Methods for treating metastatic castration-resistant prostate cancer using a bispecific anti-PSMA x anti-CD28 antibody in combination with an anti-PD-1 antibody

Abstract

The present disclosure provides methods for treating, reducing the severity of, or inhibiting the growth of cancer (e.g., prostate cancer or metastatic castration-resistant prostate cancer). The methods of the disclosure include administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody, or antigen-binding fragment thereof, that specifically binds prostate-specific membrane antigen (PSMA) and CD28, in combination with an antibody, or antigen-binding fragment thereof, that specifically binds programmed death receptor-1 (PD-1).

Description

[Technical Field]

[0001] Sequence Listing Reference This application incorporates by reference a computer readable sequence listing in ST.26 XML format entitled 11051WO01_Sequence, created on July 28, 2023, and containing 58,900 bytes.

[0002] The present invention relates to a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody that specifically binds to prostate-specific membrane antigen (PSMA) and CD28, in combination with an antibody that specifically binds to programmed death receptor-1 (PD-1). [Background technology]

[0003] Prostate-specific membrane antigen (PSMA), also known as FOLH1, glutamic acid carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamic acid peptidase I (NAALADase I), or N-acetyl-aspartylglutamic acid (NAAG) peptidase, is a homodimeric enzyme type II transmembrane protein encoded by the folate hydrolase 1 (FOLH1) gene. PSMA is an integral, non-transmembrane glycoprotein highly expressed in malignant prostate tissue and is a cell surface marker for prostate cancer, but shows limited expression on normal tissue. Its expression persists in castration-resistant prostate cancer, which has a poor prognosis and limited treatment options. Methods for treating prostate cancer by targeting PSMA are being investigated. For example, yttrium-90 capromab is a radiotherapeutic agent containing a monoclonal antibody directed against an intracellular epitope of PSMA. In another example, J591, a monoclonal antibody directed against an extracellular epitope of PSMA, is part of the radiotherapeutic agent lutetium-177 J591, and maytansinoid 1 (DM1, an anti-microtubule agent) is conjugated to J591 in MLN2704. These therapies are associated with toxicity. PSMA is also expressed within the angiogenesis of other tumors, such as bladder, kidney, stomach, and colorectal cancers.

[0004] CD28 is a type I transmembrane protein with a single extracellular IgV-like domain assembled as a homodimer and expressed on the surface of T cells. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is activated by CD80 or CD86 expressed on antigen-presenting cells (APCs). Binding of CD28 to CD80 or CD86 provides a costimulatory signal important for T cell activation and survival. CD28-mediated T cell stimulation, in addition to the T cell receptor (TCR), provides a potent signal for the production of various interleukins. CD28 also enhances cellular signals, such as pathways regulated by the NFκB transcription factor, after TCR activation. CD28 costimulation is important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death. Anti-CD28 antibodies have been proposed for therapeutic purposes, including T cell activation. One specific anti-CD28 antibody, TGN1412 (an anti-CD28 superagonist), was used in a clinical trial in 2006. Six healthy volunteers were given TGN1412 (an anti-CD28 superagonist) intravenously at a dose of 0.1 mg / kg. Within two hours, all six patients developed a significant inflammatory response (cytokine storm), and all patients developed multiple organ failure within 16 hours. The subjects were treated with corticosteroids, and cytokine levels returned to normal within two to three days (Suntharalingam, et al., "Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412," NEJM 355:1018-1028 (2006)).

[0005] Programmed death receptor-1 (PD-1) signaling in the tumor microenvironment plays a key role in enabling tumor cells to escape immunosurveillance by the host immune system. Blockade of the PD-1 signaling pathway has demonstrated clinical activity in patients with multiple tumor types, and PD-1-blocking antibody therapeutics (e.g., nivolumab and pembrolizumab) have been approved for the treatment of metastatic melanoma and metastatic squamous non-small cell lung cancer. Recent data have demonstrated the clinical activity of PD-1 blockade in patients with aggressive NHL and Hodgkin lymphoma (Lesokhin, et al. 2014, Abstract 291, 56th ASH Annual Meeting and Exposition, San Francisco, Calif.; Ansell et al. 2015, N. Engl. J. Med. 372(4):311-9).

[0006] Prostate cancer is the leading cause of new cancer diagnoses and the second leading cause of cancer-related deaths among men in the United States. In 2018, there were an estimated 1.3 million new cases of prostate cancer worldwide, resulting in 358,989 deaths. Therapies that block androgen-related pathways have been the standard of care for prostate cancer for decades. However, patients progress through androgen deprivation and / or surgical castration and develop castration-resistant prostate cancer. The prognosis is particularly poor for men with metastatic castration-resistant prostate cancer (mCRPC). Currently, metastatic prostate cancer remains incurable, and improving long-term survival remains a high unmet need. Summary of the Invention

[0007] According to certain embodiments, the present disclosure provides a method for treating, ameliorating, or inhibiting the growth of at least one symptom or sign of a PSMA-expressing cancer in a subject. The method according to this aspect of the disclosure comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody, or antigen-binding fragment thereof, that specifically binds prostate-specific membrane antigen (PSMA) and CD28, in combination with an antibody, or antigen-binding fragment thereof, that specifically binds programmed death receptor-1 (PD-1).

[0008] Certain embodiments of the present disclosure provide methods for treating, ameliorating at least one symptom or sign of, or inhibiting the growth of, a PSMA-expressing cancer in a subject. Certain embodiments of the present disclosure provide methods for slowing tumor growth or preventing tumor recurrence. Methods according to this and other aspects of the disclosure include sequentially administering to a subject in need thereof a therapeutically effective amount of one or more doses of a bispecific anti-PSMA x anti-CD28 antibody or antigen-binding fragment thereof in combination with a therapeutically effective amount of one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof.

[0009] In one aspect, the disclosure provides a method of treating a PSMA-expressing cancer in a subject in need thereof, comprising administering to the subject a combination of a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to prostate-specific membrane antigen (PSMA) on a target tumor cell and a second antigen-binding domain that specifically binds to human CD28 on a T cell, and an antibody or antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the bispecific antibody is administered to the subject at a dose of at least 0.03 mg.

[0010] In some embodiments, the PSMA-expressing cancer is prostate cancer. In some cases, the PSMA-expressing cancer is metastatic prostate cancer. In some cases, the PSMA-expressing cancer is castration-resistant prostate cancer.

[0011] In some embodiments, the subject has received at least two prior therapies for metastatic and / or castration-resistant prostate cancer. In some cases, the subject has received at least one anti-androgen therapy. In some embodiments, the anti-androgen therapy is selected from abiraterone, enzalutamide, apalutamide, or darolutamide.

[0012] In some embodiments, the subject has histologically or cytologically confirmed adenocarcinoma of the prostate that does not have pure small cell carcinoma.

[0013] In some embodiments, the subject has metastatic castration-resistant prostate cancer with a prostate-specific antigen (PSA) level of ≧4 ng / ml prior to treatment with the bispecific antibody. In some cases, the subject's cancer has progressed within 6 months prior to treatment with the bispecific antibody, with cancer progression determined by (a) a confirmed rise in PSA levels with an interval of ≧1 week between assessments, (b) soft tissue radiographic disease progression with or without a rise in PSA, and / or (c) bone radiographic disease progression with the appearance of two or more bone lesions on a bone scan with or without a rise in PSA.

[0014] In some embodiments, the subject has undergone an orchiectomy. In some embodiments, the subject has undergone luteinizing hormone-releasing hormone (LHRH) agonist or antagonist therapy and has a serum testosterone level of <50 ng / ml prior to treatment with the bispecific antibody.

[0015] In some embodiments, the first antigen-binding domain of the bispecific antibody comprises (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 9. In some cases, the first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4. In some cases, the first antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1, and an LCVR comprising the amino acid sequence of SEQ ID NO: 9.

[0016] In some embodiments, the second antigen-binding domain of the bispecific antibody comprises (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 5, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 9. In some cases, the second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some cases, the second antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 5, and an LCVR comprising the amino acid sequence of SEQ ID NO: 9.

[0017] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:13.

[0018] In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:14.

[0019] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 13, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 14, and a common light chain comprising the amino acid sequence of SEQ ID NO: 15.

[0020] In some embodiments, the first antigen-binding domain of the bispecific antibody comprises (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 16, and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 28. In some cases, the first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some cases, the first antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 31. In some cases, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 16, and an LCVR comprising the amino acid sequence of SEQ ID NO: 28.

[0021] In some embodiments, the second antigen-binding domain of the bispecific antibody comprises (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 20 of SEQ ID NO: 24, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 28. In some cases, the second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 27. In some cases, the second antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:24, and an LCVR comprising the amino acid sequence of SEQ ID NO:28.

[0022] In any of the various embodiments described above or discussed herein, the bispecific antibody may comprise a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is of the isotype IgG1. In some cases, the human IgG heavy chain constant region is of the isotype IgG4.

[0023] In any of the various embodiments described above or discussed herein, the bispecific antibody may comprise a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0024] In any of the various embodiments described above or discussed herein, the first heavy chain of the bispecific antibody or the second heavy chain of the bispecific antibody (but not both) may comprise a CH3 domain comprising the H435R (EU numbering) variant and the Y436F (EU numbering) variant.

[0025] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:32.

[0026] In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:33.

[0027] In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:34.

[0028] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and a common light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0029] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and a common light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0030] In any of the various embodiments described above or discussed herein, the antibody or antigen-binding fragment thereof that binds PD-1 comprises (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 36, and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 40. In some cases, the antibody or antigen-binding fragment thereof that binds PD-1 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some cases, the antibody or antigen-binding fragment thereof that binds PD-1 comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 41, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment thereof that binds PD-1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 36, and an LCVR comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment thereof that binds PD-1 is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the amino acid sequence of SEQ ID NO: 45.

[0031] In any of the various embodiments described above or discussed herein, the bispecific antibody or antigen-binding fragment thereof may be administered to a subject once weekly at a dose of 0.03 mg to 1000 mg. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject once weekly at a dose of 0.03 mg to 900 mg. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject once weekly at a dose of 30 mg to 900 mg. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject once weekly at a dose of 100 mg to 900 mg. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject once weekly at a dose of 300 mg to 900 mg.

[0032] In any of the various embodiments described above or discussed herein, the bispecific antibody or antigen-binding fragment thereof may be administered to a subject at a dose of 0.03 mg to 1000 mg once every three weeks. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject at a dose of 0.03 mg to 900 mg once every three weeks. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject at a dose of 30 mg to 900 mg once every three weeks. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject at a dose of 100 mg to 900 mg once every three weeks. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to a subject at a dose of 300 mg to 900 mg once every three weeks.

[0033] In any of the various embodiments described above or discussed herein, the antibody or antigen-binding fragment thereof that binds PD-1 may be administered to a subject at a dose of 300-400 mg once every three weeks. In some instances, the antibody or antigen-binding fragment thereof that binds PD-1 is administered to a subject at a dose of 350 mg once every three weeks.

[0034] In any of the various embodiments described above or discussed herein, the subject experiences stable disease, a partial response, or a complete response after at least one week of administration of the bispecific antibody or antigen-binding fragment thereof, in combination with the antibody or antigen-binding fragment thereof that binds PD-1, at a dose of between 0.03 mg and 900 mg.

[0035] In any of the various embodiments described above or herein, the subject may further be administered an IL-6R antagonist. In some cases, the IL-6R antagonist is an anti-IL-6R antibody. In some cases, the anti-IL-6R antibody is sarilumab or tocilizumab.

[0036] In any of the various embodiments described above or discussed herein, the subject, after administration of a combination of a bispecific anti-PSMAxCD28 antibody (e.g., REGN5678) or antigen-binding fragment thereof and an anti-PD-1 antibody (e.g., cemiplimab) or antigen-binding fragment thereof: ·At least a 50% reduction in prostate-specific antigen (PSA) levels in the subject; ·At least a 55% decrease in PSA levels in the subject; ·At least a 60% decrease in PSA levels in the subject; ·At least a 65% reduction in PSA levels in the subject; ·At least a 70% reduction in PSA levels in the subject; ·At least a 75% reduction in PSA levels in the subject; ·At least an 80% reduction in PSA levels in the subject; ·At least an 85% reduction in PSA levels in the subject; ·At least a 90% reduction in PSA levels in the subject; ·At least a 95% reduction in PSA levels in the subject; ·At least a 96% reduction in PSA levels in the subject; ·At least a 97% reduction in PSA levels in the subject; ·At least a 98% reduction in PSA levels in the subject; ·At least a 99% reduction in PSA levels in the subject; a reduction in the size of at least one lesion having a PSMA PET signal that is smaller than the PSMA PET signal in the subject's liver, and / or - Response in subjects after pseudoprogression,

[0037] The present disclosure also encompasses the use of bispecific antibodies and / or anti-PD-1 antibodies (and any antigen-binding fragments) in the manufacture of a medicament for treating a PSMA-expressing cancer as described above or in any of the method embodiments discussed herein. The present disclosure also encompasses bispecific antibodies and / or anti-PD-1 antibodies (and any antigen-binding fragments) for use in any of the method embodiments discussed above or herein. The present disclosure also encompasses pharmaceutical compositions comprising bispecific antibodies and / or anti-PD-1 antibodies (and any antigen-binding fragments) for use in any of the method embodiments discussed above or herein.

[0038] In one aspect, the disclosure provides a method of treating a solid tumor in a subject in need thereof, comprising administering to the subject a combination of a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds a tumor-associated antigen on tumor cells and a second antigen-binding domain that specifically binds human CD28 on T cells, and an antibody or antigen-binding fragment thereof that specifically binds programmed death receptor-1 (PD-1).

