Methods of treating metastatic castration resistant prostate cancer using bispecific anti-psmax anti-CD28 antibodies in combination with anti-pd-1 antibodies

Abstract

The present disclosure provides methods for treating, reducing the severity, or inhibiting the growth of cancer (e.g., prostate cancer or metastatic castration-resistant prostate cancer). The methods of the present disclosure comprise 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 to Programmed Death Receptor-1 (PD-1).

Description

[0001] Reference to sequence listing

[0002] This application incorporates by reference a computer-readable sequence listing in ST.26 XML format, named 11051WO01_Sequence, created on July 28, 2023, and containing 58,900 bytes. Technical Field

[0003] 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 Art

[0004] Prostate-specific membrane antigen (PSMA), also known as FOLH1, glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALAD enzyme I) or N-acetyl-aspartylglutamate (NAAG) peptidase, is a homodimeric enzyme type II transmembrane protein encoded by the folate hydrolase 1 (FOLH1) gene. PSMA is an intact non-shedding membrane glycoprotein that is highly expressed in malignant prostate tissue and is a cell surface marker for prostate cancer, but shows limited expression in normal tissue. Its expression remains unchanged in castration-resistant prostate cancer, a condition with a poor outcome and limited treatment options. Methods for treating prostate cancer by targeting PSMA have been studied. For example, yttrium-90 carlosomab is a radiotherapeutic agent comprising a monoclonal antibody to an intracellular epitope of PSMA. In another example, J591 is a monoclonal antibody directed against an extracellular epitope of PSMA, is part of the radiotherapeutic agent Lutetium-177 J591, and in MLN2704 in which maytansinoid 1 (DM1, an anti-microtubule agent) is conjugated to J591. These therapies are associated with toxicity. PSMA is also expressed in the neovasculature of other tumors such as bladder cancer, kidney cancer, gastric cancer, and colorectal cancer.

[0005] CD28 is a type I transmembrane protein, which has a single extracellular Ig-V-like domain assembled into a homodimer and expressed on the surface of T cells. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2) proteins, and is activated by CD80 or CD86 expressed on antigen presenting cells (APC). The combination of CD28 with CD80 or CD86 provides a co-stimulatory signal that is important for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation by CD28 also provides an effective signal for the production of various interleukins. CD28 also enhances cell signals (such as pathways) controlled by NFκB transcription factors after TCR activation. CD28 co-signals are important for effective T cell activation (such as T cell differentiation), proliferation, cytokine release and cell death. It has been proposed that anti-CD28 antibodies are used for therapeutic purposes involving T cell activation. A specific anti-CD28 antibody, TGN1412 (anti-CD28 superagonist), was used in a clinical trial in 2006, where TGN1412 (anti-CD28 superagonist) was administered intravenously to six healthy volunteers at a dose of 0.1 mg / kg. Within two hours, all six patients had a significant inflammatory response (cytokine storm), and within sixteen hours, all patients had multiple organ failure. Subjects were treated with corticosteroids, and cytokine levels returned to normal within 2-3 days (Suntharalingam et al., Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412, NEJM 355: 1018-1028 (2006)).

[0006] Programmed death receptor-1 (PD-1) signaling in the tumor microenvironment plays a key role in allowing tumor cells to escape immune surveillance by the host immune system. Blocking of the PD-1 signaling pathway has shown clinical activity in patients with a variety of tumor types, and antibody therapies that block PD-1 (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's lymphoma (Hodgkin's lymphoma) (Lesokhin, et al. 2014, Abstract 291, 56th ASH Annual Meeting and Exhibition, San Francisco, Calif.; Ansell et al. 2015, N. Engl. J. Med. 372 (4): 311-9).

[0007] Prostate cancer is the leading cause of new cancer diagnoses and the second most common cause of cancer-related death in men in the United States. In 2018, there were an estimated 1.3 million new cases of prostate cancer and 358,989 deaths worldwide. For decades, therapies that block androgen-related pathways have been the standard of care for prostate cancer. However, patients progress to androgen depletion and / or surgical castration and develop castration-resistant prostate cancer. For men with metastatic castration-resistant prostate cancer (mCRPC), the prognosis is particularly poor. Currently, metastatic prostate cancer remains incurable, and improving long-term survival remains a high unmet need. Summary of the invention

[0008] According to certain embodiments, the present disclosure provides methods for treating, ameliorating at least one symptom or indication of a PSMA-expressing cancer in a subject, or inhibiting its growth. The method according to this aspect of the present 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 to prostate-specific membrane antigen (PSMA) and CD28 in combination with an antibody or antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1).

[0009] In certain embodiments of the present disclosure, methods for treating, ameliorating at least one symptom or indication of a PSMA-expressing cancer in a subject, or inhibiting its growth are provided. In certain embodiments of the present disclosure, methods for delaying tumor growth or preventing tumor recurrence are provided. According to this aspect and other aspects of the present disclosure, the method comprises sequentially administering to a subject in need thereof one or more doses of a therapeutically effective amount of a bispecific anti-PSMA×anti-CD28 antibody or antigen-binding fragment thereof in combination with one or more doses of a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof.

[0010] In one aspect, the present disclosure provides a method of treating a PSMA-expressing cancer in a subject in need thereof, the method comprising administering to the subject a bispecific antibody or antigen-binding fragment thereof in combination with an antibody or antigen-binding fragment thereof that specifically binds programmed death receptor-1 (PD-1), wherein the bispecific antibody or antigen-binding fragment thereof comprises 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, wherein the bispecific antibody is administered to the subject at a dose of at least 0.03 mg.

[0011] In some embodiments, the PSMA-expressing cancer is prostate cancer. In some instances, the PSMA-expressing cancer is metastatic prostate cancer. In some instances, the PSMA-expressing cancer is castration-resistant prostate cancer.

[0012] 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.

[0013] In some embodiments, the subject has histologically or cytologically confirmed prostate adenocarcinoma without pure small cell carcinoma.

[0014] In some embodiments, prior to treatment with the bispecific antibody, the subject already had metastatic castration-resistant prostate cancer with a prostate-specific antigen (PSA) value of ≥4 ng / ml. In some cases, the subject's cancer had progressed within a 6-month period prior to treatment with the bispecific antibody, wherein cancer progression was determined by: (a) an increase in PSA levels confirmed with an interval of ≥1 week between each assessment; (b) radiographic disease progression in soft tissues with or without an increase in PSA; and / or (c) radiographic disease progression in bones with the appearance of two or more bone lesions on a bone scan with or without an increase in PSA.

[0015] In some embodiments, the subject has undergone orchiectomy.In some embodiments, 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.

[0016] In some embodiments, the first antigen binding domain of the bispecific antibody comprises: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3), the three heavy chain complementary determining regions being contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 1; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3), the three light chain complementary determining regions being contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 9. In some cases, the first antigen binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 2, a HCDR2 having an amino acid sequence of SEQ ID NO: 3, and a HCDR3 having an amino acid sequence of SEQ ID NO: 4. In some cases, the first antigen binding domain comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 10, a LCDR2 having an amino acid sequence of SEQ ID NO: 11, and a LCDR3 having an amino acid sequence of SEQ ID NO: 12. In some embodiments, the first antigen binding domain comprises a HCVR having the amino acid sequence of SEQ ID NO:1 and a LCVR having the amino acid sequence of SEQ ID NO:9.

[0017] In some embodiments, the second antigen-binding domain of the bispecific antibody comprises: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3), the three heavy chain complementary determining regions being contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 5; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3), the three light chain complementary determining regions being contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 9. In some cases, the second antigen-binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 6, a HCDR2 having an amino acid sequence of SEQ ID NO: 7, and a HCDR3 having an amino acid sequence of SEQ ID NO: 8. In some cases, the second antigen-binding domain comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 10, a LCDR2 having an amino acid sequence of SEQ ID NO: 11, and a LCDR3 having an amino acid sequence of SEQ ID NO: 12. In some embodiments, the second antigen binding domain comprises a HCVR having the amino acid sequence of SEQ ID NO:5 and a LCVR having the amino acid sequence of SEQ ID NO:9.

[0018] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:13.

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

[0020] In some embodiments, the bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO:13, a second heavy chain having the amino acid sequence of SEQ ID NO:14, and a common light chain having the amino acid sequence of SEQ ID NO:15.

[0021] In some embodiments, the first antigen binding domain of the bispecific antibody comprises: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3), the three heavy chain complementary determining regions being contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 16; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3), the three light chain complementary determining regions being contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 28. In some cases, the first antigen binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 17, a HCDR2 having an amino acid sequence of SEQ ID NO: 18, and a HCDR3 having an amino acid sequence of SEQ ID NO: 19. In some cases, the first antigen binding domain comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 29, a LCDR2 having an amino acid sequence of SEQ ID NO: 30, and a LCDR3 having an amino acid sequence of SEQ ID NO: 31. In some cases, the first antigen binding domain comprises a HCVR having the amino acid sequence of SEQ ID NO:16 and a LCVR having the amino acid sequence of SEQ ID NO:28.

[0022] In some embodiments, the second antigen-binding domain of the bispecific antibody comprises: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 20 of SEQ ID NO: 24; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 28. In some cases, the second antigen-binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25, a HCDR2 having an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 26, and a HCDR3 having an amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 27. In some cases, the second antigen binding domain comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 29, a LCDR2 having an amino acid sequence of SEQ ID NO: 30, and a LCDR3 having an amino acid sequence of SEQ ID NO: 31. In some embodiments, the second antigen binding domain comprises a HCVR having an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 24 and a LCVR having an amino acid sequence of SEQ ID NO: 28.

[0023] In any of the various embodiments discussed above or herein, the bispecific antibody can comprise a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is of isotype IgG1. In some cases, the human IgG heavy chain constant region is of isotype IgG4.

[0024] In any of the various embodiments discussed above or herein, the bispecific antibody may comprise a chimeric hinge that reduces Fcγ receptor binding relative to a wild-type hinge of the same isotype.

[0025] In any of the various embodiments discussed above or 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 a H435R (EU numbering) modification and a Y436F (EU numbering) modification.

[0026] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:32.

[0027] In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:33.

[0028] In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:34.

[0029] In some embodiments, the bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO:32, a second heavy chain having the amino acid sequence of SEQ ID NO:33, and a common light chain having the amino acid sequence of SEQ ID NO:35.

[0030] In some embodiments, the bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO:32, a second heavy chain having the amino acid sequence of SEQ ID NO:34, and a common light chain having the amino acid sequence of SEQ ID NO:35.

[0031] In any of the various embodiments discussed above or herein, the antibody or antigen-binding fragment thereof that binds to PD-1 comprises: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 36; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 40. In some cases, the antibody or antigen-binding fragment that binds to PD-1 comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 37, a HCDR2 having an amino acid sequence of SEQ ID NO: 38, and a HCDR3 having an amino acid sequence of SEQ ID NO: 39. In some cases, the antibody or antigen-binding fragment that binds to PD-1 comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 41, a LCDR2 having an amino acid sequence of SEQ ID NO: 42, and a LCDR3 having an amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment that binds to PD-1 comprises a HCVR having an amino acid sequence of SEQ ID NO: 36 and a LCVR having an amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment that binds to PD-1 is an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 44 and a light chain having an amino acid sequence of SEQ ID NO: 45.

[0032] In any of the various embodiments discussed above or herein, the bispecific antibody or its antigen-binding fragment can be administered to a subject at a dosage of 0.03 mg to 1000 mg per week. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 0.03 mg to 900 mg per week. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 30 mg to 900 mg per week. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 100 mg to 900 mg per week. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 300 mg to 900 mg per week.

[0033] In any of the various embodiments discussed above or herein, the bispecific antibody or its antigen-binding fragment can be administered to a subject at a dosage of 0.03mg to 1000mg once every three weeks. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 0.03mg to 900mg once every three weeks. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 30mg to 900mg once every three weeks. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 100mg to 900mg once every three weeks. In some cases, the bispecific antibody or its antigen-binding fragment is administered to a subject at a dosage of 300mg to 900mg once every three weeks.

[0034] In any of the various embodiments discussed above or herein, the antibody or antigen-binding fragment thereof that binds to PD-1 can be administered to the subject at a dose of 300 mg to 400 mg once every three weeks. In some cases, the antibody or antigen-binding fragment thereof that binds to PD-1 is administered to the subject at a dose of 350 mg once every three weeks.

[0035] In any of the various embodiments discussed above or herein, the subject has stable disease, partial remission, or complete remission after at least one week of administration of a dose of 0.03 mg to 900 mg of the bispecific antibody or antigen-binding fragment thereof in combination with an antibody or antigen-binding fragment thereof that binds to PD-1.

[0036] In any of the various embodiments discussed above or herein, an IL-6R antagonist can be further administered to the subject. 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.

