about 50 mg about 2 days prior to administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In other such embodiments, etanercept is subcutaneously administered to the patient at a dose of about 50 mg about 1 day prior to administration of each dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. [0073] A patient may be treated according to the methods of the invention for a set treatment period. A “treatment period” begins upon administration of a first dose of a PSMA x CD3 bispecific T-cell engaging molecule in an initiation cycle and ends upon administration of a final dose of a PSMA x CD3 bispecific T-cell engaging molecule in a maintenance cycle. The treatment period may be from about 3 months to about 36 months, from about 12 months to about 24 months, or from about 6 months to about 12 months. For instance, 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 other embodiments, the treatment period is about 9 months. In yet other embodiments, the treatment period is about 12 months. The treatment period 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 invention until the patient achieves a complete response or until evidence of prostate cancer is otherwise undetectable in the patient.

[0074] In some embodiments, the patients to be treated according to the methods of the invention may have failed or be intolerant to one or more prior 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 therapy if the patient’s cancer progresses (e.g. size of prostate tumors increases; an increase in the presence, number or size of metastatic lesions; elevations in blood levels of PSA) following a standard regimen of the therapy. A patient may also be considered to have failed a therapy if the patient is unable to tolerate the therapy or the therapy is contraindicated in the patient. As the terms are used herein, a patient is considered to be refractory or resistant to a therapy if the patient’s cancer does not respond or loses an initial response following continued administration of the therapy. As used herein, a patient is considered to have relapsed after a therapy if the signs and symptoms of prostate cancer (e.g. elevation of blood PSA levels, cancerous cells in prostate gland, appearance of metastatic lesions, etc.) return after the patient has experienced a remission from the disease.

[0075] In certain embodiments, the patients to be treated according to the methods of the invention have failed or are intolerant to one or more chemotherapy regimens. In related embodiments, the patients to be treated according to the methods of the invention are refractory or resistant to one or more chemotherapy regimens. Standard chemotherapy regimens for treating prostate cancer typically include regimens of mitoxantrone, estramustine, carboplatin, oxaliplatin, cisplatin, and taxane chemotherapy regimens, for example regimens with docetaxel, cabazitaxel, or paclitaxel. In one embodiment, the patient to be treated according to the methods of the invention has failed or is intolerant, refractory, or resistant to one or more taxane chemotherapy regimens. In another embodiment, the patient to be treated according to the methods of the invention has failed or is intolerant, refractory, or resistant to two or more taxane chemotherapy regimens. In such embodiments, the patient may have failed or is intolerant, refractory, or resistant to a docetaxel regimen and/or a cabazitaxel regimen. [0076] In other embodiments, the patients to be treated according to the methods of the invention have failed or are intolerant, refractory or resistant to one or more androgen deprivation therapies, including anti-androgen therapies. Androgen deprivation therapy includes, but is 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 anti-androgen therapies, 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 anti-androgen therapy, 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 anti-androgen therapies selected from abiraterone, enzalutamide, apalutamide, and darolutamide.

[0077] In certain other embodiments, the patients to be treated according to the methods of the invention have failed or are intolerant, refractory or resistant to a radioligand therapy. A radioligand therapy is an agent that comprises a radionuclide or radioactive isotope 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 radioligand therapy to which the patient is refractory or resistant is a PSMA-targeted radioligand therapy, for example comprising a radionuclide (e.g. lutetium- 177 (¹⁷⁷Lu), actinium-225 (²²⁵Ac), yttrium-90 (⁹⁰Y), or iodine- 131 (¹³¹I)) attached to a PSMA-targeted ligand, such as PSMA-11, PSMA-617, PSMA-1007, an anti-PSMA antibody (e.g. humanized antibody J591) or binding fragment thereof, or MIP-1095 ((S)-2-(3-((S)-l-carboxy-5-(3-(4-iodophenyl) ureido) pentyl) ureido)pentanedioic acid). PSMA-targeted radiotherapies include, but are not limited to, ¹⁷⁷Lu- PSMA-617, ²²⁵AC-PSMA-617, ²²⁵Ac-huJ591, ¹⁷⁷Lu-huJ591, ⁹⁰Y-huJ591, and ¹³¹I-MIP-1095. Other PSMA-targeted radiotherapies are described in Czerwinksa et al., Molecules, Vol.

25: 1743, 2020, which is hereby incorporated by reference in its entirety. In one embodiment, the patients to be treated according to the methods of the invention have failed or are intolerant, refractory or resistant to a ¹⁷⁷Lu-PSMA-617 radioligand therapy. In another embodiment, the patients to be treated according to the methods of the invention have failed or are intolerant, refractory or resistant to a ²²⁵Ac-PSMA-617 radioligand therapy. [0078] The methods described herein comprise administering to a patient a bispecific T-cell engaging molecule that specifically binds to PSMA and CD3. The term “T-cell engaging molecule” refers to a molecule that comprises at least one domain in which the structure is derived from or comprises the minimum structural features of an antibody, e.g., of a full-length immunoglobulin molecule, that allow for 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 comprises one or more binding domains, each of which will typically comprise the minimum structural requirements of an antibody that allow for specific target binding. This minimum requirement may, for example, be defined by the presence of at least three light chain “complementarity determining regions” or CDRs (i.e. CDRL1, CDRL2 and CDRL3 of a VL region) and/or three heavy chain CDRs (i.e. CDRH1, CDRH2 and CDRH3 of a VH region), and preferably all six CDRs from both the light and heavy chain variable regions. The T-cell engaging molecules according to the invention may comprise domains or regions (e.g. CDRs or variable regions) from monoclonal, chimeric, humanized and human antibodies.

[0079] Preferably, the T-cell engaging molecules used in the methods of the invention are proteins and comprise one or more polypeptide chains. A 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 according to the methods of the invention are single-chain polypeptides. In other embodiments, the T-cell engaging molecules administered according to the methods of the invention comprise two or more polypeptide chains - e.g. are polypeptide dimers or multimers. In certain embodiments, the T- cell engaging molecules administered according to the methods of the invention comprise four polypeptide chains, and may, e.g. have the format of an antibody or an immunoglobulin protein. [0080] As used herein, the term “antibody” generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (about 25 kDa each) and two heavy chain polypeptides (about 50-70 kDa each). 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 (K) or human lambda (X) 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), an immunoglobulin heavy chain constant domain 1 (CHI), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (p), delta (A), gamma (y), alpha (a), and epsilon (a), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG-class and IgA-class antibodies are further divided into subclasses, namely, IgGl, IgG2, IgG3, and IgG4, and IgAl and IgA2, respectively. The heavy chains in IgG, IgA, and IgD antibodies have three constant domains (CHI, CH2, and CH3), whereas the heavy chains in IgM and IgE antibodies have four constant domains (CHI, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CHI domain (i.e. between the light and heavy chain) and between the hinge regions of the two antibody heavy chains.

[0081] 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 chain and light chain pair typically are aligned by the framework regions to form a structure that binds specifically to a specific epitope on the target protein (e.g., PSMA or CD3). From N-terminus to C-terminus, naturally-occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised for assigning numbers to 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 etal., 1989, Nature 342:878-883. The CDRs and FRs of a given antibody may be identified using this system. Other numbering systems for the amino acids in 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).

[0082] The T-cell engaging molecules used in the methods of the invention are preferably at least bispecific T-cell engaging molecules. The term “bispecific T-cell engaging molecule” refers to a molecule capable of specifically binding to two different antigens. In the context of the present invention, such bispecific T-cell engaging molecules specifically bind to PSMA (e.g. human PSMA) on the cell surface of target cells and CD3 (e.g. human CD3) on the cell surface of T cells. The term “PSMA x CD3 bispecific T-cell engaging molecule” is used herein to refer to a bispecific T-cell engaging molecule that specifically binds to PSMA and CD3. A T-cell engaging molecule or binding domain thereof “specifically binds” to a target antigen when it has a significantly higher binding affinity for, and consequently is capable of distinguishing, that antigen compared to its affinity for other unrelated proteins, under similar binding assay conditions. T-cell engaging molecules or binding domains thereof that specifically bind an antigen may bind to that antigen with an equilibrium dissociation constant (KD) < 1 x 10'⁶ M. T- cell engaging molecules or binding domains thereof specifically bind antigen with “high affinity” when the KD is < 1 x 10'⁸ M. In one embodiment, the T-cell engaging molecules or binding domains thereof used in the methods of the invention bind to human PSMA and/or human CD3 with a KD of < 5 x 10'⁷ M. In another embodiment, the T-cell engaging molecules or binding domains thereof used in the methods of the invention bind to human PSMA and/or human CD3 with a KD of < 1 x 10'⁷ M. In yet another embodiment, the T-cell engaging molecules or binding domains thereof used in the methods of the invention bind to human PSMA and/or human CD3 with a KD of < 5 x 10'⁸ M. In another embodiment, the T-cell engaging molecules or binding domains thereof used in the methods of the invention bind to human PSMA and/or human CD3 with a KD of < 2 x 10'⁸ M. In certain embodiments, the T-cell engaging molecules or binding domains thereof used in the methods of the invention bind to human PSMA and/or human CD3 with a KD of < 1 x 10'⁸ M. In other embodiments, the T-cell engaging molecules or binding domains thereof used in the methods of the invention bind to human PSMA and/or human CD3 with a KD of < 1 X 10'⁹ M.

[0083] Affinity is determined using a variety of techniques, an example of which is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., BIAcore®-based assay). Using this methodology, the association rate constant (k_a in M' ¹) and the dissociation rate constant (kd in s'¹) can be measured. The equilibrium dissociation constant (KD in M) can then be calculated from the ratio of the kinetic rate constants (kd/ka). In some embodiments, affinity is determined by a kinetic method, such as a Kinetic Exclusion Assay (KinExA) as described in Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. Using a KinExA assay, the equilibrium dissociation constant (KD in M) and the association rate constant (k_a in M' ¹) can be measured. The dissociation rate constant (kd in s'¹) can be calculated from these values (KD X k_a). In other embodiments, affinity is determined by a bio-layer interferometry method, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278: 165-82, 2015 and employed in Octet® systems (Pall ForteBio). The kinetic (k_a and kd) and affinity (KD) constants can be calculated in real-time using the bio-layer interferometry method. In some embodiments, the T-cell engaging molecules or binding domains thereof described herein exhibit desirable characteristics such as binding avidity as measured by kd (dissociation rate constant) for human PSMA and/or human CD3 of about 10'², 10'³, 10'⁴, 10'⁵, 10'⁶,

10'⁹, 10'¹⁰ s'¹ or lower (lower values indicating higher binding avidity), and/or binding affinity as measured by KD (equilibrium dissociation constant) for human PSMA and/or human CD3 of about 10'⁷, 10'⁸, 10'⁹, 10'¹⁰, 10'¹¹ M or lower (lower values indicating higher binding affinity).

[0084] In some embodiments, bispecific T-cell engaging molecules used in the methods of the invention may be antibodies and have the general structure of a full-length immunoglobulin. For example, the bispecific T-cell engaging molecules may comprise two full-length antibody heavy chains and two full-length antibody light chains. In particular embodiments, the bispecific T-cell engaging molecules are heterodimeric antibodies (used interchangeably herein with “hetero immunoglobulins” or “hetero Igs”), which refer to antibodies comprising two different light chains and two different heavy chains. For instance, in some embodiments, the heterodimeric antibody comprises a light chain and heavy chain from an anti-PSMA antibody and a light chain and heavy chain from an anti-CD3 antibody.

[0085] The bispecific T-cell engaging molecules employed in the methods of the invention may also comprise 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 “r IgG” (“half antibody” consisting of a heavy chain and a light chain). Bispecific T-cell engaging molecules according to the invention may also comprise modified fragments of antibodies. Examples of such modified fragments include, but are not limited to, single-chain variable fragment (scFv), di-scFv or bi(s)- scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fabs, diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFv, tandem tri-scFv, “minibodies” exemplified by a structure which is as follows: (VH-VL-CH3)2, (scFv-CH3)2 , ((scFv)2-CH3 + CH3), ((SCFV)2-CH3) or (scFv-CH3-scFv)2, multibodies, such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies comprising merely one variable region, which might be VHH, VH or VL, that specifically binds to an antigen or target independently of other variable regions or domains.

[0086] In certain embodiments, the bispecific T-cell engaging molecules used in the methods of the invention are multivalent. The valency of the T-cell engaging molecule denotes the number of individual antigen-binding domains within the T-cell engaging molecule. For example, the terms “monovalent,” “bivalent,” and “tetraval ent” with reference to the T-cell engaging molecules in the context of the invention refer to T-cell engaging molecules with 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. a higher valency) than specificities. 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 bispecific T-cell engaging molecules used in the methods of the invention are bivalent. Thus, such bispecific, bivalent T-cell engaging molecules contain 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).