[0039] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0040] Other embodiments of the present invention will become apparent upon consideration of the detailed description that follows. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 shows an embodiment of a study flow diagram for QW dosing of REGN5678 in combination with Q3W dosing of cemiplimab, as discussed in Example 5. 1 Dose cohorts receiving QW dosing of REGN5678 may receive a 3-week monotherapy lead-in of REGN5678 QW, followed by combination therapy of REGN5678 QW and cemiplimab Q3W. 2 For some dose expansion cohorts, PSMA-PET / CT and optional whole-body FDG-PET / CT will be performed at screening, 12-24 weeks after treatment initiation, and at disease progression. These PET / CT scans are optional for dose-escalation patients. DLT = dose-limiting toxicity, PD = disease progression, Q6W = every 6 weeks, Q12W = every 12 weeks. [Figure 2]Figure 2 shows an embodiment of a study flow diagram for Q3W dosing of REGN5678 in combination with Q3W dosing of cemiplimab, as discussed in Example 5. 1 Q3W dosing interval dose cohorts may receive a 3-week monotherapy lead-in of REGN5678, followed by combination therapy of REGN5678 and cemiplimab. 2 For some dose expansion cohorts, PSMA-PET / CT and optional whole-body FDG-PET / CT will be performed at screening, 12-24 weeks after treatment initiation, and at disease progression. These PET / CT scans are optional for dose-escalation patients. DLT = dose-limiting toxicity, PD = disease progression, Q6W = every 6 weeks, Q12W = every 12 weeks. [Figure 3A] Figures 3A, 3B, 3C, and 3D are prostate-specific antigen (PSA) waterfall plots showing the results (for the first 33 patients) of the combined administration of mAb1 (0.1 mg to 300 mg) and cemiplimab to patients with metastatic castration-resistant prostate cancer. Each figure shows the best percent change in PSA level from baseline for patients receiving various doses of mAb1 in combination with cemiplimab (baseline corresponds to the PSA drawn immediately before the first combined administration). Figure 3A shows all mAb1 dose levels together. Figure 3B shows doses from 0.1 mg to 10 mg. Figure 3C shows doses from 30 mg to 300 mg. Figure 3D shows all doses for doses ≥ 30 mg versus < 30 mg. Dose levels (DL) correspond to those shown in Table 8. DL numbers are indicated above or below each bar in Figures 3A, 3B, and 3C, and bars corresponding to DLs 6 to 8 in Figure 3D are identified with an asterisk. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 4A]Figures 4A and 4B are prostate-specific antigen (PSA) waterfall plots showing the results (days for the first 35 patients) of the combined administration of mAb1 (0.1 mg to 300 mg) and cemiplimab to patients with metastatic castration-resistant prostate cancer. Each figure shows the best percent change in PSA level from baseline for patients receiving various doses of mAb1 in combination with cemiplimab (baseline corresponds to the PSA drawn immediately before the first combined administration). Figure 4A shows doses from 0.1 mg to 10 mg. Figure 4B shows doses from 30 mg to 300 mg. Dose levels (DL) correspond to those shown in Table 8. DL numbers are indicated above or below each bar in Figures 4A and 4B. [Figure 4B] Same as above. [Figure 5] Figure 5 shows the decline in PSA levels in patients treated at dose level 8 (300 mg mAb1 and 350 mg cemiplimab). PSMAxCD28 corresponds to mAb1 and Libtayo™ is cemiplimab. [Figure 6] 6 shows an embodiment of a study flow diagram of QW dosing of REGN5678 in combination with Q3W dosing of cemiplimab and sarilumab, as discussed in Example 5. As shown, the dose of sarilumab is 350 mg IV Q3W for a total of 12 weeks, starting with the first dose of REGN5678 in combination with cemiplimab (C1D1). [Figure 7] 7 shows an embodiment of a study flow diagram of Q3W dosing of REGN5678 in combination with Q3W dosing of cemiplimab and sarilumab, as discussed in Example 5. As shown, the dose of sarilumab is 350 mg IV Q3W for a total of 12 weeks, starting with the first dose of REGN5678 in combination with cemiplimab (C1D1). DETAILED DESCRIPTION OF THE INVENTION

[0042] Before describing the present invention, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Any embodiments or features of the embodiments may be combined with each other, and such combinations are expressly encompassed within the scope of the present invention. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about," when used in connection with a specific recited numerical value or range of values, means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0044] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0045] Methods for treating or inhibiting the growth of cancer The present disclosure includes methods for treating, ameliorating, or reducing the severity of at least one symptom or sign, or inhibiting the growth of cancer (e.g., metastatic castration-resistant prostate cancer) in a subject. The method according to this aspect of the disclosure includes administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody against PSMA and CD28, in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds PD-1. As used herein, the terms "treat," "treating," and the like, mean alleviating symptoms, eliminating the cause of symptoms, either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell mass or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, and / or prolonging the survival of a subject.

[0046] As used herein, the phrase "subject" or "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of cancer, and / or a human or non-human mammal diagnosed with cancer, including prostate cancer (e.g., metastatic castration-resistant prostate cancer), and in need of treatment. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may have a primary tumor or a metastatic tumor and / or be diagnosed with one or more symptoms or signs, including, but not limited to, enlarged lymph nodes, abdominal distension, unexplained pain, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, and / or an enlarged spleen. This phrase includes subjects with primary or established prostate cancer. In specific embodiments, this phrase includes human subjects with prostate cancer or another tumor that expresses PSMA and in need of treatment. In other specific embodiments, this phrase includes subjects with PSMA+ tumors (e.g., tumors with PSMA expression as determined by flow cytometry). In certain embodiments, the phrase "subject in need thereof" includes patients with prostate cancer that is resistant, refractory, or inadequately controlled to prior therapy (e.g., treatment with conventional anticancer agents, including antiandrogen therapy). For example, the phrase includes subjects treated with chemotherapy or antiandrogen therapy, such as abiraterone, enzalutamide, apalutamide, or darolutamide. The phrase also includes subjects with prostate cancer for whom conventional anticancer therapy is not advisable, e.g., due to toxic side effects. For example, the phrase includes patients who have received one or more cycles of chemotherapy or other anticancer therapy with toxic side effects. In certain embodiments, the phrase "subject in need thereof" includes patients with treated prostate cancer that has subsequently recurred or metastasized. For example, patients with prostate cancer who may have undergone treatment with one or more anticancer agents and experienced tumor regression, but who subsequently recur with cancer resistant to the one or more anticancer agents (e.g., castration-resistant prostate cancer), are treated with the methods of the present disclosure.

[0047] In certain embodiments, the methods of the present disclosure may be used to treat patients with histologically or cytologically confirmed adenocarcinoma of the prostate without pure small cell carcinoma. In certain embodiments, the methods of the present disclosure may be used to treat patients with metastatic castration-resistant prostate cancer with a prostate-specific antigen (PSA) level of ≧4 ng / ml (e.g., ≧4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 6.5 ng / ml, 7 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml, 9.5 ng / ml, or 10 ng / ml) prior to treatment with the bispecific antibody. In certain embodiments, the methods of the present disclosure may be used prior to treatment with a bispecific antibody to treat patients with prostate cancer that has progressed within a period of time (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more), where cancer progression is determined by, for example, (a) a confirmed rise in PSA levels at an interval of ≥ 1 week (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks or more), (b) radiographic (e.g., PET / CT imaging) disease progression in soft tissue with or without a rise in PSA, and / or (c) radiographic (e.g., PET / CT imaging) disease progression of bone with the appearance of two or more bone lesions on a bone scan with or without a rise in PSA. In certain embodiments, the methods of the present disclosure may be used to treat patients who have undergone orchiectomy. In certain embodiments, the methods of the disclosure may be used to treat patients having or undergoing luteinizing hormone-releasing hormone (LHRH) agonist or antagonist therapy, and having serum testosterone levels of <50 ng / ml (e.g., 1 ng / ml to 49 ng / ml, about 45 ng / ml, about 40 ng / ml, about 35 ng / ml, about 30 ng / ml, about 25 ng / ml, about 20 ng / ml, about 15 ng / ml, about 10 ng / ml, or about 5 ng / ml) prior to treatment with the bispecific antibody.

[0048] In certain embodiments, the methods of the present disclosure are used in subjects with prostate cancer. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein. As used herein, the term "prostate cancer" refers to tumors of the prostate, including metastatic tumors originating from the prostate.

[0049] According to certain embodiments, the present disclosure includes methods of treating, slowing, or inhibiting tumor growth. In certain embodiments, the present disclosure includes methods of promoting tumor regression. In certain embodiments, the present disclosure includes methods of reducing tumor cell burden or reducing tumor volume. In certain embodiments, the present disclosure includes methods of preventing tumor recurrence. Methods according to this aspect of the disclosure include administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, wherein each antibody or fragment is administered to the subject in multiple doses, e.g., as part of a particular therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering one or more doses of an anti-PSMAxCD28 antibody or antigen-binding fragment thereof to a subject approximately once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every week, once every two weeks, once every three weeks, once every four weeks, once every month, once every two months, once every three months, once every four months, or less frequently. In certain embodiments, the anti-PSMAxanti-CD28 antibody or antigen-binding fragment thereof is administered weekly. In certain embodiments, the anti-PSMAxanti-CD28 antibody or antigen-binding fragment thereof is administered once every three weeks. In certain embodiments, one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof are administered to a subject approximately once every day, every two days, every three days, every four days, every five days, every six days, every week, every two weeks, every three weeks, every four weeks, every month, every two months, every three months, every four months, or less frequently. In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to a subject once every three weeks.

[0050] In certain embodiments, the present disclosure includes a method of inhibiting, slowing, or stopping tumor metastasis or tumor invasion to peripheral organs, which method, according to this aspect, comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof.

[0051] In certain embodiments, the anti-PSMA / CD28 bispecific antibody or antigen-binding fragment thereof is administered to a subject before the anti-PD-1 antibody or antigen-binding fragment thereof. In some cases, the anti-PSMA / CD28 antibody or antigen-binding fragment thereof may be administered about 1 day, more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, 2 weeks, 3 weeks, or more before the anti-PD-1 antibody or antigen-binding fragment thereof.

[0052] In certain embodiments, the methods of the present disclosure are used to treat patients with MRD-positive disease. Minimal residual disease (MRD) refers to the small number of cancer cells that remain in a patient during or after treatment, and the patient may or may not exhibit symptoms or signs of disease. If not removed, these residual cancer cells often lead to disease recurrence. The present disclosure includes methods for inhibiting and / or removing residual cancer cells in patients at the time of MRD testing. MRD can be assayed according to methods known in the art (e.g., MRD flow cytometry). According to this aspect of the disclosure, the method includes administering a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to a subject in need thereof.

[0053] According to certain embodiments, the methods of the disclosure comprise administering to a subject a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody, or antigen-binding fragment thereof, in combination with an anti-PD-1 antibody, or antigen-binding fragment thereof, and optionally a third therapeutic agent. The third therapeutic agent may be, for example, an agent selected from the group consisting of radiation, chemotherapy, surgery, a cancer vaccine, a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody), a LAG3 inhibitor (e.g., an anti-LAG3 antibody), a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody against a tumor-specific antigen, a cytotoxin, a chemotherapeutic agent, an anti-androgen therapy, cytokines such as IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, and IL-15, anti-inflammatory agents such as corticosteroids and nonsteroidal anti-inflammatory agents, and nutritional supplements such as antioxidants. In certain embodiments, the antibody may be administered in combination with therapies including chemotherapeutic agents, radiation, and surgery. As used herein, the phrase "in combination with" means that the antibody is administered to the subject simultaneously with, immediately before, or immediately after the administration of a third therapeutic agent. In certain embodiments, the antibody and the third therapeutic agent are administered in separate formulations.

[0054] In certain embodiments, the methods of the disclosure comprise administering a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to a subject in need thereof. In certain embodiments, administration of the combination inhibits tumor growth by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to an untreated subject. In certain embodiments, administration of the combination enhances tumor regression, tumor reduction, and / or elimination. In certain embodiments, administration of the combination leads to a delay in tumor growth and progression; for example, tumor growth may be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years compared to an untreated subject. In certain embodiments, administration of the combination prevents tumor recurrence and / or increases the subject's survival, e.g., increases the duration of survival by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months compared to an untreated subject. In certain embodiments, administration of the combination increases progression-free survival or overall survival. In certain embodiments, administration of the combination increases the response and duration of response in a subject by, e.g., more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to an untreated subject. In certain embodiments, administration of the combination to a subject with prostate cancer results in the complete disappearance of all evidence of tumor cells (a "complete response"). In certain embodiments, administration of the combination to a subject with prostate cancer results in a reduction of tumor cells or tumor size by at least 30% or more ("partial response"). In certain embodiments, administration of the combination to a subject with prostate cancer results in the complete or partial disappearance of tumor cells / lesions, including new measurable lesions. Tumor reduction can be measured by any of the methods known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.In some cases, PET / CT imaging uses radioactive tracers (e.g., ) to detect lesions in patients with metastatic prostate cancer (e.g., mCRPC). 18 In certain embodiments, administration of a bispecific antibody or antigen-binding fragment thereof with an anti-PD-1 antibody or antigen-binding fragment thereof results in a synergistic anti-tumor effect that exceeds the combined effect of the two agents when administered alone.

[0055] In certain cases, a subject's response to therapy is classified as complete response (CR), partial response (PR), progressive disease (PD), or stable disease (SD). CR is defined as the disappearance of all target lesions and a reduction in the short axis of any pathological lymph nodes (target or non-target) to <10 mm (<1 cm). PR is defined as at least a 30% reduction in the sum of target lesion diameters, relative to the baseline sum of diameters. PD is defined as at least a 20% increase in the sum of target lesion diameters, relative to the minimum study sum (including the baseline sum, if that sum is the minimum study sum). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm (0.5 cm). (Note: The appearance of one or more new lesions also counts as progression.) SD is defined as neither sufficient shrinkage, relative to the minimum study sum of diameters, to qualify for PR nor sufficient increase to qualify for PD.