[0037] In any of the various embodiments discussed above or herein, following administration of a combination of a bispecific anti-PSMA×CD28 antibody (e.g., REGN5678) or an antigen-binding fragment thereof and an anti-PD-1 antibody (e.g., cemiplimab) or an antigen-binding fragment thereof:

[0038] The prostate-specific antigen (PSA) level in the subject decreases by at least 50%;

[0039] The PSA level in the subject decreases by at least 55%;

[0040] The PSA level in the subject decreases by at least 60%;

[0041] The PSA level in the subject decreases by at least 65%;

[0042] The PSA level in the subject is reduced by at least 70%;

[0043] The PSA level in the subject is reduced by at least 75%;

[0044] The PSA level in the subject is reduced by at least 80%;

[0045] The PSA level in the subject is reduced by at least 85%;

[0046] The PSA level in the subject is reduced by at least 90%;

[0047] The PSA level in the subject is reduced by at least 95%;

[0048] The subject's PSA level is reduced by at least 96%;

[0049] The PSA level in the subject is reduced by at least 97%;

[0050] The PSA level in the subject is reduced by at least 98%;

[0051] The PSA level in the subject is reduced by at least 99%;

[0052] a decrease in size of at least one lesion in the subject, the PSMA PET signal of the lesion being less than the PSMA PET signal in the subject's liver; and / or

[0053] The subject responded after a pseudoprogression.

[0054] The present disclosure also encompasses the use of bispecific antibodies and / or anti-PD-1 antibodies (and antigen-binding fragments of any of them) in the manufacture of a medicament for treating a PSMA-expressing cancer as described in any of the embodiments of the methods discussed above or herein. The present disclosure also encompasses bispecific antibodies and / or anti-PD-1 antibodies (and antigen-binding fragments of any of them) for use in any of the embodiments of the methods discussed above or herein. The present disclosure also encompasses pharmaceutical compositions comprising bispecific antibodies and / or anti-PD-1 antibodies (and antigen-binding fragments of any of them) for use in any of the embodiments of the methods discussed above or herein.

[0055] In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject a combination of a bispecific antibody or an antigen-binding fragment thereof and an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), the bispecific antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to a tumor-associated antigen on a tumor cell and a second antigen-binding domain that specifically binds to human CD28 on a T cell. In various embodiments, any features or components of the embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specified value discussed above or herein may be combined with another related value discussed above or herein to list a range, the values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0056] Other embodiments of the present invention will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 An embodiment of a study flow chart for QW dosing of REGN5678 in combination with Q3W dosing of semilazumab, as discussed in Example 5, is shown. 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 semilazumab 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 upon disease progression. For dose escalation patients, these PET / CT scans are optional. DLT = dose-limiting toxicity, PD = progressive disease, Q6W = every 6 weeks, Q12W = every 12 weeks.

[0058] Figure 2An embodiment of a study flow chart for Q3W dosing of REGN5678 in combination with Q3W dosing of semilaplimab, as discussed in Example 5, is shown. 1 Dose cohorts with a Q3W dosing interval can receive a 3-week lead-in of REGN5678 monotherapy followed by a combination of REGN5678 and semilazumab. 2 For some dose expansion cohorts, PSMA-PET / CT and optional whole-body FDG-PET / CT will be performed at screening, 12 to 24 weeks after treatment initiation, and upon disease progression. For dose escalation patients, these PET / CT scans are optional. DLT = dose-limiting toxicity, PD = progressive disease, Q6W = every 6 weeks, Q12W = every 12 weeks.

[0059] Figure 3A , 3B , 3C and 3D are prostate specific antigen (PSA) waterfall graphs showing the results of administering mAb1 (0.1 mg to 300 mg) and semaplizumab in combination to patients with metastatic castration-resistant prostate cancer (first 33 patients). Each graph shows the best percent change in PSA levels relative to baseline for patients receiving different doses of mAb1 in combination with semaplizumab (baseline corresponds to PSA plotted shortly before the first combined dose). Figure 3A All mAbl doses administered are shown. Figure 3B Doses ranging from 0.1 mg to 10 mg are shown. Figure 3C Doses ranging from 30 mg to 300 mg are shown. Figure 3D All doses in the comparison of ≥30 mg doses versus <30 mg doses are shown. The doses administered (DL) correspond to the doses administered shown in Table 8. Figure 3A , 3B and 3C show the DL number above or below each bar, and Figure 3D The bars corresponding to DL6-8 are marked with asterisks.

[0060] Figure 4A and 4B The following are prostate-specific antigen (PSA) waterfall graphs showing the results of administering mAb1 (0.1 mg to 300 mg) and semaplizumab in combination to patients with metastatic castration-resistant prostate cancer (first 35 patients to date). Each graph shows the best percent change from baseline in PSA levels for patients receiving different doses of mAb1 in combination with semaplizumab (baseline corresponds to the PSA plotted shortly before the first combined dose). Figure 4A Doses ranging from 0.1 mg to 10 mg are shown. Figure 4BDoses ranging from 30 mg to 300 mg are shown. The administered doses (DL) correspond to the administered doses shown in Table 8. Figure 4A and 4B The DL number is shown above or below each bar in the chart.

[0061] Figure 5 The decrease in PSA levels in patients treated with dose 8 (300 mg mAb1 and 350 mg semiplimab) is shown. PSMA×CD28 corresponds to mAb1, and Libtayo TM It's semimiprimab.

[0062] Figure 6 An embodiment of a study flow chart is shown for QW dosing of REGN5678 in combination with Q3W dosing of salizumab and salizumab, as discussed in Example 5. As shown, the dose of salizumab was 350 mg IV Q3W for a total of 12 weeks starting with the initial dose of REGN5678 in combination with salizumab (C1D1).

[0063] Figure 7 An embodiment of a study flow chart is shown for Q3W dosing of REGN5678 in combination with Q3W dosing of salizumab and salizumab, as discussed in Example 5. As shown, the dose of salizumab was 350 mg IV Q3W for a total of 12 weeks starting with the initial dose of REGN5678 in combination with salizumab (C1D1). DETAILED DESCRIPTION

[0064] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, and are not intended to be limited, because the scope of the present invention will only be limited by the appended claims. The features of any embodiment or embodiment can be combined with each other, and such combinations are clearly included in the scope of the present invention. Any specified value discussed above or herein can be combined with another related value discussed above or herein to list a range, and the value represents the upper and lower limits of the range, and such ranges are included in the scope of the present disclosure.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited numerical value means that the value may vary by no more than 1% 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.).

[0066] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0067] Methods for treating or inhibiting the growth of cancer

[0068] The present disclosure includes methods for treating, improving or reducing the severity of at least one symptom or indication in a subject, or inhibiting the growth of cancer (e.g., metastatic castration-resistant prostate cancer). The method according to this aspect of the present disclosure includes administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody for PSMA and CD28 in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to PD-1. The term "treatment" and the like as used herein means alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, delaying or inhibiting tumor growth, reducing tumor cell load or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and / or disappearance, preventing tumor recurrence, and / or increasing the duration of survival of a subject.

[0069] As used herein, the expression "subject" or "subject in need thereof" means a human or non-human mammal that exhibits one or more symptoms or signs of cancer, and / or has been diagnosed with cancer, including prostate cancer (e.g., metastatic castration-resistant prostate cancer), and is in need of treatment for the cancer. In many embodiments, the term "subject" can be used interchangeably with the term "patient." For example, a human subject may be diagnosed with a primary or metastatic tumor and / or one or more symptoms or signs, including, but not limited to, enlarged lymph nodes, abdominal swelling, unexplained pain, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, and / or an enlarged spleen. The expression includes subjects with primary or established prostate tumors. In specific embodiments, the expression includes human subjects with prostate cancer or another tumor that expresses PSMA and is in need of treatment. In other specific embodiments, the expression includes subjects with PSMA+ tumors (e.g., tumors with PSMA expression as determined by flow cytometry). In certain embodiments, the expression "subject in need" includes patients with prostate cancer, which is resistant to previous therapies (e.g., treatment with conventional anticancer agents, including anti-androgen therapy) or is difficult to treat with previous therapies, or cannot be adequately controlled by previous therapies. For example, the expression includes subjects who have been treated with chemotherapy or anti-androgen therapy, such as abiraterone, enzalutamide, apalutamide or darolutamide. The expression also includes subjects with prostate tumors, for which conventional anticancer therapy is undesirable, for example, due to toxic side effects. For example, the expression includes patients who have received one or more cycles of chemotherapy or other anticancer therapy with toxic side effects. In certain embodiments, the expression "subject in need" includes patients with prostate tumors, who have been treated but subsequently relapsed or metastasized. For example, patients with prostate tumors who may have been treated with one or more anticancer agents leading to tumor regression; however, cancers resistant to one or more anticancer agents (e.g., castration-resistant prostate cancer) are subsequently treated with the methods of the present disclosure.

[0070] In certain embodiments, the method of the present disclosure can be used to treat patients with prostate adenocarcinoma confirmed histologically or cytologically without pure small cell carcinoma. In certain embodiments, the method of the present disclosure can be used to treat patients with metastatic castration-resistant prostate cancer, and the prostate-specific antigen (PSA) value of the patient before treatment with the bispecific antibody is ≥4ng / ml (e.g., 4ng / ml, 4.5ng / ml, 5ng / ml, 5.5ng / ml, 6ng / ml, 6.5ng / ml, 7ng / ml, 7.5ng / ml, 8ng / ml, 8.5ng / ml, 9ng / ml, 9.5ng / ml or 10ng / ml or higher). In certain embodiments, the methods of the present disclosure can be used to treat patients with prostate cancer that progressed within a certain period of time (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer) prior to treatment with a bispecific antibody, wherein cancer progression is determined, for example, by: (a) an increase in PSA levels confirmed with an interval of ≥1 week (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks or longer) between each assessment; (b) radiographic (e.g., PET / CT imaging) disease progression in soft tissues, with or without elevated PSA; and / or (c) radiographic (e.g., PET / CT imaging) disease progression in bones, with the appearance of two or more bone lesions in a bone scan, with or without elevated PSA. In certain embodiments, the methods of the present disclosure can be used to treat patients who have undergone orchiectomy. In certain embodiments, the methods of the present disclosure can be used to treat patients who have been or are receiving luteinizing hormone releasing hormone (LHRH) agonist or antagonist therapy and whose serum testosterone levels are <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.

[0071] In certain embodiments, the methods of the present disclosure can be used for subjects suffering from prostate cancer. The terms "tumor", "cancer" and "malignant tumor" are used interchangeably herein. As used herein, the term "prostate cancer" refers to prostate tumors, including metastatic tumors derived from the prostate.

[0072] According to certain embodiments, the present disclosure includes methods for treating or delaying or inhibiting tumor growth. In certain embodiments, the present disclosure includes methods for promoting tumor regression. In certain embodiments, the present disclosure includes methods for reducing tumor cell load or reducing tumor load. In certain embodiments, the present disclosure includes methods for preventing tumor recurrence. According to this aspect of the present disclosure, the method includes administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein each antibody or fragment is administered to the subject in multiple doses, for example, as part of a specific therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering one or more doses of an anti-PSMA×CD28 antibody or an antigen-binding fragment thereof to a subject at a frequency of about once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or less frequently. In certain embodiments, anti-PSMA×anti-CD28 antibodies or antigen-binding fragments thereof are administered once a week. In certain embodiments, the anti-PSMA x anti-CD28 antibody or antigen-binding fragment thereof is administered once every three weeks. In certain embodiments, one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof are administered to the subject at a frequency of about once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or less. In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject once every three weeks.

[0073] In certain embodiments, the present disclosure includes methods of inhibiting, delaying or stopping tumor metastasis or tumor infiltration into peripheral organs. According to this aspect, the method 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.

[0074] In a specific embodiment, the anti-PSMA / CD28 bispecific antibody or antigen-binding fragment thereof is administered to the subject prior to the anti-PD-1 antibody or antigen-binding fragment thereof. In some cases, the anti-PSMA / CD28 antibody or antigen-binding fragment thereof can 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 prior to the anti-PD-1 antibody or antigen-binding fragment thereof.

[0075] In certain embodiments, the methods of the present disclosure are used to treat patients with MRD-positive diseases. Minimal residual disease (MRD) refers to a small amount of cancer cells remaining in a patient's body during or after treatment, wherein the patient may or may not show symptoms or signs of the disease. Such residual cancer cells, if not eliminated, often lead to recurrence of the disease. The present disclosure includes methods for inhibiting and / or eliminating residual cancer cells in a patient's body after an MRD test. MRD can be determined according to methods known in the art (e.g., MRD flow cytometry). According to this aspect of the present disclosure, the method comprises administering to a subject in need thereof a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0076] According to certain embodiments, the methods of the present 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 can be an agent selected from the group consisting of, for example, radiation, chemotherapy, surgery, cancer vaccines, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), LAG3 inhibitors (e.g., anti-LAG3 antibodies), CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, Ang2 inhibitors, transforming growth factor β (TGF.β.) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies to tumor-specific antigens, cytotoxins, chemotherapeutic agents, anti-androgen therapy, IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, cytokines (such as IL-2, IL-7, IL-21 and IL-15), anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs) and dietary supplements (such as antioxidants). In certain embodiments, the antibody can be administered in combination with a therapy including chemotherapeutic agents, radiation, and surgery. As used herein, the phrase "in combination with" means that the antibody is administered to the subject at the same time, just before, or just after administration of a third therapeutic agent. In certain embodiments, the antibody and the third therapeutic agent are administered in separate formulations.