[0087] In some embodiments, the bispecific T-cell engaging molecules employed in the methods of the invention comprise a first binding domain that specifically binds to PSMA (e.g. human PSMA) and a second binding domain that specifically binds to CD3 (e.g. human CD3). As used herein, the term “antigen-binding domain,” which is used interchangeably with “binding domain,” refers to the region of the T-cell engaging molecule that contains the amino acid residues that interact with the antigen and confer on the T-cell engaging molecule its specificity and affinity for the antigen. In certain embodiments, one or more binding domains of the T-cell engaging molecules may be derived from an antibody or antigen-binding fragment thereof. For instance, the binding domains of the bispecific T-cell engaging molecules used in the methods of the invention may comprise one or more CDRs from the light and heavy chain variable regions of antibodies that specifically bind to human PSMA and/or human CD3. In some embodiments, the anti-PSMA binding domain of the bispecific T-cell engaging molecules comprises all six CDRs of the heavy and light chain variable regions of an anti-PSMA antibody described herein and the anti-CD3 binding domain of the bispecific T-cell engaging molecules comprises all six CDRs of the heavy and light chain variable regions of an anti-CD3 antibody described herein. In some embodiments, the binding domains (the anti-PSMA binding domain, the anti-CD3 binding domain or both) of the bispecific T-cell engaging molecules used in the methods of the invention comprise a Fab, a Fab', a F(ab')2, a Fv, a single-chain variable fragment (scFv), or a nanobody. In one embodiment, both binding domains of the bispecific T-cell engaging molecule are Fab fragments. In another embodiment, one binding domain of the bispecific T-cell engaging molecule is a Fab fragment and the other binding domain is a scFv. In yet another embodiment, both binding domains of the bispecific T-cell engaging molecule are scFvs.

[0088] As used in the context of the invention, an “antigen-binding fragment,” 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 a full-length heavy chain and/or light chain, but which is still capable of specifically binding to an antigen. An antigen-binding fragment includes, but is not limited to, a single-chain variable fragment (scFv), a nanobody (e.g. VH domain of camelid heavy chain antibodies; VHH fragment, see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a Fd fragment, and a CDR fragment, and can be derived from any mammalian source, such as human, mouse, rat, rabbit, or camelid. Antigen-binding fragments may compete for binding of a target antigen with an intact antibody and the fragments may be produced by the modification of intact antibodies (e.g. enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technologies or peptide synthesis. In some embodiments, the antigenbinding fragment comprises at least one CDR from an antibody that binds to the antigen, for example, the heavy chain CDR3 from an antibody that binds to the antigen. In other embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds to the antigen or all three CDRs from the light chain of an antibody that binds to the antigen. In still other embodiments, the antigen-binding fragment comprises all six CDRs from an antibody that binds to the antigen (three from the heavy chain and three from the light chain).

[0089] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with 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 (CHI) of the heavy chain. Thus, a “Fab fragment” is comprised of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CHI domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The “Fd fragment” comprises the VH and CHI domains from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0090] The “Fc fragment” or “Fc region” of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. In certain embodiments, the bispecific T-cell engaging molecules used in the methods of the invention comprise an Fc region from an immunoglobulin. The Fc region may be an Fc region from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises CH2 and CH3 domains from a human IgGl or human IgG2 immunoglobulin. The Fc region may retain effector function, such as Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function.

[0091] A “Fab¹ fragment” is a Fab fragment having at the C-terminus of the CHI domain one or more cysteine residues from the antibody hinge region.

[0092] A “F(ab')2 fragment” is a bivalent fragment including two Fab' fragments linked by a disulfide bridge between the heavy chains at the hinge region.

[0093] The “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 region (VH) and one immunoglobulin light chain variable region (VL) in tight, non-covalent association. It is in this configuration that the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-VL dimer. A single light chain or heavy chain variable region (or half of an Fv fragment comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site comprising both VH and VL. [0094] A “single-chain variable fragment” or “scFv fragment” comprises the VH and VL regions of an antibody, wherein these regions are present in a single polypeptide chain, and optionally comprising a peptide linker between the VH and VL regions that 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).

[0095] A “nanobody” is the heavy chain variable region of a heavy-chain antibody. Such variable domains are the smallest fully functional antigen-binding fragment 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 are naturally occurring in certain species of animals, such as nurse sharks, wobbegong sharks and Camelidae, such as camels, dromedaries, alpacas and llamas. The antigen-binding site is reduced to a single domain, the VHH domain, in these animals. These antibodies form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only having the structure H2L2 (referred to as “heavy-chain antibodies” or “HCAbs”). Camelized VHH reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CHI domain. Camelized VHH domains have been found to bind to antigen with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and possess high stability in solution (Ewert et al., Biochemistry, Vol. 41 :3628-36, 2002). Methods for generating antibodies having camelized heavy chains are described in, for example, U.S. 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 may provide a framework for a long penetrating loop structure.

[0096] In certain embodiments, the binding domains of the bispecific T-cell engaging molecules used in the methods of the invention comprise an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) of an antibody or antibody fragment which specifically binds to the desired antigen. For instance, the anti-PSMA binding domain of the bispecific T-cell engaging molecules of the invention comprises a VH region and VL region from an anti-PSMA antibody, such as any of the anti-PSMA antibodies or fragments thereof described herein, and the anti-CD3 binding domain comprises a VH region and VL region from an anti-CD3 antibody, such as any of the anti-CD3 antibodies or fragments thereof described herein. The binding domains that specifically bind to human PSMA or human CD3 can be derived from known antibodies to these antigens or from new antibodies or antibody fragments obtained by de novo immunization methods using the antigen proteins or fragments thereof, by phage display, or other methods described herein or known in the art. The antibodies from which the binding domains for the bispecific T-cell engaging molecules are derived can be monoclonal antibodies, recombinant antibodies, chimeric antibodies, human antibodies, or humanized antibodies. In certain embodiments, the antibodies from which the binding domains are derived are monoclonal antibodies. In these and other embodiments, the antibodies are human antibodies or humanized antibodies and can be of the IgGl-, IgG2-, IgG3-, or IgG4-type. [0097] The first binding domain of the bispecific T-cell engaging molecules used in the methods of the invention specifically binds to PSMA, preferably human PSMA. This binding domain is referred to herein as an anti-PSMA binding domain. PSMA (prostate-specific membrane antigen; also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I, or NAAG peptidase) is a type II membrane glycoprotein expressed primarily on prostate epithelial cells. More preferably, the first binding domain binds to PSMA on the surface of a target cell. The “target cell” can be any prokaryotic or eukaryotic cell expressing PSMA on its surface; preferably the target cell is a cell that is part of the human or animal body, such as a specific PSMA-expressing cancer or tumor cell. It is furthermore envisaged that the first binding domain of the bispecific T-cell engaging molecules binds to human PSMA, preferably to human PSMA on the surface of a target cell. It is also envisaged that the first binding domain binds to macaque PSMA, preferably to macaque PSMA on the surface of a target cell. Exemplary amino acid sequences for the mature polypeptides and extracellular domains of human PSMA and macaque PSMA are provided in Table 1 below.

Table 1. Sequences of human and macaque PSMA polypeptides

[0098] Examples of anti-PSMA binding domains from which the first binding domain of the bispecific T-cell engaging molecules used in the methods of the invention can be constructed or derived are described in WO 2010/037836 and WO 2017/134158, both of which are hereby incorporated by reference in their entireties. Light chain and heavy chain variable regions and associated CDRs of exemplary anti-human PSMA antibodies from which the anti-PSMA binding domain of the bispecific T-cell engaging molecules can be derived or constructed are set forth in Tables 2A and 2B, respectively.

Table 2A. Exemplary Anti-Human PSMA Antibody Light Chain Variable Region Amino Acid Sequences

Table 2B. Exemplary Anti-Human PSMA Antibody Heavy Chain Variable Region Amino Acid Sequences

[0099] The domain that specifically binds to human PSMA (e.g. the anti-PSMA binding domain) of the bispecific T-cell engaging molecules suitable for use in the methods of the invention may 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 instance, in some embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 to 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 to 19; and a CDRH3 comprising the sequence of SEQ ID NO: 20.

[0100] In some embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules comprise a light chain variable region comprising a CDRL1, a CDRL2, and a CDRL3, wherein: (a) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 9, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 10, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 11, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 12, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 13, respectively; (f) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 8 and 9, respectively; or (g) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 6, 8 and 9, respectively. In these and other embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules comprise a heavy chain variable region comprising a CDRH1, a CDRH2, and a CDRH3, wherein: (a) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 17 and 20, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 15, 18 and 20, respectively; or (d) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 19 and 20, respectively.

[0101] In certain embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules suitable for use in the methods of the invention comprise a light chain variable region comprising a CDRL1, a CDRL2, and a CDRL3 and a heavy chain variable region comprising a CDRH1, a CDRH2, and a CDRH3, wherein:

(a) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 9, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively;

(b) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 10, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively;

(c) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 11, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 17 and 20, respectively;

(d) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 12, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively;

(e) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 13, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively;

(f) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 11, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 15, 18 and 20, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 7 and 11, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively;

(h) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 5, 8 and 9, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 16 and 20, respectively; or

(i) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 6, 8 and 9, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 14, 19 and 20, respectively. In a preferred embodiment, the anti-PSMA binding domain of the bispecific T- cell engaging molecules used in the methods of the invention comprises (i) a light chain variable region 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 heavy chain variable region 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.

[0102] In some embodiments, the anti-PSMA binding domain of the bispecific T-cell engaging molecules used in the methods of the invention comprise 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 the antibodies described herein. The “variable region,” used interchangeably herein with “variable domain” (variable region of a light chain (VL), variable region of a heavy chain (VH)), refers to the region in each of the light and heavy immunoglobulin chains which is involved directly in binding of the antibody to the antigen. As discussed above, the regions of variable light and heavy chains have the same general structure and each region comprises four framework (FR) regions, the sequences of which are widely conserved, connected by three CDRs. The framework regions adopt a beta-sheet conformation and the CDRs may form loops connecting the beta-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form, together with the CDRs from the other chain, the antigen binding site. Thus, in some embodiments, the anti-PSMA binding domain of the bispecific T-cell engaging molecules according to the invention may comprise a light chain variable region selected from LV-01 to LV-12 (SEQ ID NOs: 21-32), as shown in Table 2A, and/or a heavy chain variable region selected from HV-01 to HV-07 (SEQ ID NOs: 33-39), as shown in Table 2B, and binding fragments, derivatives, and variants of these light chain and heavy chain variable regions.

[0103] 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 an anti-PSMA binding domain of the bispecific T-cell engaging molecules according to the invention. Examples of such combinations include, but are not limited to: (i) HV-01 and any one of LV-01, LV-02, LV-03, LV-04, LV-05 and LV-10; (ii) LV-03 and HV-02; (iii) LV-06 and HV-03; (iv) LV-07 and HV-04; (v) LV-08 and HV-05; (vi) LV-09 and HV-05; (vii) LV-11 and HV-06; and (viii) LV-12 and HV-07.

[0104] In certain embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 still other embodiments, the anti-PSMA binding domains of the bispecific T- cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 certain embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 yet another embodiment, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 still another embodiment, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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.

[0105] In some embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise a light chain variable region comprising a sequence of contiguous amino acids that differs from the sequence of a light chain variable region in Table 2A, i.e. a VL selected from LV-01 to LV-12, at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion or substitution of one amino acid, with the deletions, insertions and/or substitutions resulting in no more than 15 amino acid changes relative to the foregoing variable domain sequences. The light chain variable region in 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 sequences of SEQ ID NOs: 21 to 32 (i.e. the light chain variable regions in Table 2A).

[0106] In one embodiment, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise a light chain variable region comprising a sequence selected from SEQ ID NOs: 21-32.

[0107] In these and other embodiments, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence of contiguous amino acids that differs from the sequence of a heavy chain variable region in Table 2B, i.e., a VH selected from HV-01 to HV-07, at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion or substitution of one amino acid, with the deletions, insertions and/or substitutions resulting in no more than 15 amino acid changes relative to the foregoing variable domain sequences. The heavy chain variable region in 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 sequences of SEQ ID NOs: 33 to 39 (i.e. the heavy chain variable regions in Table 2B).

[0108] In one embodiment, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 33-39. In another embodiment, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 33-39. In yet another embodiment, the anti-PSMA binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 33-39.

[0109] The term “identity,” as used herein, refers to a 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 the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the 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 that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening 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. U.S.A. 89: 10915-10919) can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences. In calculating percent identity, the sequences being compared are aligned in a way that gives the largest match between the sequences.

[0110] The GCG program package is a computer program that can be used to determine percent identity, which package 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 the two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span,” as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. [OHl] Recommended parameters for determining percent identity for polypeptide or nucleotide sequences 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, supra, Gap Penalty: 12 (but with no penalty for end gaps) Gap Length Penalty: 4 Threshold of Similarity: 0

[0112] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

[0113] The second binding domain of the bispecific T-cell engaging molecules used in the methods of the invention specifically binds to CD3, preferably human CD3. This binding domain is referred to herein as an anti-CD3 binding domain. “CD3” (cluster of differentiation 3) is a T cell co-receptor composed of four chains. In mammals, the CD3 protein complex contains a CD3y (gamma) chain, a CD36 (delta) chain, and two CD3s (epsilon) chains. These four chains associate with the T cell receptor (TCR) and the so-called C, (zeta) chain to form the “T cell receptor complex” and to generate an activation signal in T lymphocytes. The CD3y (gamma), CD36 (delta), and CD3s (epsilon) chains are highly related cell-surface proteins of the immunoglobulin superfamily and each contain a single extracellular immunoglobulin domain. The intracellular tails of the CD3 molecules contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif (IT AM), which is essential for the signaling capacity of the TCR. The CD3 epsilon molecule is a polypeptide, which in humans is encoded by the CD3E gene which resides on chromosome 11. [0114] The redirected lysis of target cells via the recruitment of T cells by a T-cell engaging molecule which binds to CD3 on the T cell and to a target protein (e.g. PSMA) on the target cell (e.g. tumor cell) generally involves cytolytic synapse formation and delivery of perforin and granzymes. The engaged T cells are capable of serial target cell lysis, and are not affected by immune escape mechanisms interfering with peptide antigen processing and presentation, or clonal T cell differentiation; see, for example, WO 2007/042261.