[0056] Anti-PD-1 antibodies and their antigen-binding fragments According to certain exemplary embodiments of the present disclosure, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof. As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (HCVR or VL) (referred to herein as a VL). H The heavy chain constant region comprises three domains: C H 1. C H2 and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are relatively conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an anti-IL-4R antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0057] As used herein, the term "antibody" also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard method, such as, for example, proteolytic or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable regions and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0058] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0059] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domain and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may contain domains.

[0060] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (e.g., via disulfide bonds).

[0061] The term "antibody" as used herein also includes multispecific (e.g., bispecific) antibodies. Multispecific antibodies or antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format can be adapted for use in connection with the antibodies or antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art. For example, the present disclosure includes methods comprising the use of bispecific antibodies in which one arm of the immunoglobulin is specific for PD-1 or a fragment thereof and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety. Exemplary bispecific formats that can be used in connection with the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (such as a common light chain with knobs-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab.sup.2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a discussion of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0062] The antibody used in the methods of the present disclosure may be a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0063] The antibody used in the methods of the present disclosure may be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector (described in more detail below) transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby increasing the V H Area and V L The amino acid sequence of the region is human germline V H Sequence and V LWhile derived from and related to sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0064] According to certain embodiments, the antibodies used in the methods of the present disclosure specifically bind to PD-1. The term "specifically binds" and the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, as used in the context of the present disclosure, an antibody that "specifically binds" to PD-1 has a K of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by a surface plasmon resonance assay. D However, an isolated antibody that specifically binds human PD-1 may exhibit cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

[0065] According to certain exemplary embodiments of the present disclosure, an anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR) comprising any of the amino acid sequences of the anti-PD-1 antibodies described in U.S. Patent No. 9,987,500. In certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof that may be used in the context of the methods of the present disclosure comprises a heavy chain complementarity-determining region (HCDR) of the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:36 and a light chain complementarity-determining region (LCDR) of the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:40. According to certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 37, HCDR2 comprises the amino acid sequence of SEQ ID NO: 38, HCDR3 comprises the amino acid sequence of SEQ ID NO: 39, LCDR1 comprises the amino acid sequence of SEQ ID NO: 41, LCDR2 comprises the amino acid sequence of SEQ ID NO: 42, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 43. In yet other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 36 and a LCVR comprising SEQ ID NO: 40. In certain embodiments, the methods of the disclosure involve the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 45. An exemplary antibody comprising a HCVR comprising the amino acid sequence of SEQ ID NO:36 and a LCVR comprising the amino acid sequence of SEQ ID NO:40 is the fully human anti-PD-1 antibody, also known as REGN2810 (cemiplimab; LIBTAYO®). According to certain exemplary embodiments, the methods of the disclosure comprise the use of REGN2810, or a biological equivalent thereof. As used herein, the term "bioequivalent" refers to an anti-PD-1 antibody or PD-1 binding protein, or fragment thereof, that is a pharmaceutical equivalent or pharmaceutical substitute that exhibits a rate and / or extent of absorption that does not differ significantly from that of REGN2810 when administered in either single or multiple doses at the same molar dose under similar experimental conditions.In the context of the present disclosure, the term refers to an antigen binding protein that binds to PD-1 that has no clinically meaningful differences in safety, purity and / or potency from REGN2810.

[0066] Other anti-PD-1 antibodies that may be used in the context of the methods of the present disclosure include antibodies referred to or known in the art as, for example, nivolumab (U.S. Pat. No. 8,008,449), pembrolizumab (U.S. Pat. No. 8,354,509), MEDI0608 (U.S. Pat. No. 8,609,089), pidilizumab (U.S. Pat. No. 8,686,119), or any of the anti-PD-1 antibodies described in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0067] Anti-PD-1 antibodies used in the context of the methods of the present disclosure may have pH-dependent binding characteristics. For example, anti-PD-1 antibodies for use in the methods of the present disclosure may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, anti-PD-1 antibodies of the present disclosure may exhibit enhanced binding to their antigen at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, and 5.0 or less. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0068] In certain instances, "reduced binding to PD-1 at acidic pH compared to neutral pH" refers to the K of an antibody that binds to PD-1 at acidic pH. D K values ​​of antibodies binding to PD-1 at neutral pH DFor example, an antibody or antigen-binding fragment thereof may be used where the antibody or antigen-binding fragment thereof has an acidic / neutral K value of about 3.0 or greater. D When a ratio is presented, it may be considered to indicate "reduced binding to PD-1 at acidic pH compared to neutral pH" for purposes of this disclosure. In certain exemplary embodiments, the acidic / neutral K ratio for an antibody or antigen-binding fragment of the disclosure is D The ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0069] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced (or increased) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modifying the antigen-binding domain in amino acid concentration can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, antibodies can be obtained that have reduced antigen binding at acidic pH compared to neutral pH. As used herein, the term "acidic pH" refers to a pH of 6.0 or below.

[0070] Bispecific anti-PSMA x anti-CD28 antibody According to certain exemplary embodiments of the present disclosure, the methods include administering a therapeutically effective amount of a bispecific antibody that specifically binds to CD28 and PSMA or an antigen-binding fragment thereof. Such antibodies and fragments may be referred to herein, for example, as "anti-PSMA / anti-CD28," or "anti-PSMAxCD28," or "PSMAxCD28" bispecific antibodies or antigen-binding fragments thereof, or other similar terms.

[0071] As used herein, the phrase "bispecific antibody" refers to an immunoglobulin protein comprising at least a first antigen-binding domain and a second antigen-binding domain. In the context of the present disclosure, the first antigen-binding domain specifically binds to a first antigen (e.g., PSMA), and the second antigen-binding domain specifically binds to a second, distinct antigen (e.g., CD28). Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each comprising three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen-binding domain may be designated with the prefix "A," and the CDRs of the second antigen-binding domain may be designated with the prefix "B." Thus, the CDRs of the first antigen-binding domain may be referred to herein as A-HCDR1, A-HCDR2, and A-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as B-HCDR1, B-HCDR2, and B-HCDR3.

[0072] The first antigen-binding domain and the second antigen-binding domain are each connected to a separate multimerizing domain. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or composition. In the context of this disclosure, a multimerizing component is defined as a (C H2 -C H3 The Fc portion of an immunoglobulin (including domains), for example, the Fc portion of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0073] Bispecific antibodies of the present disclosure typically comprise two multimerization domains, e.g., two Fc domains, each of which is part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0074] Any bispecific antibody format or technique may be used to generate the bispecific antibodies of the present disclosure. For example, an antibody or fragment thereof with a first antigen-binding specificity can be operatively linked (e.g., by chemical conjugation, genetic fusion, or noncovalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment with a second binding specificity, to generate the bispecific antibody. Specific exemplary bispecific formats that may be used in connection with the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (such as a common light chain with knobs-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for a discussion of the foregoing formats).

[0075] In the context of bispecific antibodies of the present disclosure, the Fc domain may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a naturally occurring version of the wild-type Fc domain. For example, the present disclosure includes bispecific antibodies containing one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., enhanced or diminished) between the Fc and FcRn. In one embodiment, the bispecific antibody comprises a C H2Area or C H3 The FcRn-binding domain contains modifications in regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in U.S. Patent Publication No. 20150266966, which is incorporated herein in its entirety.

[0076] The present disclosure also provides a first C H 3 domain and second Ig C H a bispecific antibody comprising three domains, a first and a second Ig C H In one embodiment, the three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C H The third domain contains a mutation that reduces or eliminates protein A binding, for example, an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). See, e.g., U.S. Patent No. 8,586,713. H Further modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT).

[0077] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be a chimeric Fc domain that combines Fc sequences from human IgG1, human IgG2, or human IgG4 C H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4 H The chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A particular example of a chimeric Fc domain that may be included in any of the antibodies described herein is, from N-terminus to C-terminus, [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antibodies described herein comprises, from N-terminus to C-terminus, [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains or chimeric heavy chain constant regions that can be included in any of the antibodies of the disclosure are described in U.S. Patent Application Publication No. 20140243504, which is incorporated herein in its entirety. Chimeric Fc domains and chimeric heavy chain constant regions having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, thereby affecting Fc effector function.

[0078] According to certain exemplary embodiments of the present disclosure, the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof comprises heavy chain variable regions (A-HCVR and B-HCVR), light chain variable regions (A-LCVR and B-LCVR), and / or complementarity-determining regions (CDRs) comprising any of the amino acid sequences of the bispecific anti-PSMA / anti-CD28 antibodies described in International Patent Publication No. WO2019 / 246514. In certain exemplary embodiments, a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof that can be used in connection with the methods of the present disclosure comprises: (a) a first antigen-binding arm that specifically binds PSMA, comprising heavy chain complementarity-determining regions (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 1, and light chain complementarity-determining regions (A-LCDR1, A-LCDR2, and A-LCDR3) of a light chain variable region (A-LCVR) comprising the amino acid sequence of SEQ ID NO: 9; and (b) a second antigen-binding arm that specifically binds CD28, comprising heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a HCVR (B-HCVR) comprising the amino acid sequence of SEQ ID NO: 5, and light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a LCVR (B-LCVR) comprising the amino acid sequence of SEQ ID NO: 9. According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 3, A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, A-LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, A-LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and A-LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 12.In yet other embodiments, the bispecific anti-PSMA / anti-CD28 antibody, or antigen-binding fragment thereof, comprises (a) a first antigen-binding arm comprising an HCVR comprising SEQ ID NO: 1 (A-HCVR) and an LCVR comprising SEQ ID NO: 9 (A-LCVR), and (b) a second antigen-binding arm comprising an HCVR comprising SEQ ID NO: 5 (B-HCVR) and an LCVR comprising SEQ ID NO: 9 (B-LCVR). In certain exemplary embodiments, the bispecific anti-PSMA×CD28 antibody comprises a PSMA-binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 15, and a CD28-binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15.

[0079] In certain exemplary embodiments, a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof that can be used in connection with the methods of the present disclosure comprises: (a) a first antigen-binding arm that specifically binds PSMA, comprising heavy chain complementarity-determining regions (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 16, and light chain complementarity-determining regions (A-LCDR1, A-LCDR2, and A-LCDR3) of a light chain variable region (A-LCVR) comprising the amino acid sequence of SEQ ID NO: 28; and (b) a second antigen-binding arm that specifically binds CD28, comprising heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a HCVR (B-HCVR) comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 24, and light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a LCVR (B-LCVR) comprising the amino acid sequence of SEQ ID NO: 28. According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 17, A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, A-LCDR1 comprises the amino acid sequence of SEQ ID NO: 29, A-LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, A-LCDR3 comprises the amino acid sequence of SEQ ID NO: 31, B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25, B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 26, B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 27, B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 29, B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 31. In yet other embodiments, the bispecific anti-PSMA / anti-CD28 antibody, or antigen-binding fragment thereof, comprises (a) a first antigen-binding arm comprising an HCVR comprising SEQ ID NO: 16 (A-HCVR) and an LCVR comprising SEQ ID NO: 28 (A-LCVR), and (b) a second antigen-binding arm comprising an HCVR comprising SEQ ID NO: 20, or SEQ ID NO: 24 (B-HCVR), and an LCVR comprising SEQ ID NO: 28 (B-LCVR).In certain exemplary embodiments, the bispecific anti-PSMAxCD28 antibody comprises a PSMA-binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, and a CD28-binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. In certain exemplary embodiments, the bispecific anti-PSMAxCD28 antibody comprises a PSMA-binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, and a CD28-binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0080] Combination therapy According to certain embodiments, the methods of the present disclosure involve administering to a subject an anti-PSMA / anti-CD28 bispecific antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the methods of the present disclosure involve administering antibodies with additive or synergistic activity to treat PSMA-expressing cancer, preferably prostate cancer. In some embodiments, the combination of an anti-PSMA x CD28 bispecific antibody (e.g., mAb1) and an anti-PD-1 antibody (e.g., cemiplimab) provides a synergistic therapeutic effect in the treatment of metastatic castration-resistant prostate cancer. As used herein, the phrase "in combination with" means that the anti-PSMA / anti-CD28 bispecific antibody or antigen-binding fragment thereof is administered before, after, or simultaneously with the anti-PD-1 antibody or antigen-binding fragment thereof. The term "in combination with" also includes sequential or simultaneous administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof. For example, when administered "before" the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof may be administered more than about 72 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, or about 30 minutes before administration of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof. When administered "after" the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof may be administered about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more than 72 hours after administration of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof."Concurrent administration" with the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof means that the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject in a separate dosage form within less than 30 minutes of (before, after, or simultaneously with) administration of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, or is administered to the subject as a single combined dosage formulation containing both the anti-PD-1 antibody or antigen-binding fragment thereof and the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof.

[0081] In certain embodiments, the methods of the disclosure include administering a third therapeutic agent, wherein the third therapeutic agent is an anti-cancer agent. In certain embodiments, the methods of the disclosure include administering an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-PSMA / anti-CD28 bispecific antibody or antigen-binding fragment thereof in combination with radiation therapy, surgery, or other anti-cancer therapy to generate a long-term, durable anti-tumor response and / or enhance survival in patients with PSMA-expressing cancer.

[0082] In some embodiments, the methods of the present disclosure include administering radiation therapy before, simultaneously with, or after administering the anti-PD-1 antibody or antigen-binding fragment thereof and the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof to a cancer patient. For example, radiation therapy may be administered in one or more doses to a tumor lesion after administration of one or more doses of the antibodies. In some embodiments, radiation therapy may be administered locally to a neoplastic lesion after systemic administration of the anti-PD-1 antibody or antigen-binding fragment thereof and the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof to increase the local immunogenicity of the patient's tumor (adjuvanting radiation) and / or kill tumor cells (ablative radiation).