[0077] In certain embodiments, the methods of the present disclosure include administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof. In certain embodiments, the combined administration 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 untreated subjects. In certain embodiments, the combined administration results in increased tumor regression, tumor shrinkage, and / or disappearance. In certain embodiments, the combined administration results in delayed tumor growth and development, for example, compared to untreated subjects, 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. In certain embodiments, the combined administration prevents tumor recurrence and / or extends the survival duration of the subject, for example, compared with untreated subjects, the survival duration is extended 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. In certain embodiments, the combined administration increases progression-free survival or total survival. In certain embodiments, the combined administration increases the reaction and reaction duration of the subject, for example, compared with untreated subjects, the reaction and reaction duration are increased by 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%. In certain embodiments, the combination is applied to subjects with prostate cancer, causing all signs of tumor cells to disappear completely ("complete remission"). In certain embodiments, the combination is applied to subjects with prostate cancer, causing tumor cells or tumor size to reduce by at least 30% or more ("partial remission"). In certain embodiments, administration of the combination to a subject with prostate cancer results in complete or partial disappearance of tumor cells / lesions (including new measurable lesions). Tumor reduction can be measured by any method 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, radiotracers (e.g., 18 F-DCFPyL) for PET / CT imaging to detect lesions in patients with metastatic prostate cancer (e.g., mCRPC). In certain embodiments, the administration of a bispecific antibody or antigen-binding fragment thereof with an anti-PD-1 antibody or antigen-binding fragment thereof produces a synergistic anti-tumor effect that exceeds the combined effect of the two agents when administered alone.

[0078] In certain cases, a subject's response to therapy is classified as a 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 node (whether target or non-target) to <10mm (<1cm). PR is defined as a reduction of at least 30% in the sum of diameters of target lesions, with reference to the baseline sum diameter. PD is defined as an increase of at least 20% in the sum of diameters of target lesions, with reference to the minimum sum studied (including the baseline sum if it is the minimum for the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5mm (0.5cm). (Note: The appearance of one or more new lesions is also considered progression). SD is defined as a reduction that is not sufficient to meet the requirements of PR, and an increase that is not sufficient to meet the requirements of PD, with reference to the minimum sum diameter during the study.

[0079] Anti-PD-1 antibodies and antigen-binding fragments thereof

[0080] According to certain exemplary embodiments of the present disclosure, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM) comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds. In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region contains three domains: C H 1. C H 2 and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain (C L 1). V H Area and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FRs of the 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.

[0081] The term "antibody" as used herein also includes the antigen binding fragment of a complete antibody molecule. The terms "antigen binding portion of an antibody", "antigen binding fragment of an antibody", etc. as used herein include any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen binding fragments of antibodies can be derived from full antibody molecules, for example, using any suitable standard technique (such as proteolytic digestion or the recombinant genetic engineering techniques involving the operation and expression of DNA encoding antibody variable domains and optionally antibody constant domains). Such DNA is known, and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA sequencing can be performed, and chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration, or to import codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0082] 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 that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides), or binding FR3-CDR3-FR4 peptides. Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, microbodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0083] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can have any size or amino acid composition and will generally include at least one CDR adjacent to or in frame with one or more framework sequences. H Domain and V L In the antigen-binding fragment associated with the structural domain, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomer VH or V L Structural domain.

[0084] In certain embodiments, an antigen-binding fragment of an antibody may contain 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 within an antigen-binding fragment of an antibody of the present disclosure include: (i) 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 L . In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected through a full-length or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present disclosure may comprise any of the variable domain and constant domain configurations listed above, which are connected to each other and / or to one or more monomeric V domains. H or VL The domains are in non-covalent association (eg, through disulfide bonds) as homodimers or heterodimers (or other multimers).

[0085] The term "antibody" as used herein also includes multispecific (e.g., bispecific) antibodies. Multispecific antibodies or antigen-binding fragments of antibodies generally include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format can be applied to the context of the antibodies or antigen-binding fragments of antibodies disclosed herein. For example, the present disclosure includes methods comprising the use of bispecific antibodies, wherein 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 the context of the present disclosure include, but are not limited to, for example, scFv-based or bispecific bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab.sup.2 bispecific formats (for a review of the foregoing formats, see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that are then self-assembled into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Electronic version: Dec. 4, 2012]).

[0086] The antibody used in the disclosed method can be a human antibody. As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. However, the human antibody of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies that have been implanted into human framework sequences using CDR sequences derived from the germline of another mammalian species (such as a mouse).

[0087] The antibody used in the disclosed method can be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or separated by recombinant means, such as antibodies expressed by recombinant expression vectors transfected into host cells (described further below), antibodies separated from recombinant human antibody combinatorial libraries (described further below), antibodies separated from transgenic animals (e.g., mice) relative to human immunoglobulin genes (see, e.g., Taylor et al., (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, produced or separated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using transgenic animals with respect to human Ig sequences, undergo in vivo somatic cell mutagenesis) and thus the V H and V L The amino acid sequence of the region is derived from human germline V H and V L Sequences that are related to but that may not naturally occur within the human antibody germline repertoire in vivo.

[0088] According to certain embodiments, the antibody used in the disclosed method specifically binds to PD-1. The term "specific binding" and the like means that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen 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, antibodies that "specifically bind" to PD-1 include antibodies with 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. D An antibody that binds to PD-1 or a portion thereof as measured in a surface plasmon resonance assay. However, an isolated antibody that specifically binds to human PD-1 may have cross-reactivity with other antigens, such as PD-1 molecules from other (non-human) species.

[0089] 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 amino acid sequence of the anti-PD-1 antibodies listed in U.S. Patent No. 9,987,500. In certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof useful in the context of the present disclosure comprises a heavy chain complementarity determining region (HCDR) of a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 36 and a light chain complementarity determining region (LCDR) of a light chain variable region (LCVR) having an 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 other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR having SEQ ID NO: 36 and a LCVR having SEQ ID NO: 40. In certain embodiments, the methods of the present disclosure comprise the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain having 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 having the amino acid sequence of SEQ ID NO:36 and a LCVR having the amino acid sequence of SEQ ID NO:40 is REGN2810 (also known as cemiprilimab, ) of a fully human anti-PD-1 antibody. According to certain exemplary embodiments, the methods of the present disclosure include the use of REGN2810 or a bioequivalent thereof. The term "bioequivalent" as used herein refers to an anti-PD-1 antibody or PD-1-binding protein or a fragment thereof that is a pharmaceutical equivalent or pharmaceutical substitute, and whose absorption rate and / or extent do not show significant differences from the absorption rate and / or extent of REGN2810 when administered at the same molar dose (whether single or multiple doses) 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 does not have clinically meaningful differences from REGN2810 in terms of its safety, purity and / or efficacy.

[0090] Other anti-PD-1 antibodies that can be used in the context of the disclosed methods include, for example, antibodies mentioned and known in the art, such as 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.

[0091] The anti-PD-1 antibodies used in the context of the methods of the present disclosure may have pH-dependent binding characteristics. For example, the anti-PD-1 antibodies used in the methods of the present disclosure may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, the anti-PD-1 antibodies of the present disclosure may exhibit enhanced binding to their antigens at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values ​​less than about 6.2, for example, 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, 5.0 or less. As used herein, the expression "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.

[0092] In certain instances, "reduced binding to PD-1 at acidic pH compared to neutral pH" refers to the K of the antibody binding to PD-1 at acidic pH. D The K value of the antibody binding to PD-1 at neutral pH D For example, for the purposes of this disclosure, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K of about 3.0 or greater, D In certain exemplary embodiments, the acidic / neutral K of an antibody or antigen-binding fragment of the present disclosure is preferably 0.0547 W / m , 0.0617 W / m . DThe 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.

[0093] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening antibody populations for reduced (or enhanced) binding to specific antigens at acidic pH compared to neutral pH. In addition, antibodies with pH-dependent characteristics can be produced by modifying the antigen-binding domain at the amino acid level. For example, by replacing one or more amino acids of the antigen-binding domain (e.g., inside the CDR) with histidine residues, antibodies with reduced antigen binding at acidic pH relative to neutral pH can be obtained. The expression "acidic pH" used herein refers to a pH of 6.0 or less.

[0094] Bispecific anti-PSMA × anti-CD28 antibody

[0095] According to certain exemplary embodiments of the present disclosure, the method comprises administering a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to CD28 and PSMA. Such antibodies and fragments may be referred to herein as, for example, "anti-PSMA / anti-CD28" or "anti-PSMA×CD28" or "PSMA×CD28" bispecific antibodies or antigen-binding fragments thereof or other similar terms.

[0096] As used herein, the expression "bispecific antibody" refers to an immunoglobulin 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 different 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 domain comprising three CDRs. In the context of a bispecific antibody, the CDR of the first antigen binding domain may be represented by the prefix "A", and the CDR of the second antigen binding domain may be represented by the prefix "B". Therefore, 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.

[0097] The first antigen binding domain and the second antigen binding domain are each connected to a separate multimerization domain. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid that has the ability to bind to a second multimerization domain of the same or similar structure or configuration. In the context of the present disclosure, the multimerization component is the Fc portion of an immunoglobulin (comprising a C H2 -C H3 domain), such as an Fc domain selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and IgG of any allotype within each isotype group.

[0098] The bispecific antibodies of the present disclosure generally comprise two multimerization domains, such as two Fc domains, which are respectively parts of a single antibody heavy chain. The first multimerization domain and the second multimerization domain may have the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimerization domain and the second multimerization domain may have different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0099] Any bispecific antibody format or technology can be used to prepare the bispecific antibodies of the present disclosure. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen binding specificity, to produce a bispecific antibody. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs into holes, common light chains (e.g., common light chains with knobs into holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab 2 Bispecific formats (for review of the aforementioned formats, see, eg, Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein).

[0100] In the context of the bispecific antibodies of the present disclosure, the Fc domain may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring Fc domain. For example, the present disclosure includes bispecific antibodies comprising one or more modifications in the Fc domain that result in the modified Fc domain having a modified binding interaction (e.g., enhanced or weakened) between Fc and FcRn. In one embodiment, the bispecific antibody comprises one or more modifications in the Fc domain. H2 or C H3 The Fc domain comprises a modification in the region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome at a pH range of 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.

[0101] The present disclosure also includes a first C H 3 domain and the second Ig C H 3-domain bispecific antibody, wherein the first and second Ig C H The at least one amino acid in the first Ig C domain and the second Ig C domain differs from each other, and wherein 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 3 domains bind protein A and the second Ig C H The 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may also contain a Y96F modification (according to IMGT; Y436F according to EU). See, e.g., U.S. Pat. No. 8,586,713. H Other modifications within 3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies.

[0102] In certain embodiments, the Fc domain can be chimeric, combining Fc sequences from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise Fc sequences from human IgG1, human IgG2, or human IgG4. H C in Zone 2 H 2 sequence, and C derived from human IgG1, human IgG2 or human IgG4 H 3 sequence. The chimeric Fc domain may also contain 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. Specific examples of chimeric Fc domains that may be included in any of the antibodies described herein include, from N-terminus to C-terminus: [IgG4C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can 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 antibody of the present disclosure are described in U.S. Patent Publication No. 20140243504, which is incorporated herein in its entirety. Chimeric Fc domains and chimeric heavy chain constant regions and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector function.

[0103] According to certain exemplary embodiments of the present disclosure, the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (A-HCVR and B-HCVR), a light chain variable region (A-LCVR and B-LCVR), and / or a complementarity determining region (CDR) comprising any amino acid sequence of the bispecific anti-PSMA / anti-CD28 antibody listed in WO 2019 / 246514. In certain exemplary embodiments, the bispecific anti-PSMA / anti-CD28 antibodies or antigen-binding fragments thereof useful in the context of the methods of the present disclosure comprise: (a) a first antigen-binding arm that specifically binds to 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) comprising the amino acid sequence of SEQ ID NO: 9; and (b) a second antigen-binding arm that specifically binds to 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; 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; and B-HCDR3 comprises the amino acid sequence of SEQ ID NO:8; and 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 a HCVR comprising SEQ ID NO: 1 (A-HCVR) and a LCVR comprising SEQ ID NO: 9 (A-LCVR); and (b) a second antigen-binding arm comprising a HCVR comprising SEQ ID NO: 5 (B-HCVR) and a LCVR comprising SEQ ID NO: 9 (B-LCVR). In certain exemplary embodiments, the bispecific anti-PSMA x CD28 antibody comprises: a PSMA-binding arm comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 13 and a light chain comprising an amino acid sequence of SEQ ID NO: 15; and a CD28-binding arm comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 14 and a light chain comprising an amino acid sequence of SEQ ID NO: 15.

[0104] In certain exemplary embodiments, the bispecific anti-PSMA / anti-CD28 antibodies or antigen-binding fragments thereof useful in the context of the methods of the present disclosure comprise: (a) a first antigen-binding arm that specifically binds to 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) comprising the amino acid sequence of SEQ ID NO: 28; and (b) a second antigen-binding arm that specifically binds to 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 SEQID NO:26; and B-HCDR3 comprises the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:27; and 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 a HCVR comprising SEQ ID NO: 16 (A-HCVR) and a LCVR comprising SEQ ID NO: 28 (A-LCVR); and (b) a second antigen-binding arm comprising a HCVR comprising SEQ ID NO: 20 or SEQ ID NO: 24 (B-HCVR) and a LCVR comprising SEQ ID NO: 28 (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: 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-PSMA×CD28 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.