[0115] In certain embodiments, the second binding domain of the bispecific T-cell engaging molecules used in the methods of the invention specifically binds to CD3 on the surface of a T cell, more preferably to human CD3 on the surface of a T cell. In some embodiments, the second binding domain of the bispecific T-cell engaging molecules specifically binds to CD3 epsilon, preferably human CD3 epsilon, e.g. human CD3 epsilon on the surface of a T cell. An exemplary amino acid sequence for 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 )

[0116] Examples of anti-CD3 binding domains from which the second binding domain of the bispecific T-cell engaging molecules used in the methods of the invention can be constructed or derived are described in WO 2007/042261 and WO 2008/119567, both of which are hereby incorporated by reference in their entireties. Light chain and heavy chain variable regions and associated CDRs of exemplary anti-human CD3 antibodies from which the anti-CD3 binding domain of the bispecific T-cell engaging molecules can be derived or constructed are set forth in Tables 3A and 3B, respectively.

Table 3A. Exemplary Anti-Human CD3 Antibody Light Chain Variable Region Amino Acid Sequences

Table 3B. Exemplary Anti-Human CD3 Antibody Heavy Chain Variable Region Amino Acid Sequences

[0117] The domain that specifically binds to human CD3 (e.g. the anti-CD3 binding domain) of the bispecific T-cell engaging molecules suitable for use in the methods of the invention may 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 instance, in some embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a CDRL1 comprising a sequence selected from SEQ ID NOs: 41 to 43; a CDRL2 comprising the sequence of SEQ ID NO: 44 or SEQ ID NO: 45; a CDRL3 comprising the sequence of SEQ ID NO: 46 or SEQ ID NO: 47; a CDRH1 comprising a sequence selected from SEQ ID NOs: 48 to 53; a CDRH2 comprising a sequence selected from SEQ ID NOs: 54 to 58; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 59 to 67.

[0118] In some embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules comprise a light chain variable region comprising a CDRL1, a CDRL2, and a CDRL3, wherein: (a) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 42, 45 and 46, respectively; or (c) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 43, 44 and 47, respectively. In these and other embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules comprise a heavy chain variable region comprising a CDRH1, a CDRH2, and a CDRH3, wherein: (a) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 48, 54 and 59, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 49, 55 and 60, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 50, 56 and 61, respectively; (d) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 51, 56 and 62, respectively; (e) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 52, 57 and 63, respectively; (f) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 49, 54 and 64, respectively; (g) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 53, 58 and 65, respectively; (h) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 52, 57 and 66, respectively; or (i) CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 50, 56 and 67, respectively.

[0119] In certain embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules suitable for use in the methods of the invention comprise a light chain variable region comprising a CDRL1, a CDRL2, and a CDRL3 and a heavy chain variable region comprising a CDRH1, a CDRH2, and a CDRH3, wherein:

(a) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 48, 54 and 59, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 49, 55 and

60, respectively;

(c) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 50, 56 and

61, respectively;

(d) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 51, 56 and

62, respectively;

(e) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 42, 45 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 52, 57 and

63, respectively;

(f) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 49, 54 and

64, respectively;

(g) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 42, 45 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 53, 58 and

65, respectively;

(h) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 41, 44 and 46, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 52, 57 and

66, respectively;

(i) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 43, 44 and 47, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 50, 56 and

67, respectively; or

(j) CDRL1, CDRL2, and CDRL3 have the sequence of SEQ ID NOs: 43, 44 and 47, respectively, and CDRH1, CDRH2, and CDRH3 have the sequence of SEQ ID NOs: 49, 55 and 60, respectively. In a preferred embodiment, the anti-CD3 binding domain of the bispecific T- cell engaging molecules used in the methods of the invention comprises (i) a light chain variable region 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 (ii) a heavy chain variable region 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.

[0120] The anti-CD3 binding domain of the bispecific T-cell engaging molecules according to the invention may comprise a light chain variable region selected from LV-101 to LV-103 (SEQ ID NOs: 68-70), as shown in Table 3A, and/or a heavy chain variable region selected from HV- 101 to HV-109 (SEQ ID NOs: 71-79), as shown in Table 3B, and binding fragments, derivatives, and variants of these light chain and heavy chain variable regions. Each of the light chain variable regions listed in Table 3 A may be combined with any of the heavy chain variable regions listed in Table 3B to form an anti-CD3 binding domain of the bispecific T-cell engaging molecules 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; and (x) LV-103 and HV-102.

[0121] In certain embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 still other embodiments, the anti-CD3 binding domains of the bispecific T- cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 certain embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 domains of the bispecific T-cell engaging molecules according to the invention comprise 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 preferred embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise 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.

[0122] In some embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a light chain variable region comprising a sequence of contiguous amino acids that differs from the sequence of a light chain variable region in Table 3A, i.e. a VL selected from LV-101 to LV-103, at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion or substitution of one amino acid, with the deletions, insertions and/or substitutions resulting in no more than 15 amino acid changes relative to the foregoing variable domain sequences. The light chain variable region in some anti-CD3 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 sequences of SEQ ID NOs: 68 to 70 (i.e. the light chain variable regions in Table 3A).

[0123] In one embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a light chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 68-70. In another embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a light chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 68-70. In yet another embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a light chain variable region comprising a sequence selected from SEQ ID NOs: 68-70.

[0124] In these and other embodiments, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence of contiguous amino acids that differs from the sequence of a heavy chain variable region in Table 3B, i.e., a VH selected from HV-101 to HV-109, at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion or substitution of one amino acid, with the deletions, insertions and/or substitutions resulting in no more than 15 amino acid changes relative to the foregoing variable domain sequences. The heavy chain variable region in some anti-CD3 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 sequences of SEQ ID NOs: 71 to 79 (i.e. the heavy chain variable regions in Table 3B). [0125] In one embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 71-79. In another embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 71-79. In yet another embodiment, the anti-CD3 binding domains of the bispecific T-cell engaging molecules according to the invention comprise a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 71-79.

[0126] According to certain embodiments, one or more of the binding domains of the bispecific T-cell engaging molecule used in the methods of the invention, are in the format of an scFv. In an scFv, the VH region and the VL region are arranged in the order VH-VL or VL-VH (from N- to C-terminus). It is envisaged that the VH and the VL regions of the first and/or the second binding domain are connected via a linker, preferably a peptide linker. In one embodiment of the first and/or second binding domain, the VH-region is positioned N-terminally of the linker, and the VL-region is positioned C-terminally of the linker. The linkers are preferably peptide linkers, more preferably short peptide linkers. Examples of suitable linkers include, but are not limited to: • 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)

• GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 86)

• GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87)

• GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (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)

[0127] In the present context, a “short” linker has between 2 and 50 amino acids, preferably between 3 and 35, between 4 and 30, between 5 and 25, between 6 and 20 or between 6 and 17 amino acids. The linker between 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 two variable regions of one or both binding domains may have a length between 8 and 16 amino acids, preferably between 10 and 15, and the linker between the two binding domains may have a length between 3 and 10 amino acids, preferably between 5 and 8. It is further envisaged that the peptide linkers are glycine/ serine linkers, such as those depicted in SEQ ID NOs: 81-92. In one embodiment, the anti-PSMA binding domain and/or the anti-CD3 binding domain of the bispecific T-cell engaging molecule according to the invention is an scFv comprising, from N-terminus to C-terminus, a VH region - peptide linker - VL region, where 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 and anti-CD3 binding domains (e.g. scFv domains) is the linker set forth in SEQ ID NO: 81 or SEQ ID NO: 91. Exemplary scFv domains for the anti-PSMA and anti-CD3 binding domains of the bispecific T- cell engaging molecules suitable for use in the methods of the invention are set forth in Table 4 below. Table 4. Exemplary Single-Chain Variable Fragment Binding Domains

[0128] In certain embodiments, the bispecific T-cell engaging molecules suitable for use in the methods of the invention comprise a first binding domain that specifically binds to 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 to 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 (e.g. anti-PSMA binding domain) of the bispecific T-cell engaging molecules comprises the amino acid sequence of SEQ ID NO: 104. In another preferred embodiment, the second binding domain (e.g. the anti-CD3 binding domain) of the bispecific T-cell engaging molecules comprises the amino acid sequence of SEQ ID NO: 116.

[0129] The bispecific T-cell engaging molecules according to the invention can comprise any of the anti-PSMA scFv binding domains set forth in Table 4 in combination with any of the anti- CD3 scFv binding domains set forth in Table 4. For instance, in some embodiments, the bispecific T-cell engaging molecules comprise an anti-PSMA scFv binding domain from Table 4 and an anti-CD3 scFv binding domain from Table 4, wherein the anti-PSMA scFv binding domain is connected to the anti-CD3 scFv binding domain through a peptide linker, such as the peptide linkers described herein. In certain embodiments, the bispecific 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.

[0130] The bispecific T-cell engaging molecules according to the invention may also comprise additional domains, which, e.g., can modulate the pharmacokinetic profile of the molecule. For instance, the bispecific T-cell engaging molecules may further comprise an immunoglobulin Fc region, a domain derived from serum albumin (e.g. human serum albumin), or an albuminbinding domain (e.g. comprising human albumin binding peptides), and/or be conjugated to polyethylene glycol chains to increase the serum half-life of the bispecific T-cell engaging molecule. In certain embodiments, the bispecific T-cell engaging molecules used in the methods of the invention further comprise one or more immunoglobulin Fc regions. Each immunoglobulin Fc region or “Fc monomer” typically comprises at least a CH2 domain and a CH3 domain from an immunoglobulin molecule. The Fc monomer may comprise the CH2 and CH3 domains from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. As an example, the CH2 domain comprises amino acids 231 to 340 of an IgGl immunoglobulin and the CH3 domain comprises amino acids 341 to 446 of an IgGl immunoglobulin, where the amino acid numbering is according to the EU numbering system 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). The boundaries of the CH2 and CH3 domains may vary slightly from one IgG isoform to another, but the CH2 and CH3 domains in IgG2, IgG3, and IgG4 can be ascertained by alignment with the CH2 and CH3 domains in IgGl .

[0131] In some embodiments, the Fc monomer may comprise an immunoglobulin hinge region or portion thereof. The immunoglobulin hinge region is typically the region defined by amino acids 216 to 231 (according to the EU numbering system) of IgG immunoglobulins. In certain embodiments, the Fc monomer comprises a hinge region from an IgGl immunoglobulin or a portion thereof. In some such embodiments, the IgGl hinge 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), EPKSCDTPPPCPRCP (SEQ ID NO: 121), or ELKTPLGDTTHTCPRCP (SEQ ID NO: 122), or an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 123). In certain embodiments, the Fc monomer comprises, in amino to carboxyl order, an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain.

[0132] In certain embodiments, the bispecific T-cell engaging molecules comprise a domain having one Fc monomer. In alternative embodiments, the bispecific T-cell engaging molecules comprise a domain having two or more Fc monomers. For instance, in one embodiment, the bispecific T-cell engaging molecules used in the methods of the invention comprise a domain having two Fc monomers. The two Fc monomers can be present on separate polypeptide chains and associate to form a dimer, e.g. via non-covalent interactions and/or disulfide bonds (e.g. between cysteine residues in the hinge regions of Fc monomers). In a preferred embodiment, the two Fc monomers are fused to each other via a peptide linker, preferably a linker sufficient in length to allow the Fc monomers to associate and form an intra-chain 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.

[0133] The peptide linker, by which the Fc monomers are fused to each other to form a singlechain Fc domain, preferably comprises at least 25 amino acid residues (e.g. 25, 26, 27, 28, 29, 30 or more). More preferably, this peptide linker comprises at least 30 amino acid residues (e.g. 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, and even more preferably exactly 30 amino acid residues. In certain embodiments, the peptide linker comprises glycine-serine residues, for example repeats of 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 of 5 or greater (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 connect the two Fc monomers to form a singlechain Fc domain comprises the sequence of SEQ ID NO: 86.

[0134] The Fc monomer may contain one or more amino acid substitutions relative to the native CH2 or CH3 immunoglobulin amino acid sequences, e.g. to modulate effector function, alter glycosylation, or enhance stability. For instance, in one embodiment, the glycosylation site in the CH2 domain at amino acid position 297 according to EU numbering is removed by substituting a different amino acid for the asparagine residue at this position. A N297G substitution is preferred in some embodiments. Stability-enhancing mutations include the substitution of one or more amino acids in the CH2 and/or CH3 domains with cysteine residues to promote disulfide bond formation. Preferably, specific pairs of residues are substituted with cysteine such that they preferentially form a disulfide bond with each other, thus limiting or preventing disulfide bond scrambling. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C, with the amino acid positions numbered according to the EU numbering system. In one particular embodiment, the Fc monomer(s) incorporated into the third domain of the bispecific T-cell engaging molecules comprises N297G, R292C, and V302C substitutions, with the amino acid positions numbered according to the EU numbering system.