[0083] A clinical trial was conducted in which REGN5678 (mAb1) was administered in combination with the anti-PD-1 antibody cemiplimab in patients with advanced metastatic castration-resistant prostate cancer (CRPC) who had failed multiple anti-androgen therapies. Such patients typically have an estimated life expectancy of 1-2 years and limited treatment options. Metastatic CRPC is considered an immunologically "cold" tumor, largely resistant to immune checkpoint therapy, and large-scale trials of anti-PD-1 antibodies have shown single-digit response rates. The disclosed anti-PSMA x CD28 costimulatory (costim) bispecific, REGN5678, was designed to enhance response in these tumor types, such as prostate cancer, essentially transforming these cold tumors into hot tumors.

[0084] As detailed in the Examples described herein, patients received REGN5678 weekly and cemiplimab every three weeks. The first dose of cemiplimab was not co-administered until week 4, allowing for a PSMAxCD28 lead-in period to evaluate the safety and efficacy of the monotherapy. The primary endpoints were safety, tolerability, and pharmacokinetics. Secondary endpoints were objective response rate, defined as a ≥50% decrease in prostate-specific antigen (PSA) from baseline and / or tumor shrinkage. PSA is a protein produced by the prostate gland and is commonly used as a biomarker to diagnose and track prostate cancer, as many mCRPC patients have disease limited to bone lesions and cannot be assessed using traditional RECIST criteria.

[0085] At the five lowest dose levels (cohorts 1–5), there was no evidence of antitumor activity, with 0 of 17 patients experiencing a PSA response, 16 of 17 patients experiencing a PSA increase, and no ≥ Grade 3 (Gr3) immune-related adverse events (irAEs) in these cohorts. The lack of antitumor activity among these patients was consistent with the approximately 6% response rate reported in other trials using anti-PD1 monotherapy.

[0086] The next three dose levels (cohorts 6-8) showed evidence of dose-dependent anticancer activity. Cohort 6: One of four patients experienced a 100% decline in PSA and a complete response (CR) based on RECIST criteria. The patient discontinued therapy due to a cutaneous Gr3 irAE (considered to be a recurrence of a pre-existing condition that had resolved), but maintained a 100% decline in PSA and CR for 10 months at current follow-up. Cohort 7: Three of eight patients experienced PSA declines of 99%, 44%, and 22%. Two of these three patients had Gr3 irAEs (aseptic encephalitis and seizures). Cohort 8: Three of four patients experienced a significant reduction in PSA within six weeks of starting combination therapy, with two patients experiencing a 99% reduction and one experiencing an 82% reduction. One patient with a 99% reduction in PSA experienced a Grade 3 case of acute inflammatory demyelinating polyradiculopathy.

[0087] Regarding safety, irAEs correlated with antitumor activity, with no irAEs of grade ≥ 3 among those who did not experience antitumor activity. Immune-related adverse events (also referred to herein as immune-mediated adverse events) can be managed by blockade of the interleukin-6 receptor (IL-6R) or IL-6. For example, an anti-IL-6R antibody, such as sarilumab or tocilizumab, can be administered to patients in combination with the therapeutics discussed herein (e.g., anti-PSMA x CD28 bispecific antibody and anti-PD-1 antibody) to reduce or prevent immune-mediated adverse events. As of data cutoff, no grade 4 irAEs, grade ≥ 3 cytokine release syndrome, or treatment-related deaths were observed in this study.

[0088] These data provide the first clinical evidence that costimulatory bispecific antibodies can be synergistically combined with anti-PD-1 to provide activity against a tumor class previously resistant to anti-PD-1 immunotherapy. More generally, the results observed here indicate that costimulatory bispecific antibody therapy, in which one arm binds to a tumor antigen and the other arm provides a CD28 costimulatory immune cell signal, can provide robust antitumor responses when administered in combination with checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1 antibodies, particularly in difficult-to-treat cancers.

[0089] In various embodiments, administration of a combination of an anti-PSMAxCD28 bispecific antibody (e.g., mAb1) and an anti-PD-1 antibody (e.g., cemiplimab) to a subject with mCRPC comprises: ·At least a 50% reduction in prostate-specific antigen (PSA) levels in the subject; ·At least a 55% decrease in PSA levels in the subject; ·At least a 60% decrease in PSA levels in the subject; ·At least a 65% reduction in PSA levels in the subject; ·At least a 70% reduction in PSA levels in the subject; ·At least a 75% reduction in PSA levels in the subject; ·At least an 80% reduction in PSA levels in the subject; ·At least an 85% reduction in PSA levels in the subject; ·At least a 90% reduction in PSA levels in the subject; ·At least a 95% reduction in PSA levels in the subject; ·At least a 96% reduction in PSA levels in the subject; ·At least a 97% reduction in PSA levels in the subject; ·At least a 98% reduction in PSA levels in the subject; ·At least a 99% reduction in PSA levels in the subject; a reduction in the size of at least one lesion having a PSMA PET signal that is smaller than the PSMA PET signal in the subject's liver, and / or Response in subjects after pseudoprogression, results.

[0090] Tumor biopsy, imaging, and CRS monitoring / management The methods contemplated in this disclosure may further include tumor biopsy, imaging, and cytokine release syndrome (CRS) monitoring and management to assess efficacy and safety in individual subjects or subject populations.

[0091] Tumor biopsy Patients with soft tissue disease may undergo core or excision biopsies from soft tissue lesions, if clinically accessible, at screening and / or during treatment, as discussed herein. For patients without clinically accessible soft tissue disease, bone biopsies may be performed, if possible. In addition to clinical diagnostic applications, any available tissue from samples collected at various time points (e.g., formalin-fixed, paraffin-embedded, or preserved in blocks for molecular extraction), as well as archival specimens from previous treatments, may be utilized for biomarker assays. Specifically, these samples may be evaluated using probes for gene targets associated with REGN5678 (PSMA, CD28) and cemiplimab (PD-L1), as well as in situ imaging with markers of immune activation, suppression, and function, and tumor cell phenotype. As discussed in Example 5, expression of therapeutic pathway targets for REGN5678 and cemiplimab is an exploratory endpoint.

[0092] Tumor tissue biopsies, if available, can also undergo gene expression profiling (using RNA sequencing or other methods) as a measure of the composite tumor microenvironment phenotype, whole-exome sequencing or other mutational profiling, and targeted testing for genetic variants (tumor mutations) such as those affecting DNA repair pathways. These may also be profiled using next-generation sequencing of the T-cell receptor repertoire as a measure of tumor-associated T-cell clonal expansion.

[0093] Imaging Prostate-specific membrane antigen (PSMA) PET / CT has been shown to provide sensitive measures of both PSMA expression and tumor burden in prostate cancer patients, allowing for the detection of more tumor lesions and greater specificity than conventional imaging modalities used in combination for prostate cancer, such as CT, MRI, and bone scan, thereby significantly improving the effectiveness of tumor response assessment and treatment strategy decision-making.

[0094] Fluorine F 18 DCFPyL( 18 F-DCFPyL) is a radiolabeled small molecule that binds with high affinity to the extracellular domain of PSMA. Data from enzyme inhibition assays show that DCFPyL competitively binds to PSMA-expressing LNCaP cells with a Ki of 1.1 nM. 18 F-DCFPyL has been tested in multiple Phase 1-3 trials and has been found to be well tolerated in patients with prostate cancer. 18 The biodistribution and optimal imaging time points after administration of F-DCFPyL injection were determined, and the radiation dose used was within the limits of diagnostic radiotracers for PET. 18 The physiological accumulation of F-DCFPyL was found to correspond to the distribution of PSMA-expressing organs. Accumulation was very high in primary tumors and metastatic lesions. 18These results suggest that F-DCFPyL injection can be used to detect residual tumors and local or distant metastases with high sensitivity and specificity. Therefore, the methods discussed herein are useful for assessing systemic tumor burden and the antitumor activity of the combination of REGN5678 and cemiplimab in patients with mCRPC. 18 This may include the use of F-DCFPyL PSMA PET / CT.

[0095] CRS Monitoring and Management Cytokine release has been observed with superagonist anti-CD28 bivalent antibodies, bsAbs, and similar molecules. Cytokine release syndrome (CRS) often resulted in clinical symptoms during or within hours to days after infusion. In a clinical trial of six patients treated with a bivalent anti-CD28 superagonist antibody (TGN1412), life-threatening CRS occurred acutely, with patients becoming critically ill within 12 to 16 hours. Previous experience with bispecific antibodies targeting tumor antigens and CD3 has shown that when CRS occurred, events were most pronounced after one or two initial weekly treatment doses and were typically transient, even with higher doses administered in subsequent weeks. This has also been observed in combination with cemiplimab. CRS typically occurred more frequently with the first two weekly doses in any given patient, decreasing in frequency with subsequent exposures. Based on these findings, the risk of a first CRS episode occurring after the third dose is considered low.

[0096] Subcutaneous administration of bispecific antibodies has recently been evaluated in preclinical and early clinical trials. Subcutaneous administration of a bispecific antibody targeting a tumor antigen and CD3 was tolerated without severe CRS events (no CRS of grade ≥ 3) in B-cell malignancies. In cynomolgus monkeys, SC administration of the same bispecific antibody resulted in lower C compared with IV administration. max , delayed T max and lower plasma cytokine levels. The methods discussed herein may include measures to address potential safety issues arising from cytokine release, including: (1) Cytokine monitoring, (2) the use of anti-IL-6 pathway therapy (e.g., sarilumab or tocilizumab) and corticosteroids to manage CRS, and (3) Provisions regarding premedication and use of lower doses at the first dosing visit before stepping up to the full dose at the dose level (DL) in the event of observed CRS.

[0097] Patients who develop symptoms consistent with severe CRS, including, but not limited to, persistent fever, neurological deficits (including altered mental status, obtundation, and seizures), clinical signs of toxicity (hypotension or hypoxia [PO2<90%] requiring at least one IV vasoactive pressor), may be considered for pharmacological intervention with anti-IL-6 pathway therapy (e.g., sarilumab or tocilizumab) and / or high-dose steroids. Such additions to the methods discussed herein are contemplated by the present disclosure.

[0098] Corticosteroids may also be used to manage CRS, especially if neurological symptoms are present. In general, corticosteroids should be used if: 1) IRR / CRS does not respond adequately to anti-IL-6 pathway therapy (e.g., sarilumab or tocilizumab), or 2) anti-IL-6 pathway therapy is not in the patient's best interest.

[0099] Pharmaceutical Compositions and Administration The present disclosure includes methods comprising administering to a subject a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antibody or antibody (or fragment) is contained in a separate or combined (single) pharmaceutical composition. The pharmaceutical compositions of the present disclosure may be formulated with suitable carriers, excipients, and other agents that provide for suitable transport, delivery, tolerability, etc. Numerous suitable formulations can be found in a formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0100] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure. For example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (See, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, or injection, and can also be administered together with other biologically active agents.

[0101] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. Furthermore, for subcutaneous delivery, pen delivery devices are readily adapted for delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are sold pre-filled with the pharmaceutical composition held within a reservoir within the device. Once the reservoir is emptied of pharmaceutical composition, the entire device is discarded.

[0102] A number of reusable pen delivery devices and / or auto-injector delivery devices can be used to subcutaneously deliver the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN™, to name just a few. STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having application in subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name a few.

[0103] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0104] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with appropriate solubilizers. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0105] Advantageously, the pharmaceutical composition for the above-mentioned use is prepared in a dosage form with a unit dose suitable for fitting the dose of the active ingredient, such dosage form in a unit dose includes, for example, a vial or a pre-filled syringe.

[0106] Dosing regimen The disclosure includes methods comprising administering to a subject a bispecific anti-PSMA×CD28 antibody, or antigen-binding fragment thereof, in combination with an anti-PD-1 antibody, or antigen-binding fragment thereof, at a dosing frequency of about 4 times per week, twice per week, once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 8 weeks, once per 12 weeks, or less frequently, so long as a therapeutic response is achieved.

[0107] According to certain embodiments of the present disclosure, multiple doses of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof can be administered to a subject over a defined time course. Methods according to this aspect of the disclosure include sequentially administering to a subject one or more doses of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. As used herein, "sequentially administering" means that each dose of antibody is administered to a subject at a different time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient one initial dose of an antibody (or fragment), followed by one or more secondary doses of the antibody (or fragment), and optionally then one or more tertiary doses of the antibody (or fragment).

[0108] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of antibody or antigen-binding fragment thereof (anti-PD-1 antibody or bispecific antibody). However, in certain embodiments, the amounts contained in the initial, secondary, and / or tertiary doses differ from one another (e.g., adjusted upward or downward) during the course of treatment. In certain embodiments, one or more doses (e.g., 1, 2, 3, 4, or 5) are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0109] In one exemplary embodiment of the present disclosure, each secondary dose and / or tertiary dose is administered ½ to 14 weeks after the immediately preceding dose (e.g., ½ week, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, 13.5 weeks, 14 weeks, 14.5 weeks, or more). As used herein, the phrase "immediately preceding dose" refers to the dose of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof (and / or anti-PD-1 antibody or antigen-binding fragment thereof) administered to a patient prior to the administration of the immediately subsequent dose in a multiple-dose series, with no intervening doses.

[0110] Methods according to this aspect of the disclosure can include administering to a patient any number of secondary and / or tertiary doses of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, and an anti-PD-1 antibody or antigen-binding fragment thereof. For example, in certain embodiments, a patient is administered only a single secondary dose. In other embodiments, a patient is administered two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses. Similarly, in certain embodiments, a patient is administered only a single tertiary dose. In other embodiments, a patient is administered two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses.

[0111] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1, 2, or 3 weeks (e.g., 1 or 3 weeks) after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 1 to 4 weeks (e.g., 1 or 3 weeks) after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing.

[0112] In certain embodiments, one or more doses of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof are administered more frequently (e.g., twice a week, once a week, once every two weeks, or once every three weeks) at the start of the treatment regimen as an "induction dose," followed by subsequent doses ("bolster doses" or "maintenance doses") administered at the same or less frequent frequency (e.g., once every four to twelve weeks).