[0105] Combination therapy

[0106] According to certain embodiments, the methods of the present disclosure include administering to a subject a combination of an anti-PSMA / anti-CD28 bispecific antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof. In certain embodiments, the methods of the present disclosure include administering an antibody with additional or synergistic activity to treat a cancer expressing PSMA, preferably prostate cancer. In some embodiments, the combination of an anti-PSMA×CD28 bispecific antibody (e.g., mAb1) and an anti-PD-1 antibody (e.g., semipirimab) produces a synergistic therapeutic effect in the treatment of metastatic castration-resistant prostate cancer. As used herein, the expression "in combination with" refers to the administration of an anti-PSMA / anti-CD28 bispecific antibody or an antigen-binding fragment thereof before, after, or simultaneously with the administration of an anti-PD-1 antibody or an antigen-binding fragment thereof. The term "in combination with" also includes the sequential or concomitant administration of an anti-PD-1 antibody or an antigen-binding fragment thereof and a bispecific anti-PSMA / anti-CD28 antibody or an 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 can be administered more than 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 the 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 can 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 the administration of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof. Administration "concurrently" 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 (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 in a single combined dose formulation comprising both the anti-PD-1 antibody or antigen-binding fragment thereof and the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof.

[0107] In certain embodiments, the methods of the present disclosure include administering a third therapeutic agent, wherein the third therapeutic agent is an anti-cancer drug. In certain embodiments, the methods of the present 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 produce a long-term durable anti-tumor response and / or increase the survival of patients with PSMA-expressing cancers.

[0108] In some embodiments, the methods of the present disclosure include administering radiation therapy to a cancer patient before, simultaneously with, or after administering 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, radiation therapy may be administered to a tumor lesion in one or more doses after administering one or more doses of the antibody. In some embodiments, radiation therapy may be administered locally to a tumor lesion after systemic 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 to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablative radiation).

[0109] A clinical trial is being conducted in which REGN5678 (mAb1) is administered in combination with the anti-PD-1 antibody semiprilimab to patients with advanced metastatic castration-resistant prostate cancer (CRPC) who have failed multiple anti-androgen therapies. Such patients typically have a life expectancy of only 1-2 years and limited treatment options. Metastatic CRPC is considered an immune "cold" tumor that is highly resistant to immune checkpoint therapy, with large-scale trials of anti-PD-1 antibodies showing response rates in the single digits. The anti-PSMA×CD28 co-stimulatory bispecific antibody REGN5678 disclosed herein is designed to enhance the responsiveness of these types of tumor categories, such as prostate cancer, and essentially convert these cold tumors into hot tumors.

[0110] As detailed in the examples listed herein, patients were given REGN5678 once a week and semilazumab once every three weeks. The first dose of semilazumab was not co-administered until the fourth week to allow for a period of PSMA×CD28 introduction to evaluate the safety and efficacy of monotherapy. The primary endpoints were safety, tolerability, and pharmacokinetics. The secondary endpoint was the objective response rate, which was defined as a ≥50% decrease in prostate-specific antigen (PSA) relative to baseline and / or tumor shrinkage. PSA is a protein produced by the prostate and is commonly used as a biomarker for diagnosing and tracking prostate cancer because many mCRPC patients have disease limited to bone lesions that cannot be evaluated by conventional RECIST criteria.

[0111] At the lowest 5 doses (cohorts 1-5), there was no evidence of antitumor activity, with 0 of 17 patients showing a PSA response and 16 of 17 patients showing a PSA increase; there were no grade ≥ 3 (Gr3) immune-related adverse events (irAEs) in these cohorts. The lack of antitumor activity in these patients is consistent with the approximately 6% response rate reported in other anti-PD1 monotherapy trials.

[0112] At the next three doses administered (cohorts 6-8), evidence of dose-dependent anticancer activity was observed:

[0113] Cohort 6: 1 of 4 patients achieved a 100% reduction in PSA and a complete response (CR) by RECIST criteria. The patient discontinued therapy due to a cutaneous Gr3 irAE (thought to be a recurrence of pre-existing disease, which has resolved), but has maintained a 100% reduction in PSA and CR to date at 10 months of follow-up.

[0114] Cohort 7: Three of eight patients showed PSA reductions, 99%, 44%, and 22%, respectively. Two of these three patients had Gr3 irAEs (aseptic encephalitis and seizures).

[0115] Cohort 8: 3 of 4 patients experienced substantial PSA reductions within 6 weeks of starting combination therapy, including 99% reductions in 2 patients and 82% reduction in 1 patient. One patient with a 99% PSA reduction experienced Gr3 acute inflammatory demyelinating polyradiculopathy.

[0116] In terms of safety, irAEs were associated with anti-tumor activity, and there were no grade ≥3 irAEs in patients who did not experience anti-tumor activity. Immune-related adverse events (also referred to herein as immune-mediated adverse events) can be managed by blocking interleukin 6 receptor (IL-6R) or IL-6. For example, anti-IL-6R antibodies such as salirumab or tocilizumab can be administered to patients in combination with the therapeutic agents discussed herein (e.g., anti-PSMA×CD28 bispecific antibodies and anti-PD-1 antibodies) to reduce or prevent immune-mediated adverse events. As of the data cutoff, no grade 4 irAEs, grade ≥3 cytokine release syndrome, or treatment-related deaths had been observed in the trial.

[0117] These data provide the first clinical evidence that costimulatory bispecific antibodies can be combined synergistically with anti-PD-1 to generate activity against tumor types that were previously resistant to anti-PD-1 immunotherapy. More generally, the results observed in this example suggest that costimulatory bispecific antibody therapy (one arm binds to a tumor antigen and the other arm provides a CD28 costimulatory immune cell signal) can provide a powerful anti-tumor response, especially when administered in combination with checkpoint inhibitors (such as anti-PD-1 and anti-PD-L1 antibodies) for the treatment of difficult-to-treat cancers.

[0118] In various embodiments, the results of administering an anti-PSMA×CD28 bispecific antibody (e.g., mAbl) in combination with an anti-PD-1 antibody (e.g., semilaplimab) to a patient with mCRPC are:

[0119] The prostate-specific antigen (PSA) level in the subject decreases by at least 50%;

[0120] The PSA level in the subject decreases by at least 55%;

[0121] The PSA level in the subject decreases by at least 60%;

[0122] The PSA level in the subject decreases by at least 65%;

[0123] The PSA level in the subject is reduced by at least 70%;

[0124] The PSA level in the subject is reduced by at least 75%;

[0125] The PSA level in the subject is reduced by at least 80%;

[0126] The PSA level in the subject is reduced by at least 85%;

[0127] The PSA level in the subject is reduced by at least 90%;

[0128] The PSA level in the subject is reduced by at least 95%;

[0129] The subject's PSA level is reduced by at least 96%;

[0130] The PSA level in the subject is reduced by at least 97%;

[0131] The PSA level in the subject is reduced by at least 98%;

[0132] The PSA level in the subject is reduced by at least 99%;

[0133] a decrease in size of at least one lesion in the subject, the PSMA PET signal of the lesion being less than the PSMA PET signal in the subject's liver; and / or

[0134] The subject responded after a pseudoprogression.

[0135] Tumor biopsy, imaging, and CRS monitoring / management

[0136] The methods discussed in this disclosure may additionally include tumor biopsy, imaging, and cytokine release syndrome (CRS) monitoring and management to evaluate efficacy and safety in individual subjects or within a population of subjects.

[0137] Tumor biopsy

[0138] Patients with soft tissue disease may undergo a core or excisional biopsy of soft tissue lesions if clinically feasible at screening and / or during the treatment discussed herein. For patients without clinically accessible soft tissue disease, a bone biopsy may be performed if feasible. Any available tissue from samples collected at different time points (e.g., formalin-fixed paraffin-embedded or stored in blocks for molecular extraction) and archived samples from previous treatments can be used for biomarker determination in addition to clinical diagnostic uses. Specifically, these samples can be evaluated using in situ imaging of gene targets associated with REGN5678 (PSMA, CD28) and semiprine (PD-L1) and markers of immune activation, inhibition and function, and tumor cell phenotypes by probes. As discussed in Example 5, the expression of therapeutic pathway targets of REGN5678 and semiprine is an exploratory endpoint.

[0139] If available, tumor biopsies can also undergo gene expression profiling (using RNA sequencing or other methods) (as a measure of the comprehensive tumor microenvironment phenotype), whole exome sequencing or other mutational profiling, and targeted studies of genetic variants (tumor mutations) (such as those affecting DNA repair pathways). They can also profile the T cell receptor repertoire using next-generation sequencing as an indicator of the proliferation of tumor-associated T cell clones.

[0140] Imaging

[0141] Prostate-specific membrane antigen (PSMA) PET / CT has been shown to provide a sensitive indicator of both PSMA expression and tumor burden in patients with prostate cancer. Compared with conventional imaging modalities used in combination for prostate cancer, such as CT, MRI, and bone scan, it can detect more tumor lesions with higher specificity, greatly improving the effectiveness of tumor response assessment and treatment strategy decision-making.

[0142] Fluorine F18DCFPyL( 18 F-DCFPyL) is a radiolabeled small molecule that binds to the extracellular domain of PSMA with high affinity. Data from enzyme inhibition assays showed 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 to 3 studies and found to be well tolerated in patients with prostate cancer. 18 The biodistribution and optimal imaging time point after administration of F-DCFPyL injection were determined, and the radiation dose used was within the limits of PET diagnostic radiotracers. 18 The physiological accumulation of F-DCFPyL corresponds to the distribution in organs expressing PSMA. Accumulation is very high in primary tumors and metastatic lesions, suggesting that 18 F-DCFPyL injections can be used to detect residual tumors and regional or distant metastases with high sensitivity and specificity. 18 F-DCFPyL PSMA PET / CT was used to evaluate the systemic tumor burden and the antitumor activity of REGN5678 combined with semilaprimab in patients with mCRPC.

[0143] CRS Monitoring and Management

[0144] Cytokine release has been observed with superagonist anti-CD28 bivalent antibodies, bsAbs, and similar molecules. Cytokine release syndrome (CRS) usually causes clinical symptoms during or within hours to days after infusion. In a clinical study of 6 patients treated with a bivalent anti-CD28 superagonist antibody (TGN1412), acute life-threatening CRS occurred, and the patient became critically ill within 12 to 16 hours. Previous experience with bispecific antibodies targeting tumor antigens and CD3 has shown that when CRS occurs, these events are most evident after the first 1 or 2 weekly doses of the study treatment and are generally transient, even when higher doses are administered in subsequent weeks. This has also been observed in combination with semilazumab. For any given patient, CRS usually occurs more frequently in the first 2 weekly doses and decreases in frequency in subsequent exposures. Based on these findings, the risk of a first CRS attack occurring at or after the third dose is considered low.

[0145] Subcutaneous administration of bispecific antibodies has recently been evaluated in preclinical and early clinical studies. Subcutaneous administration of bispecific antibodies targeting tumor antigens and CD3 was tolerable, with no severe CRS events (no ≥ grade 3 CRS) in B-cell tumors. In cynomolgus macaques, subcutaneous administration of the same bispecific antibody produced lower CRS compared with intravenous injection.max , delayed T max and lower plasma cytokine levels. The approaches discussed herein may include measures to address potential safety issues arising from cytokine release, including:

[0146] (1) Cytokine monitoring;

[0147] (2) management of CRS using anti-IL-6 pathway therapy (eg, salizumab or tocilizumab) and corticosteroids; and

[0148] (3) In cases where CRS is observed, provide premedication at the initial dosing visit and use a lower dose before the full dose (DL) is delivered.

[0149] Symptoms consistent with severe CRS (including but not limited to persistent fever, neurologic impairment (including altered mental status, confusion, and seizures), clinical signs of toxicity (hypotension or hypoxia requiring at least 1 intravenous vasopressor [PO 2 <90%])) may be considered for pharmacological intervention with anti-IL-6 pathway therapy (e.g., salilumab or tocilizumab) and / or high-dose steroids. The present disclosure contemplates such additions to the methods discussed herein.

[0150] Corticosteroids can also be used to manage CRS, especially in cases with neurologic symptoms. In general, corticosteroids should be used in the following situations: 1) IRR / CRS is inadequately responsive to anti-IL-6 pathway therapy (eg, salilumab or tocilizumab) or 2) anti-IL-6 pathway therapy is not in the best interest of the patient.

[0151] Pharmaceutical compositions and administration

[0152] 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 one or more of the antibodies (or fragments) are 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 suitable transfer, delivery, tolerance, etc. A wide variety of suitable formulations may be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN TM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semisolid gels and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311. Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endosomes (see, e.g., Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition can be administered by any convenient route (e.g., by infusion or bolus or by injection) and can be administered with other biologically active agents.