[0135] In certain preferred embodiments, the bispecific T-cell engaging molecules used in the methods of the invention comprise a third domain, which is a single-chain Fc domain. Accordingly, in certain such embodiments, the third domain comprises two Fc monomers, each monomer comprising an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, wherein the two Fc monomers are fused to each other via a peptide linker as described herein. Exemplary amino acid sequences for the Fc monomers and the single-chain Fc (scFc) domains are provided in Table 5 below. In some embodiments, each of the Fc monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 124-131. In other embodiments, each of the Fc monomers of the third domain has an amino acid sequence selected from SEQ ID NOs: 124-131. In a preferred embodiment, each of the Fc monomers of the third domain comprises the amino acid sequence of SEQ ID NO: 124. In another preferred embodiment, each of the Fc monomers of the third domain comprises the amino acid sequence of SEQ ID NO: 125.

Table 5. Exemplary Fc Monomer and Single-Chain Fc Domains

[0136] The third domain of the bispecific T-cell engaging molecules used in the methods of the invention can be any of the scFc domains set forth in Table 5 or a variant of these scFc domains. In one embodiment, the bispecific T-cell engaging molecules according to the invention comprise a third domain comprising an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 132-139. In another embodiment, the bispecific T-cell engaging molecules according to the invention comprise a third domain comprising an amino acid sequence selected from SEQ ID NOs: 132-139. In a preferred embodiment, the bispecific T- cell engaging molecules according to the invention comprise a third domain comprising the amino acid sequence of SEQ ID NO: 132. In another preferred embodiment, the bispecific T-cell engaging molecules according to the invention comprise a third domain comprising the amino acid sequence of SEQ ID NO: 133.

[0137] In certain embodiments, the bispecific T-cell engaging molecules used in the methods of the invention comprise, in an amino to carboxyl 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) a third domain comprising two Fc monomers.

[0138] In some embodiments, the bispecific T-cell engaging molecules comprise, 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: 16-19, 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: 9-13;

(ii) a second domain that specifically binds to human CD3 comprising a VH2 comprising a CDRH1 having a sequence selected from SEQ ID NOs: 48-53, a CDRH2 having a sequence selected from SEQ ID NOs: 54-58, and a CDRH3 having a sequence selected from SEQ ID NOs: 59-67, and a VL2 comprising a CDRL1 having a sequence selected from SEQ ID NOs: 41- 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) a third domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, wherein said two monomers are 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.

[0139] In a preferred embodiment, the bispecific T-cell engaging molecule comprises, 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, 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;

(ii) a second domain that specifically binds to 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) a third domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, wherein said two monomers are fused to each other via a peptide linker.

[0140] In certain embodiments, peptide linkers, such as those described herein, connect the first domain to the second domain and/or the second domain to the third domain. Accordingly, in some embodiments, the bispecific T-cell engaging molecule according to the invention comprises, in amino to carboxyl order: (i) a first domain that specifically binds to 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 to human CD3;

(iv) a second peptide linker having an amino acid sequence selected from SEQ ID NOs: 80-83, and 89-91;

(v) a first Fc monomer;

(vi) a third peptide linker having an amino acid sequence selected from SEQ ID NOs: 85- 88; and

(vii) a second Fc monomer.

[0141] In certain embodiments, the bispecific T-cell engaging molecule according to the invention comprises, in amino to carboxyl order:

(i) a first domain (e.g. anti-PSMA binding domain) having an amino acid sequence selected from SEQ ID NOs: 94-106;

(iii) a second domain (e.g. anti-CD3 binding domain) having an amino acid sequence selected from SEQ ID NOs: 107-116;

(v) a first Fc monomer having an amino acid sequence selected from SEQ ID NOs: 124- 131;

(vii) a second Fc monomer having an amino acid sequence selected from SEQ ID NOs: 124-131.

[0142] In a preferred embodiment, the bispecific T-cell engaging molecule according to the invention comprises, in amino to carboxyl order:

(i) a first domain (e.g. 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. 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.

[0143] In certain embodiments, the bispecific T-cell engaging molecules used in the methods of the invention are single chain polypeptides or single chain fusion proteins. As used herein, a “single chain polypeptide” or “single chain fusion protein” refers to a molecule consisting of only one polypeptide chain, i.e. all of the domains in the bispecific T-cell engaging molecule are linked together, optionally via peptide linkers, 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 an amino to carboxyl order, an anti-PSMA scFv domain, a first peptide linker, an anti-CD3 scFv domain, a second peptide linker, and an scFc domain. Exemplary PSMA x CD3 bispecific single chain polypeptides or single chain fusion proteins that can be used in the methods of the invention are set forth in Table 6 below. Other PSMA x CD3 bispecific 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 hereby incorporated by reference in its entirety.

Table 6. Exemplary PSMA x CD3 Bispecific Single Chain Polypeptides

[0144] In certain embodiments, the bispecific T-cell engaging molecule administered to a patient according to the methods of the invention comprises an amino acid sequence selected from SEQ ID NOs: 140-157. In one embodiment, the bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 141. In another embodiment, the bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 144. In yet another embodiment, the bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 147. In still another embodiment, the bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 150. In a preferred embodiment, the bispecific T-cell engaging molecule used in the methods of the invention comprises the amino acid sequence of SEQ ID NO: 140.

[0145] The PSMA x CD3 bispecific T-cell engaging molecules employed in the methods of the invention may be variants of the single chain polypeptides shown in Table 6 and comprise 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 NOs: 140-157. In one embodiment, the bispecific T-cell engaging molecule comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 140-157. In another embodiment, the bispecific T-cell engaging molecule comprises an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 140-157. In certain embodiments, the sequence variability occurs in the peptide linker regions and/or the singlechain Fc domain.

[0146] The PSMA x CD3 bispecific T-cell engaging molecules for use in the methods of the invention may be prepared by any of a number of conventional techniques. For example, the PSMA x CD3 bispecific T-cell engaging molecules described herein may be produced by recombinant expression systems, using any technique known in the art. See, e.g., 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, N.Y. (1988).

[0147] PSMA x CD3 bispecific T-cell engaging molecules or components thereof (e.g. Fv fragments, Fc monomers) can be expressed in hybridoma cell lines or in cell lines other than hybridomas. Expression vectors or constructs encoding the bispecific T-cell engaging molecules can be used to transform a mammalian, insect or microbial host cell. 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, for example, recombinant expression vectors. The term “expression vector” or “expression construct” as used herein refers to a recombinant nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the operably linked coding sequence in a particular host cell. An expression vector can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a 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 possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by the recombinant host cell, for more facile isolation of the polypeptide of interest from the cell, if desired. [0148] Recombinant expression vectors or constructs will typically comprise 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., CHI, CH2 and/or CH3); a heavy chain variable region; hinge region, Fc region, and/or another scaffold portion of an anti-PSMA antibody or anti-CD3 antibody. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In embodiments in which the bispecific T-cell engaging molecule is a single chain polypeptide or single chain fusion protein, the nucleic acid comprised in the recombinant expression vector will typically encode the full-length single chain polypeptide (e.g. full-length single chain fusion protein). The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene can occur). In some embodiments, vectors are used that employ protein-fragment complementation assays using protein reporters, such as dihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964, which is hereby incorporated 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 the T-cell engaging molecules and components thereof include those described in Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84:439-44, which is hereby incorporated by reference. Additional suitable expression vectors are discussed, for example, in Methods Enzymol., vol. 185 (D. V. Goeddel, ed.), 1990, New York: Academic Press.

[0149] Typically, expression vectors used in any of the host cells to produce a bispecific T-cell engaging molecule will contain sequences for cloning and expression of exogenous nucleotide sequences encoding the bispecific T-cell engaging molecule or components thereof. Such sequences, collectively referred to as “flanking sequences,” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. [0150] Optionally, the vector may contain a “tag”-encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the PSMA x CD3 bispecific T-cell engaging molecule coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another “tag” such as FLAG® tag, HA (hemaglutinin influenza virus), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide and can serve as a means for affinity purification or detection of the PSMA x CD3 bispecific T-cell engaging molecule from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified T-cell engaging molecule by various means such as using certain peptidases for cleavage.

[0151] Expression and cloning vectors will typically contain a promoter that is recognized by the host cell and operably linked to the nucleic acid molecule encoding a PSMA x CD3 bispecific T- cell engaging molecule. The term “operably linked” as used herein refers to the linkage of two or more nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. For example, a control sequence in a vector that is “operably linked” to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences. 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 modulates the transcription of the coding sequence in an appropriate host cell or other expression system. A large number of promoters, recognized by a variety of potential host cells, are well known to those of skill in the art. For example, 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 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus, and Simian Virus 40 (SV40). A suitable promoter is operably linked to the polynucleotide encoding e.g., a PSMA x CD3 bispecific 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.

[0152] The expression vectors for recombinant production of the PSMA x CD3 bispecific T-cell engaging molecules described herein may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the desired flanking sequences are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art. The expression vectors can be introduced into host cells to thereby produce the bispecific T-cell engaging molecules encoded by the nucleic acids present in the vectors.

[0153] After the vector has been constructed and one or more nucleic acid molecules encoding the PSMA x CD3 bispecific T-cell engaging molecule or component thereof has been inserted into the proper site(s) of the vector or vectors, the completed vector(s) may be inserted into a suitable host cell for amplification and/or polypeptide expression. The term “host cell” as used herein 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 the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present. A host cell that comprises an isolated polynucleotide or nucleic acid encoding a bispecific T-cell engaging molecule, preferably operably linked to at least one expression control sequence (e.g. promoter or enhancer), is a “recombinant host cell.”

[0154] The transformation of an expression vector for a polypeptide into a selected host cell may be accomplished 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 in part be a function of the type of host cell to be used.

[0155] A host cell, when cultured under appropriate conditions, synthesizes a bispecific T-cell engaging molecule that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

Suitable host cells include, but are not limited to, prokaryotic cells (e.g. E. coh. B. subtilis), yeast cells (Saccharmoyces cerevisiae. Pichia pasloris). and mammalian cells (e.g. Chinese hamster ovary (CHO), human embryonic kidney (HEK)). CHO cells are preferred host cells in some embodiments for expressing the PSMA x CD3 bispecific T-cell engaging molecules.

[0156] Host cells are transformed or transfected with the above-described expression vectors for production of the T-cell engaging molecules and are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The host cells used to produce the T-cell engaging molecules may 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's Medium (DMEM, Sigma) are suitable for culturing the 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. Patent 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 culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), 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 supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinary skilled artisan.

[0157] Upon culturing the host cells, the T-cell engaging molecule can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the T-cell engaging molecule is produced intracellularly, as a first step, the host cells are lysed (e.g., by mechanical shear, osmotic shock, or enzymatic methods) and the particulate debris (e.g., host cells and lysed fragments), is removed, for example, by centrifugation, microfiltration, or ultrafiltration. If the T-cell engaging molecule is secreted into the culture medium, the T-cell engaging molecule can be separated from host cells through centrifugation or microfiltration, and optionally, subsequently concentrated through ultrafiltration. The PSMA x CD3 bispecific T-cell engaging molecules can be further purified or partially purified using, for example, one or more chromatography steps, such as 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 mode chromatography.

[0158] The PSMA x CD3 bispecific T-cell engaging molecule is generally administered to the patient in a pharmaceutical composition, which can include pharmaceutically-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 employed and/or do not produce allergic or adverse reactions when administered to humans. In certain embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl- beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); 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 (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium 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 arts, and are described, for example, in REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company. Pharmaceutical compositions comprising the bispecific T-cell engaging molecules to be administered according to the methods of the invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0159] If the pharmaceutical composition has been lyophilized, the lyophilized material is reconstituted in an appropriate liquid prior to administration. The lyophilized material may be reconstituted in, e.g., bacteriostatic water for injection (BWFI), physiological saline, phosphate buffered saline (PBS), or the same formulation the protein had been in prior to lyophilization. [0160] In some embodiments, the selection of carriers and excipients for incorporation into the pharmaceutical compositions influences the physical state, stability, rate of in vivo release and rate of in vivo clearance of the bispecific T-cell engaging molecules. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or nonaqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, possibly supplemented with other materials or excipients common in compositions for parenteral administration.

[0161] In certain embodiments of the methods described herein, the PSMA x CD3 bispecific T- cell engaging molecule (e.g. a pharmaceutical composition comprising the anti-CDPSMA x anti- CD3 bispecific T-cell engaging molecule) is administered to the patient parenterally. Parenteral administration refers to administration of the molecule by routes other than through the gastrointestinal tract and can include intraperitoneal, intramuscular, intravenous, intraarterial, intradermal, subcutaneous, intracerebral, intracerebroventricular, and intrathecal administration. In some embodiments, administration of the PSMA x CD3 bispecific T-cell engaging molecule according to the methods of the invention is intravenous.