[0113] The present disclosure includes methods for treating prostate cancer (e.g., metastatic castration-resistant prostate cancer) comprising sequentially administering to a patient a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the method comprises administering one or more doses of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof before or after administering one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the method comprises administering several doses (e.g., once weekly over a 3-week lead-in period) of a bispecific anti-PSMA / anti-CD28 antibody, followed by administering one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof and one or more additional doses of an anti-PSMA×CD28 bispecific antibody or antigen-binding fragment thereof. In some embodiments, one or more doses of about 100-600 mg of an anti-PD-1 antibody or antigen-binding fragment thereof may be administered together with one or more doses of about 0.1 mg / kg to about 20 mg / kg (e.g., 0.01-1000 mg) of a bispecific antibody or antigen-binding fragment thereof to inhibit tumor growth and / or prevent tumor recurrence in a subject with prostate cancer. In some embodiments, the bispecific antibody or antigen-binding fragment thereof in combination with the anti-PD-1 antibody or antigen-binding fragment thereof results in an enhanced anti-tumor effect (e.g., greater inhibition of tumor growth or greater prevention of tumor recurrence compared to an untreated subject). In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered before, after, or simultaneously with the anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the bispecific antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof are administered as separate dosage formulations.

[0114] Dosage The amounts of bispecific anti-PSMA / anti-CD28 antibodies or antigen-binding fragments thereof and anti-PD-1 antibodies or antigen-binding fragments thereof administered to a subject according to the methods of the present disclosure are generally therapeutically effective amounts. As used herein, the phrase "therapeutically effective amount" refers to an amount of antibody (anti-PD-1 antibody or bispecific anti-PSMA / anti-CD28 antibody) or antigen-binding fragment thereof that results in one or more of the following, compared to an untreated subject: (a) a reduction in the severity of cancer (e.g., prostate cancer) or the duration of cancer symptoms; (b) inhibition of tumor growth or an increase in tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delay in tumor growth and progression; (d) inhibition, slowing, or cessation of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) increased survival of a subject with cancer (e.g., prostate cancer); and / or (g) a reduction in the use or need for conventional anti-cancer therapy (e.g., reduction or elimination of the use of chemotherapeutic or cytotoxic agents).

[0115] For a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, a therapeutically effective amount is from about 0.01 milligrams (mg) to about 2000 mg, for example, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 3 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 1 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 75 mg, about 100 mg, about 125 mg, about 1 mg, about 250 ... The bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof can be 50 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg. In certain embodiments, 0.03 mg, 0.1 mg, 0.3 mg, 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300 mg, or 900 mg of the bispecific anti-PSMA x anti-CD28 antibody or antigen-binding fragment thereof is administered to a subject (e.g., once weekly or once every three weeks) to treat a PSMA-expressing cancer or prostate cancer (e.g., metastatic and / or castration-resistant prostate cancer).

[0116] In the case of an anti-PD-1 antibody or an antigen-binding fragment thereof, the therapeutically effective amount is about 100 mg to about 600 mg, for example, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, The amount of the anti-PD-1 antibody or antigen-binding fragment thereof may be 40 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. In certain embodiments, 300 mg to 400 mg of an anti-PD-1 antibody or antigen-binding fragment thereof is administered to a subject (e.g., once every three weeks) in combination with a bispecific antibody or antigen-binding fragment thereof to treat a PSMA-expressing cancer or prostate cancer (e.g., metastatic and / or castration-resistant prostate cancer). In certain embodiments, 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof is administered to a subject (e.g., once every three weeks) in combination with a bispecific antibody or antigen-binding fragment thereof to treat a PSMA-expressing cancer or prostate cancer (e.g., metastatic and / or castration-resistant prostate cancer).

[0117] The amounts of bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof and anti-PD-1 antibody or antigen-binding fragment thereof contained in each dose may be expressed in milligrams of antibody per kilogram of subject body weight (i.e., mg / kg). In certain embodiments, the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof may be administered at a dose of about 0.1 mg / kg to about 20 mg / kg of patient body weight, and the anti-PD-1 antibody or antigen-binding fragment thereof may be administered at a dose of about 2 mg / kg to about 20 mg / kg of patient body weight.

[0118] A summary of the sequences referred to herein and their corresponding SEQ ID NOs is provided in Table 1 below. [Table 1-1] [Table 1-2] [Example]

[0119] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0120] Example 1: Generation of a bispecific antibody that binds prostate-specific membrane antigen (PSMA) and CD28 Bispecific antibodies comprising an anti-PSMA-specific binding domain and an anti-CD28-specific binding domain were constructed using standard methodologies, with the anti-PSMA antigen-binding domain and the anti-CD28 antigen-binding domain each comprising a distinct HCVR paired with a common LCVR. In some instances, bispecific antibodies were constructed utilizing a heavy chain derived from an anti-CD28 antibody, a heavy chain derived from an anti-PSMA antibody, and a common light chain (see Table 2).

[0121] The bispecific antibody generated according to this example comprises two distinct antigen-binding domains (i.e., binding arms). The first antigen-binding domain comprises a heavy chain variable region derived from an anti-CD28 antibody ("CD28-VH"), and the second antigen-binding domain comprises a heavy chain variable region derived from an anti-PSMA antibody ("PSMA-VH"). Both anti-PSMA and anti-CD28 share a common light chain. The CD28-VH / PSMA-VH pairing generates an antigen-binding domain that specifically recognizes CD28 on T cells and PSMA on tumor cells.

[0122] An overview of the antigen-binding domain components of the various anti-PSMAxCD28 bispecific antibodies constructed is provided in Table 3. The corresponding CDR sequences, as well as the full-length heavy and light chain sequences, are identified in Table 1 (with reference to the "-001," "-002," and "-003" bispecific antibodies in Table 3). [Table 2]

[0123] Example 2: Surface Plasmon Resonance-Derived Binding Affinity and Rate Constants of Anti-PSMA x CD28 Bispecific Antibodies To determine the binding kinetics of the anti-PSMAxCD28 bispecific antibody, the surface plasmon resonance derived binding affinity and rate constants of the anti-PSMAxCD28 bispecific were determined.

[0124] Binding kinetics of anti-PSMA x CD28 bispecific antibody to PSMA: The equilibrium dissociation constant (K) of 6h.hPSMA (recombinant human PSMA / FOLH1 protein, R&D, Cat. No. 4234-ZN) binding to purified anti-PSMA x CD28 bispecific antibody. D The RI values ​​were determined using a real-time surface plasmon resonance biosensor on a Biacore T-200 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody to capture purified anti-PSMAxCD28 bispecific antibody.

[0125] The Biacore binding assay was performed in a buffer consisting of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 0.5 mM MgCl2, 1.0 mM CaCl2, and 0.05% v / v surfactant P20 (HBS-P++ running buffer). Different concentrations of hPSMA with an N-terminal polyhistidine tag (6h.hPSMA, R&D) were prepared in a 3-fold serial dilution range from 10 nM to 0.4 nM for the anti-PSMA x CD28 bispecific antibody in HBS-P++ running buffer.

[0126] Different concentrations of 6h.hPSMA were injected over the monoclonal antibody capture surface at a flow rate of 50 μL / min. Binding of 6h.hPSMA to the captured monoclonal antibody was monitored for 3 min, and dissociation of 6h.hPSMA in HBS-P++ running buffer was monitored for 10 min. The kinetic binding rate constant (k) was calculated by fitting the real-time sensorgram to a 1:1 binding model using Scrubber 2.0c curve-fitting software (BioLogic Software). a ) and dissociation rate constant (k d The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the reaction rate constants as follows: K D (M)=k d / k a , and t1 / 2(min)=0.693 / k d / 60

[0127] The binding kinetic parameters for 6h.hPSMA binding to purified monoclonal antibodies at 25°C are shown in Table 3 below. [Table 3]

[0128] The binding kinetic parameters for 6h hPSMA binding to one purified exemplary monoclonal bispecific antibody at 37°C are shown below in Table 4. 1 RU (response unit) is 1 mm 2Each represents 1 pg of protein. [Table 4]

[0129] Binding kinetics of anti-PSMA×CD28 bispecific antibodies to CD28: The equilibrium dissociation constant (K) for the binding of hCD28.mmh to purified monoclonal antibodies D The RI values ​​were determined using a real-time surface plasmon resonance biosensor with a Biacore T-200 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody to capture purified anti-PSMA x CD28 bispecific antibody.

[0130] Different concentrations of hCD28.mmh were injected over the monoclonal antibody capture surface at a flow rate of 50 μL / min. Binding of hCD28.mmh to the captured monoclonal antibody was monitored for 5 min, and dissociation of hCD28.mmh in HBS-P++ running buffer was monitored for 10 min. Kinetic binding rate constants (k) were calculated by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. a ) and dissociation rate constant (k d The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the reaction rate constants as follows: K D (M)=k d / k a , and t1 / 2(min)=0.693 / k d / 60

[0131] The binding kinetic parameters for hCD28.mmh binding to purified anti-PSMAxCD28 bispecific antibodies at 25°C are shown in Table 5 below. [Table 5]

[0132] The binding kinetic parameters for hCD28.mmh binding to purified anti-PSMAxCD28 bispecific antibody at 37°C are shown in Table 6. [Table 6]

[0133] Example 3: Cellular binding of anti-PSMA x CD28 bispecific antibodies to PSMA and CD28 To assess the ability of these antibodies (anti-PSMA x CD28 antibodies) to specifically bind to cell surface proteins, in vitro binding assays were performed.

[0134] The binding of the PSMAxCD28 bispecific antibody to the surface of human T cells was examined by flow cytometry. BSPSMA / CD28-001 was 4.80 x 10 -8 It bound to all T cells with an EC50 value of 5.09 × 10 M and to both CD4+ and CD8+ T cells, respectively. -8 M and 5.89 x 10 -8 BSPSMA / CD28-003 bound with an EC50 value of 1.80 × 10 -7 It bound weakly to all T cells with an EC50 value of 1.67E-07M and to both CD4+ and CD8+ T cells with EC50 values ​​of 1.67E-07M and 1.80E-07M, respectively.

[0135] The binding of the PSMAxCD28 bispecific antibody to the surface of PSMA-expressing cell lines was examined by flow cytometry. C4-2 is a prostate cancer cell line derived from LNCaP (androgen-sensitive human prostate cancer cells derived from lymph node metastases, see Wu et al., Int. J. Cancer, 57:406-412 (1994)). Both BSPSMA / CD28-001 and BSPSMA / CD28-003 expressed 3.87 x 10 -9 M and 1.50 x 10 -9BSPSMA / CD28-001 and BSPSMA / CD28-003 bound to C4-2 cells (see Liu et al., 2004, Prostate, 60:98-108) with an EC50 value of 3.05 × 10 M. 22RV1 is an epithelial prostate cancer cell line (see In Vitro Cell Dev. Biol. Anim., 1999, 35(7):403-409). Both BSPSMA / CD28-001 and BSPSMA / CD28-003 bound to C4-2 cells (see Liu et al., 2004, Prostate, 60:98-108) with an EC50 value of 3.05 × 10 M. -9 M and 6.33 x 10 -09 It bound to 22RV1 cells with an EC50 value of M.

[0136] Example 4: Primary and modified bioassays of PSMAxCD28 bispecific antibodies T cell activation is achieved by stimulating T cell receptors (TCRs) that recognize specific peptides presented by major histocompatibility complex class I or II (MHC1 or MHCII) proteins on antigen-presenting cells (APCs) (Goldrath et al., Selecting and maintaining a diverse T-cell repertoire, Nature 402, 255-262 (1999)). The activated TCR then initiates a cascade of signaling events that are driven by various transcription factors, such as activator protein 1 (AP-1), nuclear factor of activated T cells (NFAT), or nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and can be monitored by reporter genes. T cell responses are then further refined through the engagement of coreceptors constitutively or inducibly expressed on T cells, such as CD28, CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), LAG-3 (lymphocyte activation gene 3), or other molecules (Sharpe et al., The B7-CD28 Superfamily, Nat. Rev. Immunol., 2(2):116-26 (2002)). The costimulatory molecule CD28 is activated by its endogenous ligands CD80 or CD86 expressed on APCs. CD28 enhances cellular signals, such as pathways controlled by the NFκB transcription factor, following TCR activation. CD28 co-signaling is important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death (Smeets et al., NFκB activation induced by T cell receptor / CD28 costimulation is mediated by protein kinase C-θ, PNAS, 97(7):3394-3399 (2012)).

[0137] To identify antibodies that enhance T cell activity in the presence of both a primary stimulus and PSMA target expression, anti-PSMA x CD28 bispecific antibodies were characterized in modified reporter bioassays and cell-based assays using human primary T cells. The assays evaluate the behavior of anti-PSMA / CD28 bispecific antibodies in the presence and absence of a primary stimulus and in the presence and absence of target expression. Assays were performed to identify anti-PSMA x CD28 bispecific antibodies that enhance T cell activity in the presence of a primary stimulus and target expression. Thus, the assays evaluated the behavior of bispecific antibodies in the presence and absence of a primary stimulus and target expression.