[0153] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to the delivery of 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 in the cartridge has been administered and the cartridge becomes empty, 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 a disposable pen delivery device, there is no replaceable cartridge. In fact, the disposable pen delivery device is pre-filled with the pharmaceutical composition in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0154] A variety of reusable pen delivery devices and auto-injector delivery devices are used to subcutaneously deliver the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TMI, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM pen (Abbott Labs, Abbott Park IL), etc.

[0155] In some cases, the pharmaceutical composition 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 near the target of the composition, thus requiring only a small portion 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 of Langer, 1990, Science 249: 1527-1533.

[0156] The injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by known methods. For example, the injectable preparations can be prepared by, for example, dissolving, suspending or emulsifying the antibody described above or its salt in a sterile aqueous medium or an oily medium conventionally used for injection. As aqueous media for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with appropriate solubilizers. The injection thus prepared is preferably filled in an appropriate ampoule.

[0157] Advantageously, the pharmaceutical composition for use as described above is prepared in a unit dosage form suitable for matching the dosage of the active ingredient. Such unit dosage forms include, for example, vials or prefilled syringes.

[0158] Administration regimen

[0159] The present 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 four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, as long as a therapeutic response is achieved.

[0160] According to certain embodiments of the present disclosure, multiple doses of bispecific anti-PSMA / anti-CD28 antibodies or antigen-binding fragments thereof in combination with anti-PD-1 antibodies or antigen-binding fragments thereof may be administered to a subject over a specified time course. The method according to this aspect of the present disclosure includes sequentially administering to a subject one or more doses of bispecific anti-PSMA / anti-CD28 antibodies or antigen-binding fragments thereof in combination with one or more doses of anti-PD-1 antibodies or antigen-binding fragments thereof. As used herein, "sequential administration" refers to administering each dose of the antibody to the subject at different time points, such as different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes such a method: the method comprises sequentially administering to a patient a single initial dose of an antibody (or fragment), followed by one or more second doses of the antibody (or fragment), and optionally followed by one or more third doses of the antibody (or fragment).

[0161] The terms "initial dose", "second dose" and "third dose" indicate the time sequence of administration. Thus, the "initial dose" is a dose (also referred to as a "baseline amount") administered at the beginning of a treatment regimen; the "second dose" is a dose administered after the initial dose; and the "third dose" is a dose administered after the second dose. The initial dose, the second dose, and the third dose 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 dose, the second dose, and / or the third dose differ from one another during the course of treatment (e.g., adjusted upward or downward when appropriate). In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as a "loading dose", followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency.

[0162] In an exemplary embodiment of the present disclosure, each second dose and / or third dose is administered 1 / 2 to 14 weeks (e.g., 1 / 2, 1, 11 / 2, 2, 21 / 2, 3, 31 / 2, 4, 41 / 2, 5, 51 / 2, 6, 61 / 2, 7, 71 / 2, 8, 81 / 2, 9, 91 / 2, 10, 101 / 2, 11, 111 / 2, 12, 121 / 2, 13, 131 / 2, 14, 141 / 2 weeks or longer) after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means that in a sequence of multiple administrations, a dose of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof (and / or anti-PD-1 antibody or antigen-binding fragment thereof) is administered to the patient prior to administration of the immediately next dose in the sequence without an intervening dose.

[0163] The method according to this aspect of the present disclosure may include administering any number of second and / or third 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 to a patient. For example, in certain embodiments, only a single second dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to a patient. Similarly, in certain embodiments, only a single third dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to a patient.

[0164] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 week, 2 weeks, or 3 weeks (e.g., 1 week or 3 weeks) after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 1 to 4 weeks (e.g., 1 week or 3 weeks) after the previous dose. Alternatively, during the course of the treatment regimen, the frequency of the second dose and / or third dose administered to the patient can vary. The physician can also adjust the frequency of administration during the course of treatment, depending on the needs of each patient after the clinical examination.

[0165] In certain embodiments, one or more 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 are administered more frequently (twice a week, once a week, once every 2 weeks, or once every 3 weeks) at the beginning of the treatment regimen as an "induction dose," followed by subsequent doses ("consolidation doses" or "maintenance doses") either equally or less frequently (e.g., once every 4-12 weeks).

[0166] The present disclosure includes methods 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 to treat prostate cancer (e.g., metastatic castration-resistant prostate cancer). In some embodiments, the methods of the present invention comprise administering one or more doses of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, prior to or subsequent to administering one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the methods of the present invention comprise administering several doses (e.g., once a week during a 3-week lead-in period) of a bispecific anti-PSMA / anti-CD28 antibody, followed by 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 to 600 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, and one or more doses of about 0.1 mg / kg to about 20 mg / kg (e.g., 0.01 to 1000 mg) of a bispecific antibody or an antigen-binding fragment thereof may be administered to inhibit tumor growth and / or prevent tumor recurrence in subjects with prostate cancer. In some embodiments, the bispecific antibody or its antigen-binding fragment combined with an anti-PD-1 antibody or its antigen-binding fragment increases anti-tumor efficacy (e.g., greater inhibition of tumor growth, or increased prevention of tumor recurrence) compared to untreated subjects. In some embodiments, the bispecific antibody or its antigen-binding fragment is administered before, after, or simultaneously with the anti-PD-1 antibody or its antigen-binding fragment. In certain embodiments, the bispecific antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment are administered in separate dosage formulations.

[0167] dose

[0168] The amount 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 is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to an amount of an 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: (a) reducing the severity or duration of symptoms of cancer (e.g., prostate cancer); (b) inhibiting tumor growth, or increasing tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delaying tumor growth and development; (d) inhibiting or delaying or stopping tumor metastasis; (e) preventing tumor recurrence; (f) increasing the survival of subjects with cancer (e.g., prostate cancer); and / or (g) reducing the use or need for conventional anti-cancer therapy (e.g., reducing or eliminating the use of chemotherapeutic agents or cytotoxic agents) compared to untreated subjects.

[0169] In the case of a bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof, a therapeutically effective amount can be 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 150 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 of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof. 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 (e.g., once a week or once every three weeks) to a subject to treat a PSMA-expressing cancer or prostate cancer (e.g., metastatic and / or castration-resistant prostate cancer).

[0170] In the case of an anti-PD-1 antibody or antigen-binding fragment thereof, the therapeutically effective amount can be 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 or about 600 mg of an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, 300 mg to 400 mg of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific antibody or antigen-binding fragment thereof is administered (e.g., once every three weeks) to a subject to treat 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 in combination with a bispecific antibody or antigen-binding fragment thereof is administered (e.g., once every three weeks) to a subject to treat a PSMA-expressing cancer or prostate cancer (e.g., metastatic and / or castration-resistant prostate cancer).

[0171] The amount of the bispecific anti-PSMA / anti-CD28 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof contained in a single dose can 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 can 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 can be administered at a dose of about 2 mg / kg to about 20 mg / kg of patient body weight.

[0172] A summary of the sequences cited herein and the corresponding SEQ ID NOs is shown in Table 1 below.

[0173] Table 1: Sequence summary

[0174]

[0175]

[0176] Example

[0177] The following examples are provided to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but certain experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0178] Example 1: Generation of bispecific antibodies that bind prostate-specific membrane antigen (PSMA) and CD28

[0179] Bispecific antibodies comprising an anti-PSMA specific binding domain and an anti-CD28 specific binding domain are constructed using standard methods, wherein the anti-PSMA antigen binding domain and the anti-CD28 antigen binding domain each comprise a different, unique pair of HCVRs and a common LCVR. In some cases, the bispecific antibody is constructed using a heavy chain from an anti-CD28 antibody, a heavy chain from an anti-PSMA antibody, and a common light chain (see Table 2).

[0180] The bispecific antibodies produced according to embodiments of the present invention comprise two separate 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"). Anti-PSMA and anti-CD28 both share a common light chain. The CD28-VH / PSMA-VH pairing produces an antigen binding domain that specifically recognizes CD28 on T cells and PSMA on tumor cells.

[0181] A summary of the components of the antigen binding domains of the various anti-PSMA×CD28 bispecific antibodies constructed is shown in Table 3. The corresponding CDR sequences and full-length heavy and light chain sequences are listed in Table 1 (see "-001", "-002" and "-003" bispecific antibodies in Table 3).

[0182] Table 2: Summary of components of anti-PSMA×CD28 bispecific antibodies

[0183]

[0184] Example 2: Surface plasmon resonance-derived binding affinity and kinetic constants of anti-PSMA×CD28 bispecific antibodies

[0185] To determine the binding kinetics of the anti-PSMA×CD28 bispecific antibodies, the surface plasmon resonance-derived binding affinity and kinetic constants of the anti-PSMA×CD28 bispecific antibodies were determined.

[0186] Binding kinetics of anti-PSMA×CD28 bispecific antibodies to PSMA: The equilibrium dissociation constant (K) of 6h.hPSMA (recombinant human PSMA / FOLH1 protein, R&D, catalog #4234-ZN) binding to purified anti-PSMA×CD28 bispecific antibodies was determined using a Biacore T-200 instrument using a real-time surface plasmon resonance biosensor. D The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody to capture the purified anti-PSMA×CD28 bispecific antibody.

[0187] In a medium consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.5 mM MgCl 2 , 1.0 mM CaCl 2 The Biacore binding study was performed in a buffer consisting of 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 HBS-P++ running buffer, ranging from 10 nM to 0.4 nM, and serial 3-fold dilutions were performed for the anti-PSMA×CD28 bispecific antibody.

[0188] Different concentrations of 6h.hPSMA were injected onto the monoclonal antibody captured surface at a flow rate of 50 μL / min. The association of 6h.hPSMA with the captured monoclonal antibody was monitored for 3 minutes, and the dissociation of 6h.hPSMA in HBS-P++ running buffer was monitored for 10 minutes. The kinetic association (k) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software (BioLogic Software). a ) and dissociation (k d ) rate constant. Calculate the binding and dissociation equilibrium constant (K) from the kinetic rate constant D ) and dissociation half-life (t 1 / 2 )for:

[0189] K D (M) = k d / k a , and t 1 / 2 (min)=0.693 / k d / 60

[0190] The binding kinetic parameters for hPSMA binding to purified monoclonal antibodies at 25°C for 6 h are shown in Table 3 below.

[0191] Table 3: Biacore binding affinity of monoclonal antibodies to PSMA at 25°C

[0192]

[0193] TBD: Not tested

[0194] The binding kinetic parameters for hPSMA binding to purified exemplary monoclonal bispecific antibodies at 37°C for 6 h are shown in Table 4 below. One (1) RU (Response Unit) represents 1 pg protein / mm 2 , as defined by the manufacturer.

[0195] Table 4: Biacore binding affinity of monoclonal antibodies to PSMA at 37°C

[0196]

[0197] Binding kinetics of anti-PSMA × CD28 bispecific antibody to CD28: The equilibrium dissociation constant (KD) of hCD28.mmh binding to purified monoclonal antibody was determined using a Biacore T-200 instrument using a real-time surface plasmon resonance biosensor. 值 ). The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody to capture the purified anti-PSMA×CD28 bispecific antibody.

[0198] Different concentrations of hCD28.mmh were injected onto the monoclonal antibody captured surface at a flow rate of 50 μL / min. The association of hCD28.mmh with the captured monoclonal antibody was monitored for 5 minutes, and the dissociation of hCD28.mmh in HBS-P++ running buffer was monitored for 10 minutes. The kinetic association (k) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber2.0c curve fitting software. a ) and dissociation (k d ) rate constant. Calculate the binding and dissociation equilibrium constant (K) from the kinetic rate constant D ) and dissociation half-life (t 1 / 2 ) is: K D (M) = k d / k a , and t 1 / 2 (min)=0.693 / k d / 60

[0199] The binding kinetic parameters for hCD28.mmh binding to purified anti-PSMA x CD28 bispecific antibodies at 25°C are shown in Table 5 below.

[0200] Table 5: Biacore binding affinity of monoclonal antibodies to CD28 at 25°C

[0201]

[0202] TBD: Not tested

[0203] The binding kinetic parameters for hCD28.mmh binding to purified anti-PSMA x CD28 bispecific antibodies at 37°C are shown in Table 6 below.

[0204] Table 6: Biacore binding affinity of monoclonal antibodies to CD28 at 37°C

[0205]

[0206] Example 3. Anti-PSMA×CD28 bispecific antibody binding to PSMA and CD28 cells

[0207] To evaluate the ability of these antibodies (anti-PSMA x CD28 antibodies) to specifically bind to cell surface proteins, in vitro binding assays were performed.

[0208] Flow cytometry was used to test the binding of PSMA×CD28 bispecific antibody to the surface of human T cells. BSPSMA / CD28-001 bound to all T cells (EC50 value was 4.80×10 -8 M) and both CD4+ T cells and CD8+ T cells (EC50 values ​​were 5.09×10 -8 M and 5.89×10 -8 M). BSPSMA / CD28-003 bound to all T cells (EC50 value was 1.80×10 -7 ) and weakly bound to both CD4+ T cells and CD8+ T cells (EC50 values ​​were 1.67E-07M and 1.80E-07M, respectively).