[0162] Parenteral or intravenous administration can be performed by injection (e.g. using a needle and a syringe) or by infusion (e.g. via a catheter and a pump system). It is envisaged that the administration according to the present invention is via intravenous injection or via intravenous infusion. Usually, an intravenous (IV) infusion is administered via a line, a port or a catheter (small, flexible tube), such as a central venous access or a central venous catheter (CVC), which is a catheter placed into a large vein, or a peripheral venous catheter (PVC), which is a catheter placed into a peripheral vein. In general, catheters or lines can be placed in veins in the neck (internal jugular vein), chest (subclavian vein or axillary vein), groin (femoral vein), or through veins in the arms (also known as a PICC line, or peripherally inserted central catheters). Central IV lines have catheters that are advanced through a vein and empty into a large central vein, usually the superior vena cava, inferior vena cava or even the right atrium of the heart. A peripheral intravenous (PIV) line is used on peripheral veins (the veins in the arms, hands, legs and feet). A port is a central venous line that does not have an external connector; instead, it has a small reservoir that is covered with silicone rubber and is implanted under the skin. Medication is administered intermittently by placing a small needle through the skin, piercing the silicone, into the reservoir. When the needle is withdrawn, the reservoir cover reseals itself. The cover can accept hundreds of needle sticks during its lifetime.

[0163] In certain embodiments, the PSMA x CD3 bispecific T-cell engaging molecule is administered to the patient as a short intravenous infusion, which is typically an infusion of a small volume (e.g. 20 mL to 100 mL) administered over a period of, at most three hours. Preferably, each of the doses of the bispecific T-cell engaging molecule administered to the patient during the initiation cycle and/or the maintenance cycle according to the methods of the invention is administered as an intravenous infusion of about 30 min to about 3 hours, about 30 min to about 90 min, or about 30 min to about 60 min. In one embodiment, each of the doses of the bispecific T-cell engaging molecule administered to the patient during the initiation cycle and/or the maintenance cycle according to the methods of the invention is administered as an intravenous infusion of about 60 min (e.g. 55 min to 65 min).

[0164] In embodiments in which the bispecific T-cell engaging molecule is infused, an infusion pump may be used to infuse the bi specific T-cell engaging molecule into a patient’s circulatory system. The pump is generally used intravenously, although arterial and epidural infusions with pumps are also possible. The solution for infusion may be prepared in bags for IV infusion and delivered through infusion lines. Pump systems for delivering intravenous infusions are known in the art. It is also possible that infusions are administered using only the pressure supplied by gravity.

[0165] In certain embodiments, the pharmaceutical compositions comprise an effective amount of the PSMA x CD3 bispecific T-cell engaging molecule and one or more excipients. An effective amount can be a therapeutic dose or target dose or it may be a smaller amount, such as a priming dose. Excipients can be used for a wide variety of purposes, such as adjusting physical, chemical, or biological properties of formulations, such as adjustment of viscosity, and/or to stabilize such formulations against degradation and spoilage e.g. due to stresses that occur during manufacturing, shipping, storage, pre-use preparation, and administration.

[0166] In some embodiments, the pharmaceutical composition comprising an effective amount of a PSMA x CD3 bispecific T-cell engaging molecule to be administered to a patient according to the methods of the invention comprises a buffer. Buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range from about 4.0 to about 6.5. Suitable buffers include, but are not limited to, glutamate, aspartate, 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 L-glutamate buffer. Pharmaceutical compositions 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.

[0167] The pharmaceutical composition comprising an effective amount of a PSMA x CD3 bispecific T-cell engaging molecule may further comprise a surfactant. The term “surfactant” as used herein refers to a substance that functions to reduce the surface tension of a liquid in which it is dissolved. Surfactants can be included in pharmaceutical compositions 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 the lyophilization and/or reconstitution process in lyophilized formulations. Surfactants include, for example, amphipathic organic compounds that exhibit partial solubility in both organic solvents and aqueous solutions. General 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 compositions used in the methods of the invention include both non-ionic and ionic surfactants. Suitable non-ionic 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 non-ionic surfactants include the polysorbates including, for example, polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and the like; the pol oxamers including, for example, pol oxamer 188, also known as poloxalkol or poly(ethylene oxide)-poly(propylene oxide), poloxamer 407 or polyethylene-polypropylene glycol and the like, and polyethylene glycol (PEG). Suitable ionic surfactants include, for example, anionic, cationic and zwitterionic surfactants. Anionic surfactants include, but are not limited to, sulfonate-based or carboxylate-based surfactants such as soaps, fatty acid salts, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate and other alkyl sulfate salts. Cationic surfactants include, but are not limited to, quaternary ammonium-based surfactants such as cetyl trimethylammonium bromide (CTAB), other alkyltrimethylammonium salts, cetyl pyridinium chloride, polyethoxylated tallow amine (POEA) and benzalkonium chloride. Zwitterionic or amphoteric surfactants include, for example, dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and coco ampho glycinate. In certain embodiments, the pharmaceutical compositions administered according to the methods described herein comprise a non-ionic surfactant. In one embodiment, the non-ionic surfactant is polysorbate 20. In another embodiment, the non-ionic surfactant is polysorbate 80.

[0168] In certain embodiments, the pharmaceutical composition comprising an effective amount of a PSMA x CD3 bispecific T-cell engaging molecule further comprises a stabilizing agent. As used herein, the term “stabilizing agent” refers to an excipient that stabilizes the native conformation of the polypeptide or T-cell engaging molecule and/or prevents or reduces the physical or chemical degradation of the polypeptide or T-cell engaging molecule. Suitable stabilizing agents 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 stabilizing agent. In these and other embodiments, the sugar is sucrose.

[0169] Exemplary pharmaceutical compositions comprising bispecific T-cell engaging molecules, including PSMA x CD3 bispecific T-cell engaging molecules, are described in WO 2018/141910, which is hereby incorporated by reference in its entirety. In certain embodiments, a pharmaceutical composition useful for the treatment of prostate cancer according to the methods described herein comprises about 0.5 mg/ml to about 2 mg/ml of a PSMA x CD3 bispecific T-cell engaging molecule, about 5 mM to about 20 mM L-glutamic acid, about 0.005% to about 0.015% weight/volume (w/v) polysorbate (e.g. polysorbate 20 or polysorbate 80), and about 7% to about 12% (w/v) sucrose. In other embodiments, the pharmaceutical composition comprises about 0.5 mg/ml to about 1 mg/ml of a PSMA x CD3 bispecific T-cell engaging molecule, about 8 mM to about 12 mM L-glutamic acid, about 0.008% to about 0.012% (w/v) polysorbate (e.g. polysorbate 20 or polysorbate 80), and about 8% to about 10% (w/v) sucrose. The pH of these compositions is in the range of about 4.0 to about 4.4 (e.g., pH of about 4.0, about 4.1, about 4.2, about 4.3, or about 4.4).

[0170] Any of the pharmaceutical compositions comprising the PSMA x CD3 bispecific T-cell engaging molecules described herein can be lyophilized and reconstituted with, e.g. sterile water for injection, prior to administration to the patient. Reconstitution volumes will depend on the protein content following lyophilization and the desired concentration of the bispecific T-cell engaging molecule in the reconstituted solution, but may be from about 0.5 ml to about 5 ml. The solution following reconstitution can be further diluted with a diluent (e.g. saline and/or intravenous solution stabilizer (IVSS)) prior to administration to the patient as appropriate in order to administer the doses described herein according to the methods of the invention.

[0171] Any of the PSMA x CD3 bispecific 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 x CD3 bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 140. In another preferred embodiment, the PSMA x CD3 bispecific T-cell engaging molecule comprises the amino acid sequence of SEQ ID NO: 141.

[0172] The present invention also includes kits for treating prostate cancer in a patient in need thereof. In one embodiment, the kit comprises a pharmaceutical composition of a PSMA x CD3 bispecific T-cell engaging molecule described herein and packaging material that provides instructions regarding the use of the pharmaceutical compositions. The pharmaceutical composition of the kit may be present in a container, such as a vial. The pharmaceutical composition may be provided as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. In embodiments in which the pharmaceutical composition is provided as a lyophilized powder, the kit may also comprise diluents (e.g. sterile water for injection, saline, phosphate-buffer saline, formulation buffer) necessary to reconstitute the pharmaceutical composition as well as instructions for preparing the composition for administration. In certain embodiments, the kits may further comprise one or more vials of intravenous solution stabilizer (IVSS) and instructions for using the IVSS for pre-treatment of IV bags prior to dilution of the pharmaceutical composition for delivery to the patient. IVSS does not contain an active pharmaceutical ingredient and is typically a buffered, preservative-free solution. In one embodiment, IVSS comprises citric acid (e.g. 20-30 mM), lysine hydrochloride (e.g. 1-3 M), and polysorbate 80 (0.05%-0.15% (w/v)) at pH 7.0. In a particular embodiment, IVSS comprises 25 mM citric acid, 1.25 M lysine hydrochloride, and 0.1% (w/v) polysorbate 80 at pH 7.0.

[0173] In some embodiments, the methods of the invention for treating prostate cancer or other PSMA-expressing cancer in a patient comprise administering to the patient a PSMA x CD3 bispecific T-cell engaging molecule according to the dosing schedules described herein in combination with one or more agents suitable for the treatment of prostate cancer or other PSMA-expressing cancer. The term “combination therapy” or “in combination” as used herein encompasses the administration of the two compounds (e.g. PSMA x CD3 bispecific T-cell engaging molecule and additional agent) in a sequential manner (i.e. each agent is administered on a different day in any order) as well as administration of the two agents in a substantially simultaneous manner. Substantially simultaneous administration includes concurrent administration and can be accomplished by administering a single formulation comprising both agents (e.g. a single IV bag containing both agents) or concurrently administering (e.g. on the same day) separate formulations containing each of the agents. The additional agents need not be administered at the same dosing frequency or dosing interval as the PSMA x CD3 bispecific T- cell engaging molecule. In general, the additional agent will be administered at a dose and/or on a time schedule determined for that agent. The additional agent can be administered on the same day or different days of a treatment cycle (i.e. initiation cycle and/or maintenance cycle) as the PSMA x CD3 bispecific T-cell engaging molecule.

[0174] In certain embodiments of the methods of the invention, the PSMA x CD3 bispecific T- cell engaging molecule is administered to the patient in combination with a standard prostate cancer therapy, such as chemotherapy, radiation therapy, androgen deprivation therapy, or radioligand therapy. In some embodiments, the methods of the invention comprise administering a taxane chemotherapy agent in combination with the PSMA x CD3 bispecific T-cell engaging molecule, wherein the PSMA x CD3 bispecific T-cell engaging molecule is administered according to the dosing schedules described herein. In one such embodiment, the taxane chemotherapy agent is docetaxel. In another such embodiment, the taxane chemotherapy agent is cabazitaxel. The patient may receive one or more treatment cycles of the taxane chemotherapy agent prior to being administered the PSMA x CD3 bispecific T-cell engaging molecule. In alternative embodiments, the patient may receive at least one initiation cycle of the PSMA x CD3 bispecific T-cell engaging molecule prior to being administered the taxane chemotherapy agent.

[0175] In other embodiments, the methods of the invention comprise administering an androgen deprivation therapy in combination with the PSMA x CD3 bispecific T-cell engaging molecule, wherein the PSMA x CD3 bispecific T-cell engaging molecule is administered according to the dosing schedules described herein. In some such embodiments, the patients to be treated with such a combination therapy may have been newly diagnosed with prostate cancer, or they may have been diagnosed with hormone-sensitive prostate cancer or castration-resistant prostate cancer. The androgen deprivation therapy that can be administered in combination with the PSMA x CD3 bispecific T-cell engaging molecule can include, but is not limited to, an LHRH agonist or antagonist (e.g. leuprolide, goserelin, triptorelin, histrelin, or degarelix), an antiandrogen therapy, such as an androgen biosynthesis inhibitor (e.g. abiraterone, ketoconazole) or androgen receptor antagonist (e.g. flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide). In certain embodiments, the methods of the invention comprise administering a PSMA x CD3 bispecific T-cell engaging molecule and an anti-androgen therapy, wherein the PSMA x CD3 bispecific T-cell engaging molecule is administered according to the dosing schedules described herein. Anti-androgen therapies, such as enzalutamide and abiraterone, have been reported to up-regulate PSMA expression on castration-sensitive and castration-resistant prostate cancer cells (Aggarwal et al., Eur Urol Oncol., Vol. 1 (1): 78-82, 2018; Emmett et al., J Nucl Med., Vol. 60(7):950-954, 2019). In some embodiments, the antiandrogen therapy administered in combination with the PSMA x CD3 bispecific T-cell engaging molecule according to the methods described herein is enzalutamide, abiraterone, apalutamide, or darolutamide. In some such embodiments, the patient may receive at least one dose of the PSMA x CD3 bispecific T-cell engaging molecule prior to receiving the anti-androgen therapy. In related embodiments, the anti-androgen therapy is not administered to the patient until the patient has received the target dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In other related embodiments, the patient may receive at least one initiation cycle of the PSMA x CD3 bispecific T-cell engaging molecule prior to administration of the anti -androgen therapy.