[0138] A Jurkat-derived T cell clone, JRT3.T3.5 (ATCC, #TIB-153), was transduced with an NFκB luciferase reporter construct (NFκB-Luc, SA Biosciences / Qiagen, catalog number CLS-013L). After isolation of a puromycin-resistant clone (JRT3.T3.5 / NFκB-Luc clone 1C2), the cells were further engineered to express full-length human TCR alpha (1G4A - amino acids M1 to S274) and TCR beta (1G4B - amino acids M1 to G311) subunits (Robbins et al., "Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions," J. Immunol. 180(9):6116-31 (2008)). After isolating a single clone (J.RT3-T3.5 / NFκB-Luc / 1G4AB clone 1D2), the cells were further engineered to express full-length human CD8 alpha (hCD8a - amino acids M1 to V235 of accession number NP_001139345) and human CD8 beta subunit (hCD8b - amino acids M1 to K210 of accession number P10966). A single clone was again generated (J.RT3-T3.5 / NF κB-Luc / 1G4AB / hCD8ab (clone 1D5) was further transduced with full-length human CD28 (hCD28-amino acids M1 to S220, accession number P10747). Cells were sorted for high CD28 expression and maintained in RPMI + 20% FBS + penicillin / streptomycin / glutamine (P / S / G) + NEAA + NaPyr + 1 μg / mL puromycin + 500 μg / mL L G418 + 250 μg / mL hygromycin + 10 μg / mL blasticidin. For faster proliferation, the modified reporter T cells were maintained in antibiotic-free cell culture medium and used for cell-based luciferase experiments as modified reporter T cells. Reagent information is as follows: RPMI 1640, Irvine Scientific, catalog number 9160; FBS, Seradigm, catalog number 1500-50; Penicillin / Streptomycin / Glutamine 100x (P / S / G), Thermo Fisher Scientific, catalog number 10378-016; Non-essential amino acids (NEAA), Irvine Scientific, catalog number 9304; Sodium pyruvate (NaPyr), Millipore, catalog number TMS-005-C; Puromycin, Sigma, catalog number P8833; Geneticin (G418), Thermo Fisher Scientific, catalog number 11811-098; Hygromycin; Blasticidin.

[0139] A stable HEK293 cell line (ATCC, #CRL-1573) expressing human CD20 (accession number NP_068769.2, amino acids M1 to P297) was transduced with human PSMA (accession number Q04609, amino acids M1 to A750). Human PSMA-positive cells were isolated by fluorescence-activated cell sorting (FACS), and the resulting highly sorted cell line, HEK293 / CD20 / hPSMA, was maintained in DMEM + 10% + P / S / G + NEAA supplemented with 500 μg / mL G418.

[0140] In this experiment, engineered reporter T cells are stimulated via two bispecific antibodies. The first stimulus is delivered by a T cell-activating bispecific antibody, anti-CD3xCD20 hIgG4 (see WO 14 / 047231), targeting CD3 molecules on the engineered reporter T cells and CD20 on the HEK293 cells. Here, the first stimulus bypasses the need for TCR activation by their natural ligands, which are specific peptides presented on MHC molecules. The second stimulus is driven by a CD28 bispecific antibody. This antibody mimics CD28 activation on T cells through its ligands, CD80 / CD86, expressed on APCs. Here, the antibody interacts with CD28 on the T cells and PSMA on the engineered HEK293 cells, driving CD28 activation on the engineered reporter T cells. Coactivation of the TCR and CD28 leads to enhanced transcriptional activity of NFκB, which in turn induces the production of the reporter gene luciferase.

[0141] Cell suspensions and antibody dilutions for screening of anti-PSMAxCD28 bispecific antibodies were prepared using RPMI1640 supplemented with 10% FBS and P / S / G as assay medium. The day before screening, engineered reporter T cells were cultured at 1 x 10 in cell culture medium. 6 Cells were cultured to 1000 cells / mL. Three-fold (1:3) serial dilutions of anti-PSMA x CD28 bispecific antibodies and controls were tested in the presence of a constant 50 pM anti-CD20 x CD3 or hIgG4 isotype control. Ten-point dilutions ranged from 15 pM to 100 nM, with the final dilution containing no anti-PSMA x CD28 antibody. Reagents were added in the following order: 1) serially diluted antibodies were added to corresponding wells of a 96-well white flat-bottom plate; 2) a fixed concentration of 50 pM anti-CD20 x CD3 or hIgG4 isotype control was added to each well; 3) 4 x 10 5 Resuspend APCs in 1000 cells / mL to a final concentration of 1 x 10 4 4) overnight cultured reporter T cells were added to the plate at 2 × 10 cells / well. 6 Resuspend at a final concentration of 5 x 10 / mL 4Cells / well were added to the plate. o After 4-6 hours of incubation at 37°C / 5% CO2, cells were lysed by adding 100 μL of ONE-Glo™ (Promega, Cat. No. E6051) reagent, and luciferase activity was detected. Emitted light was captured in relative light units (RLU) on a multilabel plate reader, Envision (PerkinElmer, Model 2104). All serial dilutions were tested in duplicate.

[0142] Antibody EC 50 Values ​​were determined by fitting the data to a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism™ software. The following formula was used to calculate fold induction:

number

[0143] EC2 was measured for modified reporter T cells co-incubated with HEK293 / hCD20 or HEK293 / hCD20 / hPSMA cells in addition to 50 pM of either a constant hIgG4 isotype control or an anti-CD3 x CD20 bispecific antibody (a T cell stimulatory bispecific antibody). 50 and fold induction values ​​are summarized in Tables 6 and 7. [Table 7] [Table 8]

[0144] When T cells and APCs were treated with 50 pM of the hIgG4 isotype control, none of the CD28 bispecific antibodies showed an increase in luciferase activity in the absence of TCR stimulation, regardless of the APC line used in the assay. Minimal luciferase activation was observed with one of the parental CD28 antibodies (mAb14226P2) on HEK293 / hCD20 cells (4.76x) and HEK293 / hCD20 / hSPMA cells (3.59x) shown in Table 7A. mAb14226P2, mAb14193P2, and mAb14216P2 correspond to the parental anti-CD28 antibodies BSPSMA / CD28-001, BSPSMA / CD28-002, and BSPSMA / CD28-003, respectively.

[0145] In contrast, when cells were treated with 50 pM of anti-CD3xCD20 bispecific antibody, all three anti-PSMAxCD28 bispecific antibodies, BSPSMA / CD28-001, BSPSMA / CD28-002, and BSPSMA / CD28-003, strongly induced luciferase activity when co-incubated with APCs expressing hPSMA on their surface. Regardless of APC line, very low or no activation was observed with the one-armed parental CD28 controls (mAb14226P2, mAb14193P2, and mAb14216P2). Little luciferase activation was observed for all three parental CD28 antibodies (mAb14226P2, mAb14193P2, and mAb14216P2).

[0146] Example 5: Phase 1 / 2 study of a bispecific anti-PSMA x anti-CD28 antibody administered in combination with an anti-PD-1 antibody in patients with metastatic castration-resistant prostate cancer This is an open-label, Phase 1 / 2, first-in-human study evaluating the safety, tolerability, pharmacokinetics (PK), and antitumor activity of mAb1 (REGN5678) alone and in combination with cemiplimab in previously treated metastatic castration-resistant prostate cancer (mCRPC).

[0147] Test objectives

[0148] The main objectives of this study are: To evaluate the safety, tolerability, and pharmacokinetics (PK) of mAb1 alone and in combination with cemiplimab (dose escalation) To evaluate the efficacy of mAb1 in combination with cemiplimab as measured by objective response rate (ORR) according to modified Prostate Cancer Working Group 3 (PCWG3) criteria (dose expansion)

[0149] The secondary objectives of this study are: To evaluate the efficacy of mAb1 in combination with cemiplimab (dose escalation) as measured by ORR according to modified PCWG3 criteria Characterize the safety profile in each expansion cohort (dose expansion) To characterize the PK of mAb1 in combination with cemiplimab (dose expansion) To evaluate the efficacy of mAb1 in combination with cemiplimab as measured by additional criteria (dose escalation and dose expansion) To evaluate the immunogenicity of mAb1 in combination with cemiplimab (dose escalation and dose expansion)

[0150] All primary, secondary and exploratory objectives of the study will apply to each cohort of the study, including those receiving sarilumab and those not receiving sarilumab.

[0151] Study design

[0152] This is an open-label, Phase 1 / 2, first-in-human study evaluating the safety, tolerability, PK, and antitumor activity of mAb1 (anti-PSMA x CD28) alone and in combination with cemiplimab (anti-PD-1) in previously treated metastatic castration-resistant prostate cancer (mCRPC). There are two parts to the study: dose escalation and dose expansion.

[0153] Dose escalation: During dose escalation, patients will receive a 3-week monotherapy lead-in of mAb1 intravenously (IV) once weekly (QW) at their assigned dose level (DL), followed by combination therapy with mAb1 at the assigned DL, IV, QW dose and cemiplimab 350 mg IV once every 3 weeks (Q3W). Once the lowest pharmacologically active dose level is identified, the dosing interval of mAb1 will be extended from QW to Q3W for subsequent dose escalation. Once the maximum tolerated dose (MTD) / estimated recommended phase 2 dose (estimated RP2D) of mAb1 IV is identified, subcutaneous (SC) administration of mAb1 may be explored. The tolerability of the combination of mAb1 and cemiplimab (without mAb1 monotherapy lead-in), with or without sarilumab 350 mg IV Q3W x 4 doses, will be assessed at the start of the expansion phase ( * May be investigated at MTD / estimated RP2D after cohort.

[0154] The use of sarilumab prophylaxis at 350 mg IV Q3W x 4 doses will be explored during dose escalation in combination with REGN5678 and cemiplimab. Dose level (DL) 7 (100 mg REGN5678 IV QW) cleared DLT evaluation, was deemed tolerable, and was therefore selected for the initial cohort with sarilumab. Based on safety and tolerability in this initial cohort, dose escalation may continue sarilumab prophylaxis. Eligibility criteria were specifically modified for patients in the sarilumab cohort, including increasing the absolute neutrophil count test threshold to ≥ 1.5 x 109 / L and excluding patients with active or previous tuberculosis (TB), previous opportunistic infections, intestinal perforation, severe diverticulitis, or inflammatory bowel disease. Based on ongoing evaluation of cumulative safety and tolerability data in all cohorts, escalation above 100 mg (DL7) may proceed with and / or without sarilumab prophylaxis. The addition of IL-6R blockade with sarilumab may mitigate immune-mediated adverse events (imAEs) resulting from treatment with REGN5678 in combination with cemiplimab while maintaining antitumor activity.

[0155] Dose expansion: During dose expansion, patients will receive a 3-week monotherapy lead-in of mAb1 followed by combination therapy with mAb1 at the MTD / estimated RP2D IV and cemiplimab 350 mg IV Q3W. Dose expansion cohorts utilizing SC administration of mAb1 at the MTD / estimated RP2D may also be investigated.

[0156] Dose expansion cohorts will be enrolled after identification of the mAb1 MTD in combination with cemiplimab and / or the estimated RP2D. Safety assessments will be conducted at each study drug administration visit. Radiographic response assessments will be conducted every 6 weeks from Cycle 1 (C1D42 / C2D1) through Cycle 4 (including patients receiving the initial 3-week monotherapy lead-in of mAb1), and then every 12 weeks thereafter.

[0157] Exam period

[0158] The total duration of each patient's study participation will vary based on the occurrence of one or more of the following: disease progression, intolerable adverse events (AEs), withdrawal of consent, or meeting of study discontinuation criteria.

[0159] Dose escalation and expansion cohorts (no monotherapy lead-in period) * For patients in the *cohort), the study consists of four periods: a screening period (up to 28 days), a 3-week monotherapy lead-in period of mAb1 (21 days), a combination therapy period consisting of a series of 6-week (42 day) cycles of mAb1 in combination with cemiplimab with or without sarilumab (variable time to discontinuation), and a treatment-free follow-up period (90 days).For patients in the *cohort, the study consists of three periods: a screening period (up to 28 days), a combination therapy period consisting of a series of 6-week (42 day) cycles of mAb1 in combination with cemiplimab (variable time to discontinuation), and a treatment-free follow-up period (90 days).

[0160] Dose escalation of mAb1 proceeds QW until identification of a pharmacologically active dose level (i.e., the lowest pharmacologically active dose). Once the lowest pharmacologically active dose is identified, mAb1 dose escalation proceeds to the next dose level, administered Q3W. [Table 9]

[0161] Study population

[0162] Approximately 216 patients will be enrolled (approximately 108 patients during dose escalation, and up to approximately 3-4 expansion cohorts with up to 27 patients each). The study population will include men with treatment-experienced mCRPC. To be selected for the study, patients must have received at least two lines of prior systemic therapy (in addition to androgen deprivation therapy [ADT]) approved for metastatic and / or castration-resistant disease, including second-generation antiandrogen therapy (e.g., abiraterone, enzalutamide, apalutamide, or darolutamide).