[0209] The binding of the PSMA×CD28 bispecific antibodies to the surface of PSMA-expressing cell lines was tested by flow cytometry. C4-2 is a prostate cancer cell line derived from LNCaP (androgen-sensitive human prostate adenocarcinoma cells derived from lymph node metastasis; see Wu et al., Int. J. Cancer, 57:406-412 (1994)) cells. Both BSPSMA / CD28-001 and BSPSMA / CD28-003 bound to C4-2 cells (see Liu et al., 2004, Prostate, 60:98-108), with EC50 values ​​of 3.87×10 -9 M and 1.50×10 -9 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 22RV1 cells with EC50 values ​​of 3.05×10 -9 M and 6.33×10 -09 M.

[0210] Example 4: Initial and Engineering Bioassays of PSMA×CD28 Bispecific Antibodies

[0211] T cell activation is achieved by stimulating T cell receptors (TCRs) that recognize specific peptides presented by major histocompatibility complex class I or class II (MHCI 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 in turn initiates a cascade of signaling events that can be monitored by reporter genes driven by various transcription factors such as activated protein 1 (AP-1), nuclear factor of activated T cells (NFAT), or nuclear factor kappa-light-chain enhancer of activated B cells (NFκB). The T cell response is then further refined by the engagement of co-receptors expressed constitutively or inducibly 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 co-stimulatory molecule CD28 is activated by its endogenous ligands CD80 or CD86 expressed on APCs. CD28 enhances cell signals (such as pathways) controlled by NFκB transcription factors after TCR activation. CD28 co-signaling is important for efficient T cell activation (such as T cell differentiation), proliferation, cytokine release and cell death (Smeets et al., NFκB activationinduced by T cell receptor / CD28 costimulation is mediated by protein kinase C-θ, PNAS, 97(7): 3394-3399 (2012)).

[0212] To identify antibodies that enhance T cell activity in the presence of initial stimulation and PSMA target expression, anti-PSMA×CD28 bispecific antibodies were characterized in an engineered reporter bioassay and a cell-based assay using human primary T cells. The assay evaluates the behavior of anti-PSMA / CD28 bispecific antibodies in the presence and absence of initial stimulation and in the presence and absence of target expression. This assay was performed to identify anti-PSMA×CD28 bispecific antibodies that enhance T cell activity in the presence of initial stimulation and target expression. Therefore, this assay evaluates the behavior of bispecific antibodies in the presence and absence of initial stimulation and target expression.

[0213] The Jurkat-derived T cell clone JRT3.T3.5 (ATCC, #TIB-153) was transduced with a NFκB luciferase reporter gene construct (NFκB-Luc, SA Biosciences / Qiagen, catalog #CLS-013L). After isolating a puromycin-resistant clone (JRT3.T3.5 / NFκB-Luc clone 1C2), the cells were further engineered to express full-length human TCR α subunits (1G4A-amino acids M1 to S274) and TCR β subunits (1G4B-amino acids M1 to G311) (Robbins et al., Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific TCell 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 CD8α subunit (hCD8a - amino acids M1 to V235 of accession #NP_001139345) and human CD8β subunit (hCD8b - amino acids M1 to K210 of accession #P10966). κB-Luc / 1G4AB / hCD8ab clone 1D5) and further transduced monoclones with full-length human CD28 (hCD28-accession # P10747 amino acids M1 to S220). Cells were sorted for high CD28 expression and maintained in RPMI+20% FBS+penicillin / streptomycin / glutamine (P / S / G)+NEAA+NaPyr+1μg / mL puromycin+500μg / mL G418+250μg / mL hygromycin+10μg / mL blasticidin. For faster growth, engineered reporter T cells were maintained in antibiotic-free cell culture medium and used as engineered reporter T cells for cell-based luciferase experiments. Reagent information is as follows: RPMI 1640, Irvine Scientific, catalog #9160; FBS, Seradigm, catalog #1500-50; Penicillin / Streptomycin / Glutamine 100× (P / S / G), ThermoFisher Scientific, catalog #10378-016; Non-essential Amino Acids (NEAA), Irvine Scientific, catalog #9304; Sodium Pyruvate (NaPyr), Millipore, catalog #TMS-005-C; Puromycin, Sigma, catalog #P8833; Geneticin (G418), Thermo Fisher Scientific, catalog #11811-098; Hygromycin; Blasticidin.

[0214] A stable HEK293 cell line (ATCC, #CRL-1573) expressing human CD20 (amino acids M1 to P297 of accession number NP_068769.2) was transduced with human PSMA (amino acids M1 to A750 of accession number Q04609). 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.

[0215] In this experiment, engineered reporter T cells were stimulated by two bispecific antibodies. The first stimulation was delivered by the bispecific antibody anti-CD3×CD20 hIgG4 (see WO14 / 047231) that activates T cells, which targets CD3 molecules on engineered reporter T cells and CD20 on HEK293 cells. Here, the first stimulation bypasses the need to activate TCR by natural ligands, which are specific peptides displayed on MHC molecules. The second stimulation is driven by the CD28 bispecific antibody. This antibody mimics the activation of CD28 on T cells by the ligand CD80 / CD86 expressed on APCs. Here, the antibody interacts with CD28 on T cells and PSMA on engineered HEK293 cells, and drives the activation of CD28 on engineered reporter T cells. Simultaneous activation of TCR and CD28 leads to enhanced NFκB transcriptional activity, which in turn induces the production of reporter gene luciferase.

[0216] RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium to prepare the cell suspension and antibody dilution for screening of anti-PSMA×CD28 bispecific antibodies. The engineered reporter T cells were cultured to 1×10 6 cells / mL of cell culture medium. Three-fold (1:3) serial dilutions of anti-PSMA×CD28 bispecific antibodies and controls were tested in the presence of a constant 50pM anti-CD20×CD3 or hIgG4 isotype control. The 10-point dilution range was between 15pM and 100nM, with the final dilution containing no anti-PSMA×CD28 antibody. Reagents were added in the following order: 1) serial dilutions of antibody were added to the corresponding wells of a 96-well white flat-bottom plate; 2) a fixed concentration of 50pM of anti-CD20×CD3 or hIgG4 isotype control was added to each well; 3) the cells were resuspended to 4×10 5 APCs were added to the plate at a final concentration of 1×10 4 cells / well; 4) The reporter T cells cultured overnight were cultured at 2×10 6 / mL and added to the plate to a final concentration of 5×10 4 Incubate the plate at 37°C / 5% CO 2 Incubate for 4-6 hours, then add 100 μL ONE-Glo TM (Promega, catalog # E6051) reagent to lyse cells and detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer, model 2104). All serial dilutions were tested in duplicate.

[0217] By using GraphPad Prism TM The software fits the data to a four-parameter logistic equation on a 10-point dose-response curve to determine the EC of the antibody. 50 The induction fold was calculated using the following equation:

[0218]

[0219] ECs of engineered reporter T cells co-cultured with HEK293 / hCD20 or HEK293 / hCD20 / hPSMA cells 50 The induction folds and 50 pM constant hIgG4 isotype control or anti-CD3×CD20 bispecific antibody (bispecific antibody for stimulating T cells) are summarized in Tables 6 and 7.

[0220] Table 7A: Luciferase activity in engineered reporter T cells in the absence of TCR stimulation

[0221]

[0222]

[0223] Table 7B: Luciferase activity in engineered reporter T cells in the presence of TCR stimulation

[0224]

[0225] When T cells and APCs were treated with 50 pM hIgG4 isotype control, all CD28 bispecific antibodies showed no increase in luciferase activity in the absence of TCR stimulation, regardless of which APC line was used in the assay. As shown in Table 7A, slight luciferase activation was observed using one parental CD28 antibody (mAb14226P2) on HEK293 / hCD20 cells (4.76×) and HEK293 / hCD20 / hSPMA cells (3.59×). mAb14226P2, mAb14193P2, and mAb14216P2 correspond to the parental anti-CD28 antibodies of BSPSMA / CD28-001, BSPSMA / CD28-002, and BSPSMA / CD28-003, respectively.

[0226] In contrast, if cells were treated with 50 pM anti-CD3×CD20 bispecific antibodies, all three anti-PSMA×CD28 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. Very low or no activation was observed in the single-arm parental CD28 controls (one arm of mAb14226P2, mAb14193P2, and mAb14216P2), regardless of the APC line used. Slight luciferase activation was observed in all three parental CD28 antibodies (mAb14226P2, mAb14193P2, and mAb14216P2).

[0227] Example 5: Phase 1 / 2 Study of Bispecific Anti-PSMA×Anti-CD28 Antibody Administered in Combination with Anti-PD-1 Antibody in Patients with Metastatic Castration-Resistant Prostate Cancer

[0228] 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 semilazumab in previously treated metastatic castration-resistant prostate cancer (mCRPC).

[0229] Purpose

[0230] The main objectives of the study are to:

[0231] To evaluate the safety, tolerability and pharmacokinetics (PK) of mAb1 alone and in combination with semilazumab (dose escalation)

[0232] To evaluate the efficacy of mAb1 in combination with semilazumab (based on objective response rate (ORR) measured according to modified Prostate Cancer Working Group 3 (PCWG3) criteria) (dose expansion)

[0233] The secondary objectives of the study are to:

[0234] To evaluate the efficacy of mAb1 in combination with semilazumab (based on ORR measured according to the revised PCWG3 criteria) (dose escalation)

[0235] Characterize the safety profile in each expansion cohort (dose expansion)

[0236] Characterize the PK of mAb1 in combination with Cemimumab (dose expansion)

[0237] Evaluate the efficacy of mAb1 in combination with semilaprimab (based on additional criteria) (dose escalation and dose expansion)

[0238] Evaluation of the immunogenicity of mAb1 in combination with semilaplimab (dose escalation and dose expansion)

[0239] All primary, secondary, and exploratory objectives of the study will apply to each cohort in the study, including patients who received salilumab and those who did not.

[0240] Study Design

[0241] This is an open-label, phase 1 / 2, first-in-human study evaluating the safety, tolerability, PK, and antitumor activity of mAb1 (anti-PSMA×CD28) alone and in combination with semilazumab (anti-PD-1) in previously treated metastatic castration-resistant prostate cancer (mCRPC). The study was divided into 2 parts: dose escalation; and dose expansion.

[0242] Dose escalation: During the dose escalation period, patients will receive a 3-week monotherapy lead-in of mAb1 at the designated dosing dose (DL), intravenously (IV), once weekly (QW), followed by a combination of mAb1 at the designated DL, IV, QW, and salitropin, 350 mg IV, once every three weeks (Q3W). Once the minimum pharmacologically active dosing dose is determined, the dosing interval of mAb1 will be extended from QW to Q3W for subsequent dose escalation. Once the maximum tolerated dose (MTD) / putative recommended Phase 2 dose (putative RP2D) of mAb1 IV is determined, subcutaneous (SC) dosing of mAb1 can be studied. After the start of the expansion phase (*cohort), the tolerability of concomitant administration of mAb1 and salitropin (without monotherapy lead-in of mAb1) in the presence or absence of 350 mg IV Q3W × 4 doses of salitropin can be studied at the MTD / putative RP2D.

[0243] The use of 350 mg IV Q3W × 4 doses of salilumab prophylaxis in combination with REGN5678 and salipolizumab will be investigated during dose escalation. Administration dose (DL) 7 (100 mg REGN5678 IV QW) has been evaluated for DLT and deemed tolerable and was therefore selected for the initial cohort of salilumab. Based on the safety and tolerability of the initial cohort, dose escalation with salilumab prophylaxis may continue. For patients in the salilumab cohort, eligibility criteria were specifically modified, including increasing the absolute neutrophil count study inclusion threshold to ≥1.5×109 / L and excluding patients with active or prior tuberculosis (TB), prior opportunistic infections, intestinal perforation, severe diverticulitis, or inflammatory bowel disease. Based on the ongoing evaluation of cumulative safety and tolerability data in all cohorts, escalations greater than 100 mg (DL7) may be performed with or without salilumab prophylaxis. The addition of an IL-6R blockade to salizumab mitigated immune-mediated adverse events (imAEs) caused by treatment with REGN5678 in combination with salizumab while preserving antitumor activity.

[0244] Dose expansion: During the dose expansion period, patients will receive a 3-week monotherapy lead-in of mAb1, followed by a combination of mAb1 at the MTD / putative RP2D IV and semilazumab 350 mg IV Q3W. Dose expansion cohorts may also be studied using subcutaneous administration of mAb1 at the MTD / putative RP2D.

[0245] Dose expansion cohorts will be enrolled after the MTD of mAb1 in combination with semilazumab and / or presumed RP2D is determined. Safety assessments will be performed at each study drug dosing visit. Radiographic response assessments will be performed every 6 weeks from Cycle 1 (C1D42 / C2D1) until Cycle 4 (including patients who received an initial 3-week monotherapy run-in of mAb1) and every 12 weeks thereafter.

[0246] Study duration

[0247] The total duration of study participation for each patient varied based on the occurrence of 1 or more of the following: disease progression, intolerable adverse events (AEs), withdrawal of consent, or meeting study withdrawal criteria.