[0176] In certain other embodiments, the methods of the invention comprise administering an immune checkpoint inhibitor, such as an antagonist of programmed death receptor 1 (PD-1)/PD- 1-ligand 1 (PD-L1) signaling, in combination with the PSMA x CD3 bispecific T-cell engaging molecule, wherein the PSMA x CD3 bispecific T-cell engaging molecule is administered according to the dosing schedules described herein. Accordingly, the present invention includes methods of treating prostate cancer or other PSMA-expressing cancers in a patient in need thereof comprising administering to the patient: (i) an initiation cycle and one or more maintenance cycles of a PSMA x CD3 bispecific T-cell engaging molecule according to the dosing schedules described herein; and (ii) a PD-1 antagonist antibody or a PD-L1 antagonist antibody during the initiation cycle and/or one or more maintenance cycles. The term “PD-1 antagonist antibody” refers to an antibody that specifically binds to PD-1 and decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 and one or more of its ligands, such as PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1 and/or PD-L2. The term “PD-L1 antagonist antibody” refers to an antibody that specifically binds to PD-L1 and decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with the PD-1 receptor. In some embodiments, a PD-L1 antagonist antibody inhibits the binding of PD-L1 to PD-1. Suitable PD-L1 antagonist antibodies for use in combination with PSMA x CD3 bispecific T-cell engaging molecules according to the methods of the invention include, but are not limited to, atezolizumab, avelumab, or durvalumab. Examples of PD-1 antagonist antibodies suitable for use in the methods of the invention include, but are not limited to pembrolizumab, nivolumab, cemiplimab, pidilizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, and any of the PD-1 antagonist antibodies described in WO 2019/140196, which is hereby incorporated by reference in its entirety. In some embodiments, the PD-1 antagonist is any one of the antibodies described in Table 7 below. In one embodiment, the PD-1 antagonist antibody is pembrolizumab. In another embodiment, the PD-1 antagonist antibody is nivolumab. In yet another embodiment, the PD-1 antagonist antibody is cemiplimab. In still another embodiment, the PD-1 antagonist antibody is antibody 20C1.9, for which the amino acid sequences of the CDRs, variable regions, and full light and heavy chains are provided in Table 7 below. For instance, in one such embodiment, the PD-1 antagonist antibody to be administered in combination with the PSMA x CD3 bispecific T-cell engaging molecule according to the methods of the invention comprises a heavy chain variable region comprising a CDRH1 having the sequence of SEQ ID NO: 161, a CDRH2 having the sequence of SEQ ID NO: 168, and a CDRH3 having the sequence of SEQ ID NO: 163, and a light chain variable region comprising a CDRL1 having the sequence of SEQ ID NO: 158, a CDRL2 having the sequence of SEQ ID NO: 159, and a CDRL3 having the sequence of SEQ ID NO: 160. In another such embodiment, the PD-1 antagonist antibody to be administered in combination with the PSMA x CD3 bispecific T-cell engaging molecule according to the methods of the invention comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 169 and a light chain variable region comprising the sequence of SEQ ID NO: 164. In another embodiment, the PD-1 antagonist antibody to be administered in combination with the PSMA x CD3 bispecific T-cell engaging molecule according to the methods of the invention comprises a heavy chain comprising the sequence of SEQ ID NO: 170 and a light chain comprising the sequence of SEQ ID NO: 166.

Table 7. Amino Acid Sequences for Exemplary PD-1 Antagonist Antibodies

[0177] In certain embodiments in which a PD-1 antagonist antibody or PD-L1 antagonist antibody is administered to the patient in combination with the PSMA x CD3 bispecific T-cell engaging molecule, the PD-1 antagonist antibody or PD-L1 antagonist antibody is administered once per cycle (initiation and/or maintenance cycles) at a fixed dose. In some embodiments, the cycle duration is about 28 days and the PD-1 antagonist antibody or PD-L1 antagonist antibody is administered once every 28 days or once every four weeks (Q4W) at a fixed dose. In other embodiments, the PD-1 antagonist antibody or PD-L1 antagonist antibody is administered twice per cycle (initiation and/or maintenance cycles) at a fixed dose, for example once every 14 days or once every two weeks (Q2W) at a fixed dose for a cycle duration of about 28 days. In some embodiments of the methods of the invention, the PD-1 antagonist antibody or PD-L1 antagonist antibody is first administered during the first maintenance cycle of the PSMA x CD3 bispecific T-cell engaging molecule. In one particular embodiment, the PD-1 antagonist antibody or PD-L1 antagonist antibody is first administered to the patient on the first day the patient receives the target dose of the PSMA x CD3 bispecific T-cell engaging molecule during the initiation cycle. In such embodiments, the PD-1 antagonist antibody or PD-L1 antagonist antibody may be administered after the target dose of the PSMA x CD3 bispecific T-cell engaging molecule is administered to the patient, for example, following a post-infusion flush.

[0178] The fixed dose and route of administration of the PD-1 antagonist antibody or PD-L1 antagonist antibody will depend on the specific PD-1 antagonist antibody or PD-L1 antagonist antibody employed. In some embodiments, the PD-1 antagonist antibody is pembrolizumab, wherein pembrolizumab is intravenously administered once per cycle (e.g. once every 4 weeks for a 28-day cycle) at a fixed dose of 200 mg. In other embodiments, the PD-1 antagonist antibody is nivolumab, wherein nivolumab is intravenously administered once per cycle (e.g. once every 4 weeks for a 28-day cycle) at a fixed dose of 480 mg. In yet other embodiments, the PD-1 antagonist antibody is nivolumab, wherein nivolumab is intravenously administered twice per cycle (e.g. once every 2 weeks for a 28-day cycle) at a fixed dose of 240 mg. In some embodiments, the PD-L1 antagonist antibody is atezolizumab, wherein atezolizumab is intravenously administered once per cycle (e.g. once every 4 weeks for a 28-day cycle) at a fixed dose of 1200 mg. In other embodiments, the PD-L1 antagonist antibody is atezolizumab, wherein atezolizumab is intravenously administered twice per cycle (e.g. once every 2 weeks for a 28-day cycle) at a fixed dose of 840 mg.

[0179] In some embodiments, the methods of the invention comprise administering during a 28- day initiation cycle: a priming dose (e.g. 10 pg) of the bispecific T-cell engaging molecule on day 1 (DI) and a target dose (e.g. 30 pg or 90 pg) on day 8 (D8) and day 22 (D22) and a fixed dose of a PD-1 antagonist antibody or PD-L1 antagonist antibody on D8. Following a treatment- free period of 7 days, in such embodiments, the methods further comprise administering during a 28-day maintenance cycle: the target dose (e.g. 30 pg or 90 pg) of the bispecific T-cell engaging molecule on day 1 (DI) and day 15 (D15) and the fixed dose of the PD-1 antagonist antibody or PD-L1 antagonist antibody on DI. Thus, according to this combination therapy dosing regimen, for the 56-day period encompassing both the 28-day initiation cycle and the 28-day maintenance cycle and starting with the first dose of the initiation cycle, the patient would be administered the bispecific T-cell engaging molecule on each of DI, D8, D22, D36, and D50 and would be administered the PD-1 antagonist antibody or PD-L1 antagonist antibody on D8 and D36.

[0180] In other embodiments, the methods of the invention comprise administering during a 28- day initiation cycle: a first priming dose (e.g. 10 pg) of the bispecific T-cell engaging molecule on DI, a second priming dose (e.g. 90 pg) of the bispecific T-cell engaging molecule on D8, and a target dose (e.g. 300 pg) of the bispecific T-cell engaging molecule and a fixed dose of a PD-1 antagonist antibody or PD-L1 antagonist antibody on DI 5. In such embodiments, the methods further comprise administering during a 28-day maintenance cycle: the target dose (e.g. 300 pg) of the bispecific T-cell engaging molecule on DI and DI 5 and the fixed dose of the PD-1 antagonist antibody or PD-L1 antagonist antibody on DI 5, wherein the maintenance cycle is administered the following day after completing the 28-day initiation cycle. Thus, according to this dosing regimen, for the 56-day period encompassing both the 28-day initiation cycle and the 28-day maintenance cycle and starting with the first dose of the initiation cycle, the patient would be administered the bispecific T-cell engaging molecule on each of DI, D8, DI 5, D29, and D43 and would be administered the PD-1 antagonist antibody or PD-L1 antagonist antibody on D 15 and D43.

[0181] In certain other embodiments, the methods of the invention comprise administering during a 28-day initiation cycle: a first priming dose (e.g. 10 pg) of the bispecific T-cell engaging molecule on DI, a second priming dose (e.g. 30 pg) of the bispecific T-cell engaging molecule on D8, a third priming dose (e.g. 90 pg) of the bispecific T-cell engaging molecule on D15, and a target dose (e.g. 300 pg or 900 pg) of the bispecific T-cell engaging molecule and a fixed dose of a PD-1 antagonist antibody or PD-L1 antagonist antibody on D22. Following a treatment-free period of 7 days, in such embodiments, the methods further comprise administering during a 28-day maintenance cycle: the target dose (e.g. 300 pg or 900 pg) of the bispecific T-cell engaging molecule on DI and D15 and the fixed dose of the PD-1 antagonist antibody or PD-L1 antagonist antibody on DI 5. Thus, according to this dosing regimen, for the 56-day period encompassing both the 28-day initiation cycle and the 28-day maintenance cycle and starting with the first dose of the initiation cycle, the patient would be administered the bispecific T-cell engaging molecule on each of DI, D8, D15, D22, D36, and D50 and would be administered the PD-1 antagonist antibody or PD-L1 antagonist antibody on D22 and D50.

[0182] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims.

EXAMPLES

Example 1. A Phase 1 Study Evaluating the Safety, Tolerability, Pharmacokinetics, and Efficacy of AMG 160 Monotherapy in Patients with Metastatic Castration-Resistant Prostate Cancer

[0183] AMG 160 is a half-life extended (HLE) BiTE® (bispecific T-cell engager) molecule that binds both PSMA and CD3 and comprises a single chain IgG Fc region. The amino acid sequence of AMG 160 is set forth in SEQ ID NO: 140. AMG 160 is designed to engage a patient’s T cells to kill prostate cancer cells via binding of CD3 on T cells and PSMA on cancer cells. Study objectives were to evaluate safety, tolerability, pharmacokinetics, and anti -tumor activity of AMG 160 in adult patients with metastatic castration-resistant prostate cancer (mCRPC).

[0184] After signing informed consent, patients entered the screening period (up to 28 days), during which eligibility of the patients was assessed. Eligible patients had mCRPC refractory to prior novel hormonal therapy and 1 to 2 taxane regimens and evidence of progressive disease. Specifically, patients were enrolled in the study if they met all of the following key inclusion criteria:

• histologically or cytologically confirmed mCRPC who were refractory to a novel anti-androgen therapy (e.g., abiraterone, enzalutamide, darolutamide, and/or apalutamide) and had failed at least 1 (but not more than 2) taxane regimens (or who were deemed medically unsuitable to be treated with a taxane regimen or actively refused treatment with a taxane regimen);

• had undergone bilateral orchiectomy or were on continuous androgen-deprivation therapy (ADT) with a gonadotropin-releasing hormone (GnRH) agonist or antagonist;

• had a total serum testosterone level < 50 ng/dL or 1.7 nmol/L; and

• had evidence of progressive disease, defined by one or more of the following Prostate Cancer Working Group 3 (PCWG3; Scher et al., J. Clin, Oncol, Vol. 34: 1402-1418, 2016) criteria:

■ prostate-specific antigen (PSA) level > 1 ng/mL that has increased on at least 2 successive occasions at least 1 week apart

■ nodal or visceral progression as defined by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 with PCGW3 modifications

■ appearance of 2 or more new lesions in bone scan

[0185] Patients were excluded from the study if they: (i) had active autoimmune disease requiring immunosuppressive therapy; (ii) received a prior PSMA-targeted therapy with the exception of a PSMA radioligand therapy, or (iii) had CNS metastases, leptomeningeal disease, or spinal cord compression. [0186] AMG 160 was administered as a short IV infusion (approximately 60 minutes) every two weeks (Q2W)(e.g. on days 1 and 15) after target dose was reached in a 28-day cycle at target doses ranging from 0.003 to 1.8 mg. The date of the first dose of AMG 160 was defined as day 1 in the cycle. To reduce the incidence of cytokine release syndrome (CRS), the cycle 1 dosing schedule was adjusted to include single-step, two-step, and three-step dosing schedules. Single- step dosing involved a run-in dose (e.g. a priming dose) of AMG 160 administered on cycle 1 day 1 followed by administration of the target dose of AMG 160 on days 8 and 22 of a 28-day cycle (plus a 7-day infusion-free interval before start of cycle 2). A two-step dosing schedule entailed administration of a run-in dose (e.g. a first priming dose) of AMG 160 on cycle 1 day 1 followed by administration of a higher run-in dose (e.g. a second priming dose) of AMG 160 on cycle 1 day 8, and then administration of the target dose of AMG 160 on cycle 1 day 15 of a 28- day cycle. A three-step dosing schedule involved administration of a run-in dose (e.g. a first priming dose) of AMG 160 on cycle 1 day 1 followed by administration of a higher run-in dose (e.g. a second priming dose) of AMG 160 on cycle 1 day 8 followed by administration of another higher run-in dose (e.g. a third priming dose) of AMG 160 on cycle 1 day 15, and then administration of the target dose of AMG 160 on cycle 1 day 22 of a 28-day cycle (plus a 7-day infusion-free interval before start of cycle 2). After cycle 1, cycle 2 and all subsequent cycles entailed the administration of the target dose of AMG 160 on days 1 and 15 of the 28-day cycle. Table 8 below summarizes the different dosing cohorts that have been evaluated to date. For cohorts that were dosed according to a no-step dosing regimen or a two-step dosing regimen, cycle 2 was initiated immediately following the 28-day cycle 1 - that is, study day 29 was day 1 of cycle 2. For cohorts dosed according to a single-step or three-step dosing regimen, cycle 2 was initiated 7 days after the 28-day cycle 1 - i.e. study day 36 was day 1 of cycle 2. All patients were pre-treated with 8 mg PO dexamethasone 6-16 hours prior to all doses of AMG 160 in cycle 1. Additionally, dexamethasone 8 mg IV was administered within 1 hour prior to all doses of AMG 160 in cycle 1. Patients received treatment cycles of AMG 160 until disease progression or unacceptable toxicities.