[0163] Selection Criteria : Patients must meet the following criteria to be included in the study: 1. Male ≥ 18 years old 2. Histologically or cytologically confirmed adenocarcinoma of the prostate without pure small cell carcinoma 3. Metastatic castration-resistant prostate cancer (mCRPC) with a PSA level of ≥ 4 ng / mL at screening and progression within 6 months prior to screening according to one of the following: a. PSA progression defined by a confirmed rise in PSA level with an interval of ≥ 1 week between assessments. b. Radiographic disease progression in soft tissues based on RECIST version 1.1 criteria, with or without PSA progression c. Radiographic bone disease progression defined as the appearance of two or more new bone lesions on a bone scan, with or without PSA progression 4. Received ≥ 2 lines of prior systemic therapy approved in the metastatic and / or castration-resistant setting (in addition to androgen deprivation therapy [ADT]), including at least the following: One second-generation antiandrogen therapy (e.g., abiraterone, enzalutamide, apalutamide, or darolutamide) Note: Non-taxane chemotherapy regimens given for metastatic prostate cancer with mixed histology are acceptable and included when evaluating treatment options 5. Able and willing to provide archival or freshly obtained tumor tissue. Note: For dose escalation only, if archival or fresh tissue is not available, a pathology report confirming the diagnosis of prostate cancer may be submitted. 6. Have undergone orchiectomy or are receiving luteinizing hormone-releasing hormone (LHRH) agonist therapy or antagonist therapy with a serum testosterone level <50 ng / dL and agree to continue LHRH agonist or antagonist therapy during the study. Eastern Cooperative Oncology Group (ECOG) performance status of 7.0 or 1 8. Adequate organ and bone marrow function documented by: Hemoglobin ≥ 8.5 g / dL b. Absolute neutrophil count ≥ 1.0 × 10 9 / L (sarilumab cohort only: ≥ 1.5 × 10 9 / L) c. Platelet count ≧100×10 9 / L 9. Serum creatinine ≤ 1.5 × ULN or estimated glomerular filtration rate > 50 mL / min / 1.73 m 2 A 24-hour urine creatinine collection may substitute for calculated creatinine clearance to meet eligibility criteria. 10. Proper Liver Function: a. Total bilirubin ≦1.5×ULN (≦3×ULN if tumor is present in the liver) b.AST ≤ 2.5 × ULN (≤ 5 × ULN if tumor is present in the liver) c. ALT ≦2.5×ULN (≦5×ULN if tumor is present in the liver) d. Alkaline phosphatase (ALP) ≤ 2.5 x ULN (≤ 5 x ULN if tumor involves liver or bone) Notes: In patients with tumor-related liver involvement, AST levels ≥ 3 × ULN or ALT levels ≥ 3 × ULN and bilirubin levels ≥ 2 × ULN are excluded regardless of the above criteria. Patients with Gilbert syndrome do not need to meet the total bilirubin requirement unless their total bilirubin exceeds past levels. Gilbert syndrome must be properly documented in the past medical history. 11. Willing and able to comply with clinic visit and study-related procedures 12. Subject provides signed informed consent 13. Able to understand and complete exam-related questionnaires

[0164] Exclusion criteria : Patients who meet any of the following criteria will be excluded from the study. 1.Currently receiving treatment in another study 2. Participating in a study of an investigational drug or device within 4 weeks of first dose of study treatment 3. Have received treatment with approved systemic therapy (including Sipulcel-T) within 3 weeks of dosing or have not yet recovered from any acute toxicity (i.e., grade ≤ 1 or baseline) as described in the inclusion criteria and excluding laboratory changes as described below: a. Patients with neuropathy of grade 2 or less 4. Underwent radiation therapy or major surgery within 14 days of the first dose of study drug or has not recovered from an AE (i.e., grade ≤ 1 or baseline), excluding inclusion criteria and laboratory changes listed below: a. Patients with neuropathy of grade 2 or less 5. Has received any prior systemic biologic therapy within 5 half-lives of the first dose of study treatment. Exception: Patients previously treated with bevacizumab or other non-immunomodulatory antibodies with a half-life longer than 7 days are permitted after discussion with the sponsor if at least 30 days have elapsed since the last treatment. 6. Previously received PSMA-targeted therapy 7. Dose escalation: Previous anti-cancer immunotherapy (other than Sipulcel-T) within 5 half-lives prior to study treatment. Examples of immunomodulatory agents include blockers of CTLA-4, 4-1BB (CD137), or OX-40, therapeutic vaccines, anti-PD-1 / PD-L1, phosphoinositide 3-kinase (PI3K) delta inhibitors, or cytokine anti-cancer treatments. Note: Patients who have previously received study cell-based therapy (e.g., CAR-T cells) will be excluded. 8. Dose Expansion: Previous anti-cancer immunotherapy. Examples of immunomodulatory agents include blockers of CTLA-4, 4-1BB (CD137), or OX-40, therapeutic vaccines, anti-PD-1 / PD-L1, PI3K delta inhibitors, CAR-T cells, or cytokine anti-cancer therapies. Note: Prior treatment with Sipulcel-T is permitted. 9. Patients who have not recovered from immune-mediated AEs (i.e., grade ≤1 or baseline) within 3 months prior to initiation of study medication, except for endocrine disorders adequately controlled with hormone replacement. 10.Patients who have permanently discontinued anti-cancer immunomodulatory therapy due to immune-related AEs 11. Another malignancy that is ongoing or requiring active treatment, except for the following: Non-melanoma skin cancer that has received potentially curative therapy b. Any tumor deemed to be effectively treated with definitive local control (with or without continuation of adjuvant hormonal therapy) 12. Any condition requiring or continuing corticosteroid therapy (prednisone >10 mg / day or equivalent anti-inflammatory agent) within 1 week prior to the first dose of study treatment. Patients requiring short-term steroids (maximum 2 days in the week prior to enrollment) or physiological replacement will not be excluded. 13. Current or recent (within 5 years) evidence of significant autoimmune disease requiring systemic immunosuppressive treatment. The following are not excluded: vitiligo, resolved childhood asthma, endocrine disorders requiring hormone replacement only (e.g., hypothyroidism, type 1 diabetes), or psoriasis not requiring systemic treatment. 14. History of CNS metastases, including previously treated metastases 15. Encephalitis, meningitis, neurodegenerative disease (excluding mild dementia not interfering with activities of daily living [ADL]), or uncontrolled seizures within 1 year prior to the first dose of study treatment. 16. Known history or any evidence (past 5 years) of interstitial lung disease or active non-infectious pneumonia. A history of radiation pneumonitis in the radiation field is acceptable. 17. Uncontrolled human immunodeficiency virus (HIV) infection, hepatitis B or C infection, or diagnosed immunodeficiency Notes: Patients will be tested for Hepatitis C virus (HCV) and Hepatitis B virus (HBV) at screening. Patients with known HIV infection whose infection is controlled (either spontaneously or on a stable antiviral regimen, with an undetectable viral load (HIV RNA polymerase chain reaction [PCR]) and a CD4 count >350) are admitted. Patients with controlled HIV infection will be monitored according to local standards. Patients with controlled hepatitis B infection (HepBsAg+) (serum hepatitis B virus DNA PCR below the limit of detection and receiving antiviral therapy for hepatitis B) are accepted. Patients with controlled infection must undergo regular HBV DNA monitoring. Patients must continue receiving antiviral therapy for at least 6 months after the last dose of study drug. Patients who are hepatitis C antibody positive (HCV Ab+) and have a controlled infection (HCV RNA undetectable by PCR, either spontaneously or in response to a previous successful course of anti-HCV therapy) are accepted. 18. Any infection requiring hospitalization or treatment with IV anti-infectives within 2 weeks of the first dose of study treatment 19. Received a live vaccine within 4 weeks of planned study drug initiation. 20. Previous allogeneic stem cell transplant, or organ transplant recipient at any time, or autologous stem cell transplant within 12 weeks prior to initiation of study treatment 21. Known allergy or hypersensitivity to cemiplimab or any component of the study drug. 22. Known psychiatric or substance abuse disorder that would interfere with participation in this study 23. Any medical condition, comorbidity, physical examination finding, metabolic dysfunction, or laboratory test abnormality that, in the opinion of the investigator, makes the patient unsuitable for clinical study participation because it poses a high safety risk and / or may affect the interpretation of the study results, including, but not limited to, significant cardiovascular disease (e.g., New York Heart Association Class III or IV heart disease, myocardial infarction within the past 6 months, unstable arrhythmia or unstable angina pectoris), and / or significant pulmonary disease (e.g., history of obstructive pulmonary disease and symptomatic bronchospasm). 24. Sarilumab cohort only: Exclusion criteria for tuberculosis (TB) a. History of active or incompletely treated TB b. QuantiFERON-positive patients (without active disease) will be excluded from the study unless they have a history of prior documented completed chemoprophylaxis for latent tuberculosis infection (LTBI) (e.g., acceptable treatment is 9 months of isoniazid 300 mg orally daily or an equivalent proven regimen according to local guidelines) or treatment for active tuberculosis infection (TBI) and have undergone expert consultation to rule out active disease. 25. Sarilumab cohort only: Patients with a history of invasive opportunistic infections, including but not limited to histoplasmosis, coccidioidomycosis, Pneumocystis pneumoniae, or aspergillosis, or JC virus (progressive multifocal leukoencephalopathy). 26. Sarilumab cohort only: History of intestinal perforation, severe diverticulitis, or inflammatory bowel disease

[0165] Test treatment

[0166] mAb1 at the assigned dose level will be administered QW or Q3W by either infusion or SC injection over 30 minutes to 2 hours. Cemiplimab 350 mg will be administered Q3W by infusion over 30 minutes. Sarilumab 350 mg Q3W will be administered Q3W by infusion over 60 minutes for 4 doses (total of 12 weeks), starting with the initial dose of REGN5678 in combination with cemiplimab. If both mAb1 and cemiplimab are administered on the same day, mAb1 will be administered first. For sarilumab IV cohorts, it must be administered before mAb1 and cemiplimab. Selected patients will have a sarilumab scan at selected sites for the experimental PSMA PET / CT imaging procedure. 18 F-DCFPyL is administered.

[0167] Test evaluation items

[0168] The primary endpoints of this study are as follows: Dose escalation: Safety as measured by the incidence and severity of treatment-emergent adverse events (TEAEs) / adverse events of special interest (AESIs) / serious adverse events (SAEs) and grade ≥ 3 laboratory abnormalities during treatment and up to 90 days after the last dose of mAb1 and cemiplimab or until the start of a new therapy for the treatment of the patient's tumor, whichever occurs first. Tolerability as measured by the incidence of dose-limiting toxicities (DLTs) from the first dose to the end of the DLT observation period for mAb1 alone and for mAb1 in combination with cemiplimab. Serum mAb1 concentrations over time when administered either alone or in combination with cemiplimab Dose expansion: ORR by modified PCWG3 criteria is defined as the proportion of patients who achieved a response by modified PCWG3 criteria based on: - A ≥ 50% decrease in prostate-specific antigen (PSA) from baseline and from the start of combination therapy, confirmed by a second PSA test ≥ 4 weeks later, and / or - Confirmed radiographic response of complete response (CR) or partial response (PR) All primary endpoints will apply to each cohort in the study, including those receiving sarilumab and those not receiving sarilumab.

[0169] The secondary endpoints of the study were as follows: Dose escalation: ORR is defined as the proportion of patients who achieved a response according to modified PCWG3 criteria based on: A ≥ 50% decline in PSA from baseline and from the start of combination therapy confirmed by a second PSA test ≥ 4 weeks later, and / or - Confirmed radiographic response of CR or PR Dose expansion: Safety as measured by the incidence and severity of treatment-emergent adverse events (AEs) / AESIs / SAEs and grade ≥ 3 laboratory abnormalities during treatment and up to 90 days after the last dose of mAb1 in combination with cemiplimab or until the start of a new therapy for the treatment of the patient's tumor, whichever occurs first. Serum mAb1 concentrations over time when administered in combination with cemiplimab Dose Escalation and Dose Expansion: ORR based on PSA response, defined as the proportion of patients achieving a PSA decline of ≥ 50% from baseline and from the start of combination therapy, confirmed by a second PSA test ≥ 4 weeks later Proportion of patients with a ≥ 90% decline in PSA from baseline and from the start of combination therapy, confirmed by a second PSA test ≥ 4 weeks later The proportion of patients who achieved a change in circulating tumor cell (CTC) count from a baseline of ≥ 5 cells / 7.5 mL and from the start of combination therapy to < 5 cells / 7.5 mL Presence or absence of antibodies against mAb1 and cemiplimab All secondary endpoints apply to each cohort in the study, including those receiving sarilumab and those not receiving sarilumab.

[0170] Exploratory endpoints of the study include: Percent change in PSA, defined as the maximum percent change in PSA decline from baseline and from the start of combination therapy Percent change in PSA during 3 weeks of mAb1 monotherapy, defined as the maximum percent change in PSA decline from baseline during the mAb1 monotherapy lead-in period Imaging-based progression-free survival (rPFS) (per modified PCWG3 and iRECIST), defined as the time from first study treatment administration to first radiographic progression or death from any cause. Radiographic progression includes soft tissue and bone progression per modified PCWG3. If there is no radiographic progression or death before the analysis cutoff date or the start of further anti-cancer treatment, rPFS will be censored at the date of the last valid radiographic response assessment without radiographic progression performed on the analysis cutoff date or before the start of further anti-cancer treatment, whichever occurs first. PSA progression-free survival (PFS), defined as the time from first study treatment administration and from initiation of combination therapy to first PSA progression or death from any cause. PSA progression is defined as: -After a decline from baseline: PSA increase ≥ 25% and ≥ 2 ng / mL above the nadir and confirmed by a second PSA test ≥ 4 weeks later; - No decline from baseline: PSA progression as a ≥ 25% increase from baseline and an increase of ≥ 2 ng / mL over 12 weeks In the absence of PSA progression or death before the analysis cutoff date or the start of further anticancer treatment, PSA PFS will be censored at the date of the last valid PSA test not showing PSA progression performed on the analysis cutoff date or before the start of further anticancer treatment, whichever occurs first. Radiographic response-based ORR (per modified PCWG3 and iRECIST), defined as the proportion of patients achieving a radiographic response of CR or PR Time to response based on radiographic response, defined as the time from first study treatment administration to first radiographic response of CR or PR for patients with a confirmed radiographic response of CR or PR (per modified PCWG3 and iRECIST) Time to response based on PSA response, defined as the time from first study treatment administration to first PSA response, for patients with a confirmed PSA response Radiographic response-based DOR (rDOR) (per modified PCWG3 and iRECIST), defined as the time from first radiographic response of CR or PR to first radiographic progression or death for patients with a confirmed radiographic response of CR or PR, using the same censoring rules as for rPFS. DOR based on PSA response, defined as the time from first PSA response to first PSA progression for patients with a confirmed PSA response. Uses the same censoring rules as for PSA PFS. Time to radiographic progression (per modified PCWG3 and iRECIST), defined as the time from first study treatment administration to first radiographic progression. In the absence of radiographic progression before the analysis cutoff date or the start of further anti-cancer treatment, time to progression will be censored at the date of the last valid radiographic response assessment not showing radiographic progression performed on the analysis cutoff date or before the start of further anti-cancer treatment, whichever occurs first. Time to progression based on PSA progression, defined as the time from the start of the first study treatment or combination therapy to first PSA progression. In the absence of PSA progression before the analysis cutoff date or the start of further anti-cancer treatment, time to progression will be censored at the date of the last valid PSA test not showing PSA progression performed on the analysis cutoff date or before the start of further anti-cancer treatment, whichever occurs first. Disease control rate (DCR), defined as the proportion of patients achieving a radiographic response of CR, PR, SD, or non-CR / non-PD (per modified PCWG3 and iRECIST) OS, defined as the time from first study treatment administration to death from any cause. If there are no survival events, OS will be censored at the last day the patient is known to be alive. Change in CTC count in quantifiable samples (cells / 7.5 mL) Changes in serum cytokines and other biomarkers of inflammation Time to pain progression (TTPP), assessed by BPI-SF item 3 ("worst pain in 24 hours") and lactate analgesic use Change from baseline in pain severity and pain interference measured by the BPI-SF Change from baseline in GHS / QoL as measured by EORTC QLQ-C30 GHS / QoL scale score Change from baseline in physical function as measured by the EORTC QLQ-C30 physical function scale score Change from baseline in urinary symptoms as measured by the EORTC QLQ-PR25 urinary symptom scale score Change from baseline in Patient Global Impression of Severity Score (PGIS) Change from baseline in Patient Global Impression of Change (PGIC) score Correlation of baseline tumor tissue biomarker levels of mAb1 and cemiplimab target pathways (e.g., PD-L1, PSMA, and CD28) with treatment activity in available tissue biopsies Association of oncogene variants, including HDR mutations and TMB, with ORR Changes in PSMA and FDG PET / CT tumor signal intensity from baseline after treatment initiation Correlation between baseline PSMA PET / CT tumor positivity and clinical activity of mAb1 and cemiplimab All exploratory endpoints will be applied to each cohort in the study, including those receiving sarilumab and those not receiving sarilumab.