[0248] For patients in the dose-escalation and expansion cohorts (except the *cohort which had no monotherapy run-in period), the study consisted of 4 phases: a screening phase (up to 28 days), a 3-week monotherapy run-in phase for mAb1 (21 days), a combination therapy phase (consisting of a series of 6-week (42-day) cycles of mAb1 in combination with cemiplimab, with or without salizumab) (variable duration until discontinuation), and an off-treatment follow-up phase (90 days). For patients in the *cohort, the study consisted of 3 phases: a screening phase (up to 28 days), a combination therapy phase (consisting of a series of 6-week (42-day) cycles of mAb1 in combination with cemiplimab) (variable duration until discontinuation), and an off-treatment follow-up phase (90 days).

[0249] Dose escalation of mAb1 will be performed QW until the pharmacologically active dose (ie, the minimum pharmacologically active dose) is determined. Once the minimum pharmacologically active dose is determined, mAb1 dose escalation will proceed to the next dose and will be administered Q3W.

[0250] Table 8: Planned dose escalation cohorts and inclusion

[0251] Dosage mAb1 Cemimuzumab Initial inclusion DL1a 0.03mg 350mg 0-4 DL1 0.1mg 350mg 1-2 DL2 0.3mg 350mg 1-2 DL3 1mg 350mg 3-4 DL4 3mg 350mg 3-4 DL4a 6mg 350mg 0-4 DL5 10mg 350mg 3-4 DL5a 20mg 350mg 0-4 DL6 30mg 350mg 3-4 DL6a 60mg 350mg 0-4 DL7 100mg 350mg 3-12 DL7a.1 150mg 350mg 0-12 DL7a 200mg 350mg 3-12 DL7a.2 250mg 350mg 0-12 DL8 300mg 350mg 3-12 DL8a.1 450mg 350mg 0-12 DL8a 600mg 350mg 3-12 DL8a.2 750mg 350mg 0-12 DL9 900mg 350mg 3-12

[0252] Cohorts labeled “a.1” or “a.2” are intermediate cohorts that can be used when smaller dose escalations are needed.

[0253] Study population

[0254] Approximately 216 patients will be enrolled (approximately 108 patients during dose escalation and approximately up to 3-4 expansion cohorts with up to 27 patients per cohort). The study population includes treatment-experienced men with mCRPC. For inclusion in this study, patients must have received at least 2 lines of prior systemic therapy (other than androgen deprivation therapy [ADT]) approved for metastatic and / or castration-resistant disease, including second-generation antiandrogen therapy (e.g., abiraterone, enzalutamide, apalutamide, or darolutamide).

[0255] Inclusion criteria : Patients must meet the following criteria to be eligible for inclusion in this study:

[0256] 1. Males aged ≥ 18 years

[0257] 2. Patients with histologically or cytologically confirmed prostate adenocarcinoma without pure small cell carcinoma.

[0258] 3. Metastatic castration-resistant prostate cancer (mCRPC) with a PSA value ≥ 4 ng / mL at screening, with progression within 6 months prior to screening according to one of the following:

[0259] a.PSA progression was defined as an increase in PSA level confirmed by an interval of ≥1 week between each assessment.

[0260] b. Radiographic disease progression in soft tissues based on RECIST version 1.1 criteria (with or without PSA progression)

[0261] c. Radiographic disease progression in bone was defined as the presence of 2 or more bone lesions on bone scan with or without PSA progression

[0262] 4. Has received ≥2 lines of systemic therapy (other than androgen deprivation therapy [ADT]) approved for use in the metastatic and / or castration-resistant setting, including at least

[0263] a. A second-generation antiandrogen therapy (eg, abiraterone, enzalutamide, apalutamide, or darolutamide)

[0264] NOTE: For metastatic prostate cancer with mixed histologic features, non-taxane-based chemotherapy regimens may be given and will be included in the evaluation of lines of treatment.

[0265] 5. Able and willing to provide tumor tissue, either archived or newly obtained. Note: For dose escalation only, if archived or fresh tissue is not available, a pathology report confirming the diagnosis of prostate cancer may be submitted.

[0266] 6. Patients who have undergone orchiectomy or are currently receiving luteinizing hormone-releasing hormone (LHRH) agonist or antagonist therapy (serum testosterone <50 ng / dL) and agree to continue receiving LHRH agonist or antagonist therapy during the study

[0267] 7. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1

[0268] 8. Adequate organ and bone marrow function documented by the following data:

[0269] a. Hemoglobin ≥8.5 g / dL

[0270] b. Absolute neutrophil count ≥ 1.0 × 10 9 / L(Only for the salilumab cohort: ≥1.5×10 9 / L)

[0271] c. Platelet count ≥100×10 9 pcs / L

[0272] 9. Serum creatinine ≤1.5×ULN or estimated glomerular filtration rate>50mL / min / 1.73m 2 . A 24-hour urine creatinine collection may be used as an alternative to calculating creatinine clearance for purposes of meeting eligibility criteria.

[0273] 10. Adequate Liver Function:

[0274] a. Total bilirubin ≤1.5×ULN (≤3×ULN if liver involvement of tumor occurs)

[0275] b.AST ≤ 2.5 × ULN (≤ 5 × ULN if liver involvement of the tumor occurs)

[0276] c. ALT ≤ 2.5 × ULN (≤ 5 × ULN if liver involvement of the tumor occurs)

[0277] d. Alkaline phosphatase (ALP) ≤ 2.5 × ULN (≤ 5 × ULN if liver or bone involvement of the tumor occurs)

[0278] Notice:

[0279] Patients with liver involvement from neoplasia will be excluded if their AST ≥ 3 × ULN or ALT ≥ 3 × ULN and their bilirubin level ≥ 2 × ULN regardless of the above criteria

[0280] Patients with Gilbert's syndrome do not need to meet the total bilirubin requirement as long as their total bilirubin does not exceed their historical levels. Gilbert's syndrome must be appropriately documented as part of past medical history

[0281] 11. Willing and able to comply with clinical visits and study-related procedures

[0282] 12. Provide the signed informed consent form of the research patient

[0283] 13. Able to understand and complete research-related questionnaires

[0284] Exclusion criteria Patients who meet any of the following criteria will be excluded from the study:

[0285] 1. Currently receiving treatment in another study

[0286] 2. Participated in a study of an investigational agent or device within 4 weeks of the first dose of study therapy

[0287] 3. Has received treatment with approved systemic therapy (including Siprussin-T) within 3 weeks of dosing, or has not recovered from any acute toxicity (i.e., ≤ Grade 1 or baseline), except for inclusion criteria and laboratory changes described below:

[0288] a. Patients with neuropathy ≤ grade 2

[0289] 4. Has received radiation therapy or major surgery within 14 days of the first administration of study drug, or has not recovered from an AE (i.e., ≤ Grade 1 or baseline), except for inclusion criteria and laboratory changes described below:

[0290] a. Patients with neuropathy ≤ grade 2

[0291] 5. Have received any previous systemic biological therapy within 5 half-lives of the first dose of study therapy

[0292] Exceptions: Patients may be included after discussion with the sponsor if they have previously received treatment with bevacizumab or other non-immunomodulatory antibodies with a half-life greater than 7 days and at least 30 days have passed since their last treatment.

[0293] 6. Received previous PSMA targeted therapy

[0294] 7. Dose escalation: Patients who have received previous anticancer immunotherapy (except Siprussin-T) within 5 half-lives before the study therapy. Examples of immunomodulators include blockers of CTLA-4, 4-1BB (CD137), or OX-40, therapeutic vaccines, anti-PD-1 / PD-L1, phosphoinositide 3-kinase (PI3K) δ inhibitors, or cytokine anticancer therapy. Note: Patients who have received investigational cell-based therapies (e.g., CAR-T cells) are excluded.

[0295] 8. Dose expansion: Patients who have received previous anticancer immunotherapy. Examples of immunomodulators include CTLA-4, 4-1BB (CD137), or OX-40 blockers, therapeutic vaccines, anti-PD-1 / PD-L1, PI3Kδ inhibitors, CAR-T cells, or cytokine anticancer therapy. Note: Previous treatment with sipuleucel-T is allowed

[0296] 9. Patients who have not recovered (i.e., ≤ Grade 1 or baseline) from an immune-mediated AE within 3 months prior to starting study drug therapy, except for endocrine disease adequately managed by hormone replacement therapy

[0297] 10. Patients who permanently discontinued anticancer immunomodulatory therapy due to immune-related AEs

[0298] 11. Another malignant tumor that is progressing or requires active treatment, except for the following:

[0299] a. Non-melanoma skin cancer that has undergone potentially curative therapy

[0300] b. Any tumor that is considered to be effectively treated by definitive local control (with or without continued adjuvant hormonal therapy)

[0301] 12. Any condition requiring ongoing / continuous corticosteroid therapy (>10 mg prednisone / day or anti-inflammatory equivalent) within 1 week prior to the first dose of study therapy. Patients requiring short-term use of steroids or physiologic replacement therapy (up to 2 days in the week prior to inclusion) will not be excluded

[0302] 13. Continuous or recent (within 5 years) evidence of severe autoimmune disease requiring treatment with systemic immunosuppressive therapy. The following conditions are not excluded: vitiligo, resolved childhood asthma, endocrine diseases requiring only hormone replacement therapy (such as hypothyroidism or type 1 diabetes), or psoriasis that does not require systemic treatment

[0303] 14. History of CNS metastases, including those that have received previous treatment

[0304] 15. Encephalitis, meningitis, neurodegenerative disease (except mild dementia that does not affect activities of daily living [ADL]), or uncontrolled epilepsy within one year before the first dose of study therapy

[0305] 16. Known history or any evidence of interstitial lung disease or active non-infectious lung inflammation (past 5 years). History of radiation pneumonitis is allowed

[0306] 17. Uncontrolled human immunodeficiency virus infection (HIV), hepatitis B, or hepatitis C infection; or diagnosed with immunodeficiency.

[0307] Notice:

[0308] Patients will be tested for Hepatitis C virus (HCV) and Hepatitis B virus (HBV) at screening.

[0309] Patients with known HIV infection whose infection is controlled (undetectable viral load (HIV RNA polymerase chain reaction [PCR]) and CD4 count above 350, either spontaneously or while receiving a stable antiviral regimen) are permitted to be included. Patients whose HIV infection is controlled will be monitored according to local standards.

[0310] Patients with hepatitis B (HepBsAg+) whose infection has been controlled (serum HBV DNA PCR below the limit of detection and receiving HBV antiviral therapy) are allowed to be included. Patients with controlled infection must have HBV DNA monitored regularly. Patients must also continue to receive antiviral therapy for at least 6 months after receiving the last dose of investigational study drug.

[0311] Patients who were hepatitis C antibody positive (HCV Ab+) and whose infection was controlled (undetectable HCV RNA by PCR either spontaneously or as a response to previous successful anti-HCV therapy) were allowed to be included.

[0312] 18. Any infection requiring hospitalization or intravenous anti-infective medication within 2 weeks of the first dose of study therapy.

[0313] 19. Administration of a live vaccine within 4 weeks of planned initiation of study drug treatment.

[0314] 20. Received a previous allogeneic stem cell transplant or organ transplant at any time, or received an autologous stem cell transplant within 12 weeks of starting study treatment

[0315] 21. Known allergy or hypersensitivity to components of semilazumab or study drugs.

[0316] 22. Known psychiatric or substance addiction conditions that would interfere with the requirements for participation in the study

[0317] 23. Any medical condition, comorbidity, physical examination findings, metabolic dysfunction, or clinical laboratory abnormality that, in the opinion of the investigator, makes the patient unsuitable for participation in the clinical trial due to high safety risks and / or may affect the interpretation of the study results, including but not limited to severe cardiovascular disease (e.g., New York Heart Association class III or IV heart disease, myocardial infarction, unstable arrhythmia, or unstable angina within the past 6 months) and / or severe pulmonary disease (e.g., obstructive pulmonary disease and history of symptomatic bronchospasm).

[0318] 24. Salilumab cohort only: Exclusion criteria related to tuberculosis (TB)

[0319] a. Active TB or history of incompletely treated TB

[0320] b. QuantiFERON-positive patients (without active disease) will be excluded from the study unless there is documented completion of chemoprevention for latent tuberculosis infection (LTBI) (e.g., acceptable treatment is isoniazid 300 mg orally daily for 9 months, or an equivalent validated regimen according to local guidelines) or treatment for active tuberculosis infection (TBI), and active disease is excluded by expert consultation

[0321] 25. Talilumab cohort only: patients with a history of invasive opportunistic infections including but not limited to histoplasmosis, coccidioidomycosis, Pneumocystic jirovecii, or aspergillosis or John Cunningham virus (progressive multifocal leukoencephalopathy).

[0322] 26. Only in the salilumab cohort: history of intestinal perforation, severe diverticulitis, or inflammatory bowel disease

[0324] Study Treatment

[0325] mAb1 will be administered at the designated dosing doses QW or Q3W by intravenous infusion over 30 minutes to 2 hours or by subcutaneous injection. Cemizumab 350 mg will be administered by intravenous infusion Q3W over 30 minutes. Salilumab 350 mg Q3W will be administered by intravenous infusion over 60 minutes in four doses (12 weeks total), starting with an initial dose of REGN5678 in combination with cemizumab. When mAb1 and cemizumab are administered on the same day, mAb1 will be administered first. For cohorts receiving salilumab IV, it should be administered before mAb1 and cemizumab. In selected patients at selected centers, salilumab will be administered 18 F-DCFPyL for experimental PSMA PET / CT imaging procedures.

[0326] Study End Points

[0327] The primary endpoints of the study are:

[0328] Dose escalation:

[0329] Safety, 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 the treatment period and up to 90 days after the last dose of mAb1 and semilazumab, or until the start of a new therapy to treat the patient's tumor, whichever occurs first.

[0330] Tolerability, measured as the incidence of dose-limiting toxicities (DLTs) from the first dose to the end of the DLT observation period for mAb1 alone and in combination with semilaplimab

[0331] Changes in serum mAb1 concentrations over time when administered alone or in combination with semipirimab

[0332] Dose expansion:

[0333] ORR measured according to modified PCWG3 criteria, defined as the percentage of patients who had a response according to modified PCWG3 criteria based on the following metrics:

[0334] - Prostate-specific antigen (PSA) decrease of ≥50% relative to baseline and relative to the start of combination therapy, confirmed by a second PSA test ≥4 weeks later, and / or

[0335] - Radiographically confirmed response of complete remission (CR) or partial remission (PR)

[0336] All primary endpoints of the study will apply to each cohort in the study, including patients who received salilumab and those who did not.

[0337] The secondary endpoints of the study are:

[0338] Dose escalation:

[0339] ORR, defined as the percentage of patients who responded according to the modified PCWG3 criteria based on the following:

[0340] - PSA decrease of ≥50% relative to baseline and relative to the start of combination therapy, confirmed by a second PSA test ≥4 weeks later, and / or

[0341] - Confirmed radiographic response of CR or PR

[0342] Dose expansion:

[0343] Safety, measured by the incidence and severity of treatment-emergent adverse events (AEs) / AESIs / SAEs and grade ≥3 laboratory abnormalities during the treatment period and up to 90 days after the last dose of mAb1 in combination with semilazumab or until the start of a new therapy to treat the patient's tumor, whichever occurs first

[0344] Changes in serum mAb1 concentration over time when administered in combination with semipirimab

[0345] Dose escalation and dose expansion:

[0346] ORR based on PSA response, defined as the percentage of patients achieving a ≥50% decrease in PSA relative to baseline and relative to the start of combination therapy, confirmed by a second PSA test ≥4 weeks later

[0347] Percentage of patients with a ≥90% decrease in PSA relative to baseline and relative to the start of combination therapy, confirmed by a second PSA test ≥4 weeks later

[0348] Percentage of patients who achieved a conversion in circulating tumor cell (CTC) counts from ≥5 cells / 7.5 mL to <5 cells / 7.5 mL relative to baseline and relative to the start of combination therapy

[0349] Presence or absence of antibodies against mAb1 and semilaprimab

[0350] All secondary endpoints of the study will apply to each cohort in the study, including patients who received salilumab and those who did not.

[0351] The exploratory endpoints of the study are:

[0352] Percent change in PSA, defined as the maximum percent change in PSA decrease from baseline and from the start of combination therapy

[0353] Percent change in PSA during the 3-week monotherapy period of mAb1, defined as the maximum percent change in PSA decline from baseline during the mAb1 monotherapy run-in period

[0354] Radiographic progression-free survival (rPFS) (by modified PCWG3 and iRECIST), defined as the time from the first administration of study treatment to the first radiographic progression or death from any cause. Radiographic progression includes progression of soft tissue lesions and bone lesions according to modified PCWG3. If radiographic progression or death did not occur before the analysis cutoff date or the date of initiation of further anticancer treatment, rPFS will be censored to the date of the last valid radiographic response assessment that did not show radiographic progression before the analysis cutoff date or the date of initiation of further anticancer treatment, whichever is earlier

[0355] PSA progression-free survival (PFS), defined as the time from the first administration of study treatment and from the start of combination therapy to the first PSA progression or death from any cause. PSA progression was defined as:

[0356] - After a decline from baseline: PSA increase of ≥25% and ≥2 ng / mL above nadir, confirmed by a second PSA test ≥4 weeks later;

[0357] - No decrease from baseline: PSA progression after 12 weeks with an increase of ≥25% from baseline and ≥2 ng / mL

[0358] If PSA progression or death did not occur before the analysis cutoff date or the date of initiation of further anticancer treatment, PSA PFS was censored at the date of the last valid PSA test that did not show PSA progression before the analysis cutoff date or the date of initiation of further anticancer treatment, whichever was earlier.

[0359] ORR based on radiographic response, defined as the percentage of patients achieving a radiographic response of CR or PR (by revised PCWG3 and iRECIST)

[0360] Duration of response based on radiographic response (by modified PCWG3 and iRECIST), defined as the time from the first administration of study treatment to the first radiographic response of CR or PR for patients with a confirmed radiographic response of CR or PR

[0361] 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

[0362] DOR based on radiographic response (rDOR) (by modified PCWG3 and iRECIST), defined as the time from the first radiographic response of CR or PR to the first radiographic progression or death for patients with a confirmed radiographic response of CR or PR. The same censoring rules as for rPFS will be used

[0363] DOR based on PSA response, defined as the time from first PSA response to first PSA progression for patients with a confirmed PSA response. The same censoring rules as for PSA PFS will be used

[0364] Time to progression based on radiographic progression (by modified PCWG3 and iRECIST), defined as the time from the first administration of study treatment to the first radiographic progression. If radiographic progression did not occur before the analysis cutoff date or the date of initiation of further anticancer treatment, the time to progression will be censored to the date of the last valid radiographic response assessment that did not show radiographic progression before the analysis cutoff date or the date of initiation of further anticancer treatment, whichever is earlier

[0365] Time to progression based on PSA progression, defined as the time from the first study treatment administration or initiation of combination therapy to the first PSA progression. If PSA progression did not occur before the analysis cutoff date or the date of initiation of further anticancer treatment, the time to progression will be censored to the date of the last valid PSA test that did not show PSA progression, performed before the analysis cutoff date or the date of initiation of further anticancer treatment, whichever is earlier

[0366] Disease control rate (DCR) (by revised PCWG3 and iRECIST), defined as the percentage of patients achieving a radiographic response of CR, PR, SD, or non-CR / non-PD

[0367] OS, defined as the time from the first administration of study treatment to death from any cause. In the absence of a survival event, OS will be censored at the last date the patient was known to be alive.

[0368] ·Can quantify the changes in CTC count (cells / 7.5mL) in samples

[0369] Changes in serum cytokines and other biomarkers of inflammation

[0370] Time to pain progression (TTPP) assessed by BPI-SF item 3 ("worst pain in the past 24 hours") and opioid analgesic use

[0371] Change from baseline in pain severity and pain interference as measured by the BPI-SF

[0372] Change from baseline in GHS / QoL as measured by the EORTC QLQ-C30 GHS / QoL scale score

[0373] Change from baseline in physical function, as measured by the EORTC QLQ-C30 physical function scale score

[0374] Change from baseline in urinary symptoms as measured by the EORTC QLQ-PR25 urinary symptom scale score

[0375] Change from baseline in Patient Global Impression of Severity Score (PGIS)

[0376] Change from baseline in Patient Global Impression of Change (PGIC) score

[0377] Correlation of baseline tumor tissue biomarker levels of mAb1 and semiplimab target pathways (e.g., PD-L1, PSMA, and CD28) with therapeutic activity in available tissue biopsies

[0378] Correlation between tumor gene variants (including HDR mutations and TMB) and ORR

[0379] Changes from baseline in PSMA and FDG PET / CT tumor signal intensity after treatment initiation

[0380] Correlation of baseline PSMA PET / CT tumor positivity with clinical activity of mAb1 and semilazumab

[0381] All exploratory endpoints of the study will apply to each cohort in the study, including patients who received salilumab and those who did not.

[0382] Results - Anti-tumor activity was observed in patients treated with the combination of mAb1 and semilazumab, and the safety profile was manageable. Prostate cancer (e.g., metastatic castration-resistant prostate cancer) patients treated with 30 mg to 300 mg weekly (to date) of mAb1 and semilazumab (350 mg every three weeks) showed anti-tumor activity as measured by a decrease in prostate-specific antigen (PSA) levels (e.g., a decrease of more than 50%) and / or a RESIST response. Results as Figure 3A , 3B , 3C and 3D and as shown in Table 9 below.

[0383] Table 9: Summary of antitumor activity and observed AEs at doses up to 300 mg mAb1

[0384]

[0385] Figure 4A and 4B The latest anti-tumor data observed in 35 patients (17 for doses 1-5 and 18 for doses 6-8) are shown. It is worth noting that patients treated with doses 1-5 did not show ≥ grade 3 immune-mediated adverse events (imAEs), and only 1 of the 16 evaluable patients treated with doses 1-5 showed a decrease in PSA levels. In contrast, patients treated with doses 6-8 showed efficacy signs related to imAEs. In dose 6, 1 of 4 evaluable patients showed a response (PSA levels decreased by 100% and complete remission of target lesions was maintained for about 12 months) and grade 3 imAEs. In dose 7, 3 of 8 evaluable patients showed responses (PSA levels decreased by 99%, 44% and 22%), two of whom had grade 3 imAEs. In dose 8, 3 of 4 evaluable patients showed responses (PSA levels decreased by 99%, 99% and 82%), one of whom had an imAE leading to death. Mitigation strategies to prevent imAEs associated with treatment response, including blockade of IL-6R, are under investigation.

[0386] In each of the three patients who received dose 8 and observed responses, PSA levels continued to rise during the lead-in period of mAb1 until the start of semilazumab administration. Advanced metastatic castration-resistant prostate cancer (mCRPC) shows a response rate of approximately 5% to anti-PD-1 monotherapy, so the rise in PSA levels observed before the start of semilazumab administration confirms that mAb1 and semilazumab have a synergistic effect in patients with mCRPC.

[0387] One patient treated with dose 7 (99% decrease in PSA level) showed pseudoprogression in the liver, followed by a response, as evidenced by a decrease in PSMA PET-positive lesions. Of note, the same patient showed a response in tumor lesions with low PSMA PET signal, and this patient would not have been expected to respond to Pluvicto based on the eligibility criteria. TM Pluvicto™ contains a PSMA-binding ligand conjugated to a DOTA chelator radiolabeled with Lutetium-177.

[0388] Preliminary results showed minimal antitumor activity at lower doses, as predicted by preclinical models. However, antitumor activity increased as semiplimab was initiated. At dose 8, three of the four patients showed significant PSA responses when starting combination therapy ( Figure 5 ). In addition, one patient who received dose 6 experienced a response with PSA levels below the limit of detection, normalization of bone scans, negative PSMA PET scans, and resolution of soft tissue disease. However, this patient discontinued therapy due to a grade 3 irAE of the skin, which resolved after treatment. Grade ≥3 immune-related adverse events occurred only in some patients with antitumor activity.

[0389] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present 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 fall within the scope of the appended claims.

[0390] *******

[0391] Table 10: Sequences excluded from ST.26 format sequence listing

[0392] SEQ ID NO: sequence 30 AAS 42 AAS

Claims

1. A method of treating a PSMA-expressing cancer in a subject in need thereof, the method comprising administering to the subject a combination of a bispecific antibody or antigen-binding fragment thereof and an antibody or antigen-binding fragment thereof that specifically binds programmed death receptor-1 (PD-1), wherein the bispecific antibody or antigen-binding fragment thereof comprises 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, wherein the bispecific antibody is administered to the subject at a dose of at least 0.03 mg.

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

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 received at least two prior therapies for metastatic and / or castration-resistant prostate cancer.

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

7. The method of claim 6, wherein the anti-androgen 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 prostate adenocarcinoma without pure small cell carcinoma.

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) value ≥ 4 ng / ml prior to treatment with the bispecific antibody.

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

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

12. 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 method of any one of claims 1 to 12, wherein the first antigen binding domain comprises: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 1; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

9.

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

4.

15. The method of claim 13 or claim 14, wherein the first antigen binding domain comprises LCDR1 having the amino acid sequence of SEQ ID NO: 10, LCDR2 having the amino acid sequence of SEQ ID NO: 11, and LCDR3 having 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 a HCVR having the amino acid sequence of SEQ ID NO: 1 and a LCVR having the amino acid sequence of SEQ ID NO:

9.

17. The method of any one of claims 1 to 16, wherein the second antigen binding domain comprises: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 5; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

9.

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

8.

19. The method of claim 17 or claim 18, wherein the second antigen binding domain comprises LCDR1 having the amino acid sequence of SEQ ID NO: 10, LCDR2 having the amino acid sequence of SEQ ID NO: 11, and LCDR3 having 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 a HCVR having the amino acid sequence of SEQ ID NO:5 and a LCVR having 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. The method of claim 21, wherein the human IgG heavy chain constant region is of isotype IgGl.

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

24. The method of claim 22 or claim 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. 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 a 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 having 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 having the amino acid sequence of SEQ ID NO:

14.

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

15.

29. The method of any one of claims 1 to 12, wherein the first antigen binding domain comprises: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 16; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

28.

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

19.

31. The method of claim 29 or claim 30, wherein the