[0187] Anti -turn or activity of AMG 160 was evaluated by several measures, including objective response per RECIST 1.1 criteria with PCWG3 modifications, PSA response, circulating tumor cells (CTC) response, radiographic response as measured by ⁶⁸Gallium (⁶⁸Ga)-PSMA-l 1 positron emission tomography(PET)/computed tomography (CT) and ¹⁸F-fluorodeoxy glucose (FDG) PET/CT scans, progression-free survival (radiographic 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 and then every 12 weeks thereafter. Tumor burden assessments were performed based on RECIST 1.1 with PCWG3 modifications (see Eisenhauer et al., European Journal of Cancer, Vol. 45: 228-247, 2009; Scher et al., J. Clin, Oncol, Vol. 34: 1402- 1418, 2016). To confirm disease progression (PD), a second MRI/CT scan was performed 4-6 weeks after the first detection of radiographical progression. Responses (partial response (PR) and complete response (CR)) were confirmed by a repeat consecutive assessment at least 4 weeks after the first detection of radiographical response.

[0188] PSA30/50/70/90 responses were defined as 30%, 50%, 70%, and 90% reduction, respectively, in serum PSA levels. CTC response was defined as CTC0 (reduction of CTCs > 0 to 0) or CTC conversion (> 5 CTCs/7.5 mL blood to < 4 CTCs/7.5 mL blood) measured in whole blood. ⁶⁸Ga-PSMA-l 1 PET/CT scans were performed at baseline to assess PSMA-positive tumor burden and every 12 weeks during treatment for response assessment. To identify PSMA- negative disease burden, ¹⁸F-FDG PET/CT scans were performed at baseline and every 12 weeks during treatment for response assessment during the dose expansion phase.

Table 8. Summary of Dosing Regimen Cohorts

'Measured from day 1 (DI), which was the first day the patient received the first dose of AMG 160 [0189] At the time of data analysis, 33 patients had received > 1 dose of AMG 160 monotherapy at 6 target dose levels up to 0.9 mg, and 9 patients remained on treatment. Six patients received treatment for > 6 months. Of the 33 men enrolled in the study, most (75.8%) were white. Mean age of patients was 65.2 years (range: 49 to 78 years) with baseline Eastern Cooperative Oncology Group (ECOG) status score of 0 or 1. Twenty-one patients (63.6%) had received > 3 prior lines of therapy.

[0190] Treatment-emergent adverse events were reported for 32 patients (97.0%) at the time of data analysis. The most common adverse event was CRS, which presented with fever, transient transaminitis, hypotension, nausea/vomiting, and/or diarrhea (Table 9). CRS was reversible and occurred primarily in cycles 1 and 2. There were no grade 4 or 5 CRS events (assessed by the Lee criteria; Lee et al., Blood, Vol. 124: 188-195, 2014). One treatment-related discontinuation (0.3 mg target dose) and 2 reversible dose-limiting toxicities were reported: rash (0.03 mg target dose) and GI hemorrhage (0.9 mg target dose). Maximum tolerated dose (MTD) was not reached. Six out of thirty patients (20.0%) assessed at the time of data analysis developed antidrug antibodies affecting exposure of AMG 160 between cycles 1 and 10. No adverse events clearly associated with the anti-drug antibodies were observed.

Table 9. Treatment-Emergent Adverse Events

- I l l -

[0191] At the time of data analysis, preliminary evidence of efficacy and clinical benefit of AMG 160 were observed in some patients. RECIST 1.1 responses among the 22 patients with measurable disease included 2 confirmed partial responses (PR; at target doses of 0.03 mg and 0.09 mg), 8 stable disease (SD), and 5 progressive disease (PD). See Figure 1. PSA reductions occurred in 22 of 31 evaluable patients (71.0%). See Figure 2. Evaluable patients included those who had received > 1 dose of AMG 160 and had measurable baseline PSA levels. PSA reductions > 50% as a best response occurred in 10 out of 31 (32.2%) evaluable patients (Figure 2). Two patients, who exhibited PSA reductions with AMG 160 treatment, had failed prior treatment with ¹⁷⁷Lu-PSMA-617 radioligand therapy (see patients denoted by solid triangles in Figure 2). Overall, 7 patients out of 22 patients with 2 postbaseline PSA results (31.8%) had confirmed PSA responses: 1 PSA90 (0.09 mg target dose), 1 PSA70 (0.9 mg target dose), 2 PSA50 (target doses of 0.03 mg and 0.3 mg), and 3 PSA30 (target doses of 0.03 mg, 0.3 mg, and 0.9 mg). See Figure 1. An additional 3 patients out of 31 patients with measurable PSA levels at baseline had unconfirmed PSA responses at the time of data analysis: 2 PSA50 and 1 PSA30 (all at a target dose of 0.9 mg). See Figure 1. Three patients out of 13 patients with baseline CTC > 0 and postbaseline CTC assessment (23.1%) had a CTCO response (Figure 1).

[0192] The results of the study to date show that AMG160 monotherapy had a manageable safety profile as CRS, the most common adverse event, was reversible and there was no grade 4 or grade 5 CRS events. In patients who were refractory to multiple prior therapies, AMG 160 showed preliminary efficacy with 71% of patients showing any PSA decline across all monotherapy dose cohorts and 32.2% having a greater than 50% reduction in PSA. Among the patients with RECIST 1.1 measurable disease, 2 confirmed PR and 8 SD were observed.

Example 2. A Phase 1 Study Evaluating the Safety, Tolerability, Pharmacokinetics, and Efficacy of AMG 160 in Combination with Pembrolizumab in Patients with Metastatic Castration-Resistant Prostate Cancer

[0193] It has been previously shown that the mechanism of action of bispecific T-cell engaging molecules leads to an upregulation of immune checkpoint molecules, such as programmed death receptor 1 (PD-1) on immune cells and PD-ligand 1 (PD-L1) on tumor cells (Kobold et al., Front Oncol., Vol. 8:285, 2018). The combination of the BiTE molecule blinatumomab with the PD-1 antagonist antibody nivolumab was reported to be safe and tolerable in patients with acute lymphoblastic leukemia with evidence of antitumor activity (Webster et al., Blood, Vol. 132: 557, 2018). The main objectives of this study are to evaluate the safety and tolerability of AMG 160 given in combination with the PD-1 antagonist antibody pembrolizumab with additional objectives to explore pharmacokinetics, pharmacodynamics, immunogenicity, and anti -turn or activity of AMG 160 when given in combination with pembrolizumab.

[0194] After signing informed consent, patients entered the screening period (up to 28 days), during which eligibility of the patients was assessed. Eligibility criteria were the same as those described in Example 1 except that patients: (i) on a prior PD-1 or PD-L1 inhibitor who had experienced a grade 3 or higher immune-related adverse event prior to first day of dosing, (ii) had a known hypersensitivity or allergy to pembrolizumab, or (iii) had a history or evidence of interstitial lung disease or active, non-infectious pneumonitis were excluded from the study. [0195] AMG 160 was administered as a short IV infusion (approximately 60 minutes) every two weeks (Q2W)(e.g. on days 1 and 15) after target dose was reached in a 28-day cycle. In the first cohort, AMG 160 was dosed according to a single-step dosing regimen with 0.01 mg dosed on day 1 and 0.03 mg dosed on day 8 and day 22 of the first 28-day cycle and then at 0.03 mg dosed on day 1 and day 15 of cycle 2 and each subsequent cycle as in cohort 3b described in Example 1. In the second cohort, AMG 160 was dosed according to a two-step dosing regimen with 0.01 mg dosed on day 1, 0.09 mg dosed on day 8, and 0.3 mg dosed on day 15 of the first 28-day cycle and then at 0.3 mg dosed on day 1 and day 15 of cycle 2 and each subsequent cycle as in cohort 5 described in Example 1. Pembrolizumab was dosed at 200 mg IV (approximately 30- minute infusion) once every 4 weeks on AMG 160 dosing days with the first pembrolizumab administration given on the first day of target dose administration of AMG 160 (e.g. day 8 of cycle 1 in cohort 1 and day 15 of cycle 1 in cohort 2). Pembrolizumab infusion occurred after AMG 160 infusion and post-infusion flush. All patients were pre-treated with 8 mg PO dexamethasone 6-16 hours prior to all doses of AMG 160 in cycle 1. Additionally, dexamethasone 8 mg IV was administered within 1 hour prior to all doses of AMG 160 in cycle 1. Anti -tumor activity was evaluated as described in Example 1. Additional cohorts may be enrolled with AMG 160 dosed according to single-step, two-step, or three-step dosing regimens with target doses up to 1.8 mg, such as the dosing regimens described in Example 1 and Table 8. Pembrolizumab dose will remain fixed at 200 mg once every 4 weeks with the first administration of pembrolizumab given on the first day of target dose administration of AMG 160.

[0196] Six patients were enrolled in cohort 1 and three patients were enrolled in cohort 2. Five out of the nine patients remained on treatment as of the data cut-off date. Preliminary data showed that four out of the six patients in cohort 1 and all three patients in cohort 2 had grade 2 CRS as assessed by the Lee criteria. There were no grade 3, 4, or 5 CRS events in either cohort. Grade 3 adverse events were reported for 7 patients (77.8%). Grade 4 adverse events were reported for 4 patients (44.4%). There were no fatal adverse events. One patient (11.1%) had an adverse event leading to withdrawal of AMG 160. At the time of data cut-off, RECIST 1.1 responses among the 5 patients with measurable disease included 3 patients with stable disease. Six evaluable patients (66.6%) receiving AMG 160 in combination with pembrolizumab had PSA responses; PSA reductions > 50% occurred in 3 evaluable patients. One patient (25.0%) out of 4 with baseline CTC > 0 and post-baseline CTC assessment had a CTC0 response.

[0197] At the time of a second data cut-off date, there were 5 patients in cohort 1 and 3 patients in cohort 2 that had measurable disease at baseline per RECIST 1.1 with PCWG3 modifications and had received at least 1 dose of AMG 160 or pembrolizumab. All 5 patients in cohort 1 and 2 out of 3 patients in cohort 2 had a stable disease response. The remaining patient in cohort 2 had unconfirmed progressive disease.

[0198] All publications, patents, and patent applications discussed and cited herein are hereby incorporated by reference in their entireties. It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the appended claims.

[0199] 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. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed:

1. A method for treating prostate cancer in a patient in need thereof, comprising administering to the patient an initiation cycle and at least one maintenance cycle of a bispecific T-cell engaging molecule that specifically binds to human prostate-specific membrane antigen (PSMA) and human CD3, wherein the initiation cycle comprises administering one or more priming doses and a target dose of the bispecific T-cell engaging molecule at a dosing interval of at least 7 days for a first period of time, wherein the maintenance cycle comprises administering the target dose of the bispecific T-cell engaging molecule once every 14 days for a second period of time, wherein the target dose is greater than said one or more priming doses and is about 30 pg to about 1800 pg, and wherein the maintenance cycle is administered after the initiation cycle.

2. The method of claim 1, wherein said one or more priming doses are about 10 pg to about 300 pg.

3. The method of claim 1 or 2, wherein the first period of time is about 21 days to about 28 days.

4. The method of any one of claims 1 to 3, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose and the target dose, wherein the target dose is greater than the first priming dose and is administered about 7 days after the first priming dose.

5. The method of claim 4, wherein the target dose is administered a second time at least 14 days after the first administration of the target dose.

6. The method of any one of claims 1 to 3, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, a second priming dose, and the target dose, wherein the second priming dose is greater than the first priming dose and the target dose is greater than the second priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose and the target dose and is administered about 7 days after the second priming dose.

7. The method of any one of claims 1 to 3, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, a second priming dose, a third priming dose, and the target dose, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose, the third priming dose is administered about 7 days after the second priming dose, and the target dose and is administered about 7 days after the third priming dose.

8. The method of any one of claims 4 to 7, wherein the first priming dose is from about 10 pg to about 60 pg.

9. The method of any one of claims 6 to 8, wherein the second priming dose is from about 30 pg to about 180 pg.

10. The method of any one of claims 7 to 9, wherein the third priming dose is from about 60 pg to about 300 pg.

11. The method of any one of claims 1 to 10, wherein the target dose is about 90 pg to about 1800 pg.

12. The method of claim 11, wherein the target dose is about 300 pg to about 900 pg.

13. The method of claim 11, wherein the target dose is about 300 pg to about 600 pg.

14. The method of claim 6, wherein the first priming dose is about 10 pg to about 30 pg, the second priming dose is about 90 pg to about 180 pg, and the target dose is about 300 pg to about

900 pg.

15. The method of claim 7, wherein the first priming dose is about 10 pg to about 45 pg, the second priming dose is about 30 pg to about 110 pg, the third priming dose is about 90 pg to about 180 pg, and the target dose is about 300 pg to about 900 pg.

16. The method of claim 15, wherein the first priming dose is about 10 pg, the second priming dose is about 30 pg, the third priming dose is about 90 pg, and the target dose is about 900 pg.

17. The method of any one of claims 1 to 16, wherein the second period of time is about 28 days.

18. The method of any one of claims 1 to 17, wherein each of the doses of the bispecific T- cell engaging molecule administered during the initiation cycle and/or the maintenance cycle is administered as an intravenous infusion of about 30 min to about 90 min.

19. The method of any one of claims 1 to 18, wherein the maintenance cycle is administered the following day after completing the initiation cycle.

20. The method of any one of claims 1 to 18, wherein the maintenance cycle is administered about 7 days following completion of the initiation cycle.

21. The method of any one of claims 1 to 20, wherein two or more maintenance cycles are administered to the patient.

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22. The method of any one of claims 1 to 21, further comprising administering to the patient a glucocorticoid prior to administration of each dose of the bispecific T-cell engaging molecule during the initiation cycle.

23. The method of claim 22, wherein the glucocorticoid is dexamethasone.

24. The method of any one of claims 1 to 23, further comprising administering to the patient a tumor necrosis factor alpha (TNF-alpha) antagonist prior to administration of each dose of the bispecific T-cell engaging molecule during the initiation cycle.

25. The method of claim 24, wherein the TNF-alpha antagonist is etanercept.

26. The method of any one of claims 1 to 25, further comprising administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody during the initiation cycle and/or one or more maintenance cycles.

27. The method of claim 26, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, cemiplimab, or antibody 20C1.9.

28. The method of claim 26, wherein the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab.

29. The method of any one of claims 1 to 28, wherein the prostate cancer is metastatic prostate cancer.

30. The method of claim 29, wherein the prostate cancer is metastatic castration-resistant prostate cancer.

31. The method of any one of claims 1 to 30, wherein the patient has total serum testosterone levels of 50 ng/dL or less.

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32. The method of any one of claims 1 to 31, wherein the patient has failed or is intolerant to one or more chemotherapy regimens.

33. The method of claim 32, wherein the patient has failed or is intolerant to a taxane chemotherapy regimen.

34. The method of any one of claims 1 to 33, wherein the patient is refractory to one or more anti-androgen therapies.

35. The method of claim 34, wherein the anti-androgen therapy is abiraterone, enzalutamide, apalutamide, or darolutamide.

36. The method of any one of claims 1 to 35, wherein the patient has failed or is intolerant to a radioligand therapy.

37. The method of claim 36, wherein the radioligand therapy is ¹⁷⁷Lu-PSMA-617.

38. The method of any one of claims 1 to 37, wherein the bispecific T-cell engaging molecule comprises, in an amino to carboxyl order:

(i) a first domain that specifically binds to human PSMA comprising 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;

(ii) a second domain that specifically binds to human CD3 comprising 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; and

- 120 - (iii) a third domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, wherein said two monomers are fused to each other via a peptide linker.

39. The method of claim 38, wherein VH1 comprises the sequence of SEQ ID NO: 33 and VL1 comprises the sequence of SEQ ID NO: 30.

40. The method of claim 38 or 39, wherein VH2 comprises the sequence of SEQ ID NO: 72 and VL2 comprises the sequence of SEQ ID NO: 70.

41. The method of any one of claims 38 to 40, wherein the first and second binding domains are single-chain variable fragment (scFv) domains.

42. The method of any one of claims 38 to 41, wherein the first binding domain comprises the sequence of SEQ ID NO: 104.

43. The method of any one of claims 38 to 42, wherein the second binding domain comprises the sequence of SEQ ID NO: 116.

44. The method of any one of claims 38 to 43, wherein each of said Fc monomers of the third domain comprises the sequence of SEQ ID NO: 124.

45. The method of any one of claims 38 to 44, wherein the third domain comprises the sequence of SEQ ID NO: 132.

46. The method of any one of claims 38 to 45, wherein the bispecific T-cell engaging molecule is a single chain polypeptide.

47. The method of claim 46, wherein the bispecific T-cell engaging molecule comprises the sequence of SEQ ID NO: 140.

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48. A bispecific T-cell engaging molecule that specifically binds to human PSMA and human CD3 for use in a method for treating prostate cancer in a patient in need thereof, wherein the method comprises administering to the patient an initiation cycle and at least one maintenance cycle of the bispecific T-cell engaging molecule, wherein the initiation cycle comprises administering one or more priming doses and a target dose of the bispecific T-cell engaging molecule at a dosing interval of at least 7 days for a first period of time, wherein the maintenance cycle comprises administering the target dose of the bispecific T-cell engaging molecule once every 14 days for a second period of time, wherein the target dose is greater than said one or more priming doses and is about 30 pg to about 1800 pg, and wherein the maintenance cycle is administered after the initiation cycle.

49. The bispecific T-cell engaging molecule for use according to claim 48, wherein said one or more priming doses are about 10 pg to about 300 pg.

50. The bispecific T-cell engaging molecule for use according to claim 48 or 49, wherein the first period of time is about 21 days to about 28 days.

51. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 50, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose and the target dose, wherein the target dose is greater than the first priming dose and is administered about 7 days after the first priming dose.

52. The bispecific T-cell engaging molecule for use according to claim 51, wherein the target dose is administered a second time at least 14 days after the first administration of the target dose.

53. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 50, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, a second priming dose, and the target dose, wherein the second priming dose

- 122 - is greater than the first priming dose and the target dose is greater than the second priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose and the target dose and is administered about 7 days after the second priming dose.

54. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 50, wherein the initiation cycle comprises administering the bispecific T-cell engaging molecule at a first priming dose, a second priming dose, a third priming dose, and the target dose, wherein the second priming dose is greater than the first priming dose, the third priming dose is greater than the second priming dose, and the target dose is greater than the third priming dose, and wherein the second priming dose is administered about 7 days after the first priming dose, the third priming dose is administered about 7 days after the second priming dose, and the target dose and is administered about 7 days after the third priming dose.

55. The bispecific T-cell engaging molecule for use according to any one of claims 51 to 54, wherein the first priming dose is from about 10 pg to about 60 pg.

56. The bispecific T-cell engaging molecule for use according to any one of claims 53 to 55, wherein the second priming dose is from about 30 pg to about 180 pg.

57. The bispecific T-cell engaging molecule for use according to any one of claims 54 to 56, wherein the third priming dose is from about 60 pg to about 300 pg.

58. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 57, wherein the target dose is about 90 pg to about 1800 pg.

59. The bispecific T-cell engaging molecule for use according to claim 58, wherein the target dose is about 300 pg to about 900 pg.

60. The bispecific T-cell engaging molecule for use according to claim 58, wherein the target dose is about 300 pg to about 600 pg.

- 123 -

61. The bispecific T-cell engaging molecule for use according to claim 53, wherein the first priming dose is about 10 pg to about 30 pg, the second priming dose is about 90 pg to about 180 pg, and the target dose is about 300 pg to about 900 pg.

62. The bispecific T-cell engaging molecule for use according to claim 54, wherein the first priming dose is about 10 pg to about 45 pg, the second priming dose is about 30 pg to about 110 pg, the third priming dose is about 90 pg to about 180 pg, and the target dose is about 300 pg to about 900 pg.

63. The bispecific T-cell engaging molecule for use according to claim 62, wherein the first priming dose is about 10 pg, the second priming dose is about 30 pg, the third priming dose is about 90 pg, and the target dose is about 900 pg.

64. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 63, wherein the second period of time is about 28 days.

65. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 64, wherein each of the doses of the bispecific T-cell engaging molecule administered during the initiation cycle and/or the maintenance cycle is administered as an intravenous infusion of about 30 min to about 90 min.

66. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 65, wherein the maintenance cycle is administered the following day after completing the initiation cycle.

67. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 65, wherein the maintenance cycle is administered about 7 days following completion of the initiation cycle.

68. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 67, wherein two or more maintenance cycles are administered to the patient.

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69. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 68, wherein the method further comprises administering to the patient a glucocorticoid prior to administration of each dose of the bispecific T-cell engaging molecule during the initiation cycle.

70. The bispecific T-cell engaging molecule for use according to claim 69, wherein the glucocorticoid is dexamethasone.

71. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 70, wherein the method further comprises administering to the patient a TNF-alpha antagonist prior to administration of each dose of the bispecific T-cell engaging molecule during the initiation cycle.

72. The bispecific T-cell engaging molecule for use according to claim 71, wherein the TNF- alpha antagonist is etanercept.

73. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 72, wherein the method further comprises administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody during the initiation cycle and/or one or more maintenance cycles.

74. The bispecific T-cell engaging molecule for use according to claim 73, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, cemiplimab, or antibody 20C1.9.

75. The bispecific T-cell engaging molecule for use according to claim 73, wherein the PD- L1 antagonist antibody is atezolizumab, avelumab, or durvalumab.

76. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 75, wherein the prostate cancer is metastatic prostate cancer.

77. The bispecific T-cell engaging molecule for use according to claim 76, wherein the prostate cancer is metastatic castration-resistant prostate cancer.

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78. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 77, wherein the patient has total serum testosterone levels of 50 ng/dL or less.

79. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 78, wherein the patient has failed or is intolerant to one or more chemotherapy regimens.

80. The bispecific T-cell engaging molecule for use according to claim 79, wherein the patient has failed or is intolerant to a taxane chemotherapy regimen.

81. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 80, wherein the patient is refractory to one or more anti-androgen therapies.

82. The bispecific T-cell engaging molecule for use according to claim 81, wherein the antiandrogen therapy is abiraterone, enzalutamide, apalutamide, or darolutamide.

83. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 82, wherein the patient has failed or is intolerant to a radioligand therapy.

84. The bispecific T-cell engaging molecule for use according to claim 83, wherein the radioligand therapy is ¹⁷⁷Lu-PSMA-617.

85. The bispecific T-cell engaging molecule for use according to any one of claims 48 to 84, wherein the bispecific T-cell engaging molecule comprises, in an amino to carboxyl order:

(ii) a second domain that specifically binds to human CD3 comprising a second immunoglobulin heavy chain variable region (VH2) comprising a CDRH1 having the sequence

- 126 - 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; and

86. The bispecific T-cell engaging molecule for use according to claim 85, wherein VH1 comprises the sequence of SEQ ID NO: 33 and VL1 comprises the sequence of SEQ ID NO: 30.

87. The bispecific T-cell engaging molecule for use according to claim 85 or 86, wherein VH2 comprises the sequence of SEQ ID NO: 72 and VL2 comprises the sequence of SEQ ID NO: 70.

88. The bispecific T-cell engaging molecule for use according to any one of claims 85 to 87, wherein the first and second binding domains are single-chain variable fragment (scFv) domains.

89. The bispecific T-cell engaging molecule for use according to any one of claims 85 to 88, wherein the first binding domain comprises the sequence of SEQ ID NO: 104.

90. The bispecific T-cell engaging molecule for use according to any one of claims 85 to 89, wherein the second binding domain comprises the sequence of SEQ ID NO: 116.

91. The bispecific T-cell engaging molecule for use according to any one of claims 85 to 90, wherein each of said Fc monomers of the third domain comprises the sequence of SEQ ID NO: 124.

92. The bispecific T-cell engaging molecule for use according to any one of claims 85 to 91, wherein the third domain comprises the sequence of SEQ ID NO: 132.

93. The bispecific T-cell engaging molecule for use according to any one of claims 85 to 92, wherein the bispecific T-cell engaging molecule is a single chain polypeptide.

94. The bispecific T-cell engaging molecule for use according to claim 93, wherein the bispecific T-cell engaging molecule comprises the sequence of SEQ ID NO: 140.

95. Use of a bispecific T-cell engaging molecule that specifically binds to human PSMA and human CD3 for the manufacture of a medicament for the treatment of prostate cancer in a patient in need thereof, wherein the treatment comprises administering to the patient an initiation cycle and at least one maintenance cycle of the bispecific T-cell engaging molecule, wherein the initiation cycle comprises administering one or more priming doses and a target dose of the bispecific T-cell engaging molecule at a dosing interval of at least 7 days for a first period of time, wherein the maintenance cycle comprises administering the target dose of the bispecific T-cell engaging molecule once every 14 days for a second period of time, wherein the target dose is greater than said one or more priming doses and is about 30 pg to about 1800 pg, and wherein the maintenance cycle is administered after the initiation cycle.