[0171] result Antitumor activity has been observed in patients treated with the combination of mAb1 and cemiplimab, with a manageable safety profile. Patients with prostate cancer (e.g., metastatic castration-resistant prostate cancer) treated with mAb1 at 30 mg to 300 mg weekly (to date) and cemiplimab (350 mg every three weeks) have demonstrated antitumor activity as measured by a reduction in prostate-specific antigen (PSA) levels (e.g., a greater than 50% reduction) and / or a RESIST response. Results are shown in Figures 3A, 3B, 3C, and 3D and in Table 9 below. [Table 10]

[0172] Updated antitumor data observed in 35 patients (17 in dose levels 1–5 and 18 in dose levels 6–8) are shown in Figures 4A and 4B. Notably, no patients treated at dose levels 1–5 experienced a grade ≥3 immune-mediated adverse event (imAE), and only one of 16 evaluable patients at dose levels 1–5 experienced a decline in PSA levels. In contrast, patients treated at dose levels 6–8 demonstrated signs of efficacy associated with imAEs. At dose level 6, one of four evaluable patients experienced a response (a 100% decline in PSA levels and a complete target lesion response maintained for approximately 12 months) along with a grade 3 imAE. At dose level 7, three of eight evaluable patients experienced a response (99%, 44%, and 22% declines in PSA levels), two of whom experienced grade 3 imAEs. At dose level 8, three of four evaluable patients responded (99%, 99%, and 82% reductions in PSA levels), one of whom had a fatal imAE. Mitigation strategies to prevent imAEs that appear to be associated with treatment response are under investigation and include blockade of the IL-6R.

[0173] In each of the three patients treated at dose level 8 in whom a response was observed, PSA levels continued to rise during the lead-in administration of mAb1 until cemiplimab administration was initiated. Progressive metastatic castration-resistant prostate cancer (mCRPC) exhibits a response rate of approximately 5% to anti-PD-1 monotherapy, and the resulting rise in PSA levels observed until the initiation of cemiplimab administration is evidence of synergy between mAb1 and cemiplimab in patients with mCRPC.

[0174] One patient treated at dose level 7 (99% reduction in PSA level) showed pseudoprogression in the liver followed by a response confirmed by a decrease in PSMA PET-positive lesions. Notably, this same patient demonstrated a response in tumor lesions with low PSMA PET signal that, based on the eligibility criteria, would not have been expected to respond to Pluvicto™ (lutetium Lu 177 bipibotide tetraxetane), which comprises a PSMA-binding ligand conjugated to a DOTA chelator radiolabeled with lutetium-177.

[0175] Initial results suggest minimal antitumor activity at low doses, as predicted by preclinical models. However, antitumor activity was amplified with the initiation of cemiplimab. At dose level 8, three of four patients demonstrated a significant PSA response at the initiation of combination therapy (Figure 5). Additionally, one patient at dose level 6 experienced a response with PSA levels below the limit of detection, normalized bone scans with negative PSMA PET scans, and resolution of soft tissue disease. However, this patient discontinued treatment due to a grade 3 irAE of the skin that resolved with treatment. Grade ≥ 3 immune-related adverse events occurred only in select patients with antitumor activity.

[0176] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. *******

[0177]

Table 11

Claims

1. 1. A method of treating a PSMA-expressing cancer in a subject in need thereof, comprising administering to the subject a combination of a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds prostate-specific membrane antigen (PSMA) on target tumor cells and a second antigen-binding domain that specifically binds human CD28 on T cells, and an antibody or antigen-binding fragment thereof that specifically binds programmed death receptor-1 (PD-1), wherein the bispecific antibody is administered to the subject at a dose of at least 0.03 mg.

2. 10. The method of claim 1, wherein the PSMA-expressing cancer is prostate cancer.

3. 3. The method of claim 1 or 2, wherein the PSMA-expressing cancer is metastatic prostate cancer.

4. The method of any one of claims 1 to 3, wherein the PSMA-expressing cancer is castration-resistant prostate cancer.

5. The method of any one of claims 1 to 4, wherein the subject has undergone at least two prior treatments for metastatic and / or castration-resistant prostate cancer.

6. 6. The method of claim 5, wherein the subject is receiving at least one anti-androgen therapy.

7. 7. The method of claim 6, wherein the antiandrogen therapy is selected from abiraterone, enzalutamide, apalutamide, or darolutamide.

8. The method of any one of claims 1 to 7, wherein the subject has histologically or cytologically confirmed adenocarcinoma of the prostate that does not have pure small cell carcinoma.

9. 9. The method of any one of claims 1 to 8, wherein the subject has metastatic castration-resistant prostate cancer with a prostate-specific antigen (PSA) level of ≧4 ng / ml prior to treatment with the bispecific antibody.

10. 10. The method of claim 9, wherein the subject's cancer has progressed within 6 months prior to treatment with the bispecific antibody, and cancer progression is determined by (a) a confirmed rise in PSA levels with an interval of ≥ 1 week between assessments, (b) soft tissue radiographic disease progression with or without a rise in PSA, and / or (c) bone radiographic disease progression with the appearance of two or more bone lesions on a bone scan with or without a rise in PSA.

11. The method of any one of claims 1 to 10, wherein the subject has undergone an orchiectomy.

12. 11. The method of any one of claims 1 to 10, wherein the subject is receiving luteinizing hormone-releasing hormone (LHRH) agonist or antagonist therapy and has a serum testosterone level of <50 ng / ml prior to treatment with the bispecific antibody.

13. the first antigen-binding domain (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

9.

14. 14. The method of claim 13, wherein the first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:

4.

15. The method of claim 13 or 14, wherein the first antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

12.

16. The method of any one of claims 13 to 15, wherein the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO:

9.

17. the second antigen-binding domain (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:5; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

9.

18. 18. The method of claim 17, wherein the second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:

8.

19. The method of claim 17 or 18, wherein the second antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

12.

20. The method of any one of claims 17 to 19, wherein the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 5 and an LCVR comprising the amino acid sequence of SEQ ID NO:

9.

21. The method of any one of claims 1 to 20, wherein the bispecific antibody comprises a human IgG heavy chain constant region.

22. 22. The method of claim 21, wherein the human IgG heavy chain constant region is of isotype IgG1.

23. 22. The method of claim 21, wherein the human IgG heavy chain constant region is of isotype IgG4.

24. 24. The method of claim 22 or 23, wherein the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

25. 25. The method of any one of claims 21 to 24, wherein the first heavy chain or the second heavy chain, but not both, comprises a CH3 domain comprising an H435R (EU numbering) modification and a Y436F (EU numbering) modification.

26. The method of any one of claims 1 to 20, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:

13.

27. The method of any one of claims 1 to 20, wherein the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:

14.

28. 21. The method of any one of claims 1 to 20, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 13, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 14, and a common light chain comprising the amino acid sequence of SEQ ID NO:

15.

29. the first antigen-binding domain (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 16; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

28.

30. 30. The method of claim 29, wherein the first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:

19.

31. The method of claim 29 or 30, wherein the first antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

31.

32. The method of any one of claims 29 to 31, wherein the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 16 and an LCVR comprising the amino acid sequence of SEQ ID NO:

28.

33. the second antigen-binding domain (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 24; and (b) the method of any one of claims 29 to 32, comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

28.

34. 34. The method of claim 33, wherein the second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO:

27.

35. The method of claim 33 or 34, wherein the second antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

31.

36. The method of any one of claims 33 to 35, wherein the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 24, and an LCVR comprising the amino acid sequence of SEQ ID NO:

28.

37. The method of any one of claims 29 to 36, wherein the bispecific antibody comprises a human IgG heavy chain constant region.

38. 38. The method of claim 37, wherein the human IgG heavy chain constant region is of isotype IgG1.

39. 38. The method of claim 37, wherein the human IgG heavy chain constant region is of isotype IgG4.

40. 40. The method of claim 38 or 39, wherein the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

41. 41. The method of any one of claims 37 to 40, wherein the first heavy chain or the second heavy chain, but not both, comprises a CH3 domain comprising an H435R (EU numbering) modification and a Y436F (EU numbering) modification.

42. The method of any one of claims 29 to 36, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:

32.

43. The method of any one of claims 29 to 36, wherein the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:

33.

44. The method of any one of claims 29 to 36, wherein the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:

34.

45. 37. The method of any one of claims 29 to 36, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and a common light chain comprising the amino acid sequence of SEQ ID NO:

35.

46. 37. The method of any one of claims 29 to 36, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and a common light chain comprising the amino acid sequence of SEQ ID NO:

35.

47. The antibody or antigen-binding fragment thereof that binds PD-1 is (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 36; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

40.

48. 48. The method of claim 47, wherein the antibody or antigen-binding fragment thereof that binds PD-1 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:

39.

49. The method of claim 47 or 48, wherein the antibody or antigen-binding fragment thereof that binds PD-1 comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 41, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

43.

50. 50. The method of any one of claims 47 to 49, wherein the antibody or antigen-binding fragment thereof that binds PD-1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 36 and an LCVR comprising the amino acid sequence of SEQ ID NO:

40.

51. The method of claim 50, wherein the antibody or antigen-binding fragment thereof that binds PD-1 is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the amino acid sequence of SEQ ID NO:

45.

52. 52. The method of any one of claims 1 to 51, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once a week at a dose of 0.03 mg to 1000 mg.

53. 53. The method of claim 52, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once weekly at a dose of 0.03 mg to 900 mg.

54. 53. The method of claim 52, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once weekly at a dose of 30 mg to 900 mg.

55. 53. The method of claim 52, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once weekly at a dose of 100 mg to 900 mg.

56. 53. The method of claim 52, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once weekly at a dose of 300 mg to 900 mg.

57. 52. The method of any one of claims 1 to 51, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once every three weeks at a dose of 0.03 mg to 1000 mg.

58. 58. The method of claim 57, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject once every three weeks at a dose of 0.03 mg to 900 mg.

59. 58. The method of claim 57, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 30 mg to 900 mg once every three weeks.

60. 58. The method of claim 57, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 100 mg to 900 mg once every three weeks.

61. 58. The method of claim 57, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 300 mg to 900 mg once every three weeks.

62. 62. The method of any one of claims 1-61, wherein the antibody or antigen-binding fragment thereof that binds PD-1 is administered to the subject at a dose of 300-400 mg once every three weeks.

63. 63. The method of claim 62, wherein the antibody or antigen-binding fragment thereof that binds PD-1 is administered to the subject at a dose of 350 mg once every three weeks.

64. 64. The method of any one of claims 1-63, wherein the subject exhibits stable disease, a partial response, or a complete response after at least 1 week of administration of the bispecific antibody or antigen-binding fragment thereof, in combination with the antibody or antigen-binding fragment thereof that binds PD-1, at a dose of 0.03 mg to 900 mg.

65. The method of any one of claims 1 to 64, further comprising administering to the subject an IL-6R antagonist.

66. 66. The method of claim 65, wherein the IL-6R antagonist is an anti-IL-6R antibody, optionally wherein the anti-IL-6R antibody is sarilumab or tocilizumab.

67. the subject, following administration of the bispecific anti-PSMA x anti-CD28 antibody, or antigen-binding fragment thereof, and the anti-PD-1 antibody, or antigen-binding fragment thereof, - at least a 50% reduction in prostate-specific antigen (PSA) levels in said subject; - at least a 55% reduction in PSA levels in said subject; - at least a 60% reduction in PSA levels in said subject; - at least a 65% reduction in PSA levels in said subject; - at least a 70% reduction in PSA levels in said subject; - at least a 75% reduction in PSA levels in said subject; - at least an 80% reduction in PSA levels in said subject; - at least an 85% reduction in PSA levels in said subject; - at least a 90% reduction in PSA levels in said subject; - at least a 95% reduction in PSA levels in said subject; - at least a 96% reduction in PSA levels in said subject; - at least a 97% reduction in PSA levels in said subject; - at least a 98% reduction in PSA levels in said subject; - at least a 99% reduction in PSA levels in said subject; a decrease in the size of at least one lesion having a PSMA PET signal that is smaller than the PSMA PET signal in the liver of the subject; and / or - a response in the subject after pseudoprogression.

68. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject a combination of a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds a tumor-associated antigen on tumor cells and a second antigen-binding domain that specifically binds human CD28 on T cells, and an antibody or antigen-binding fragment thereof that specifically binds programmed death receptor-1 (PD-1).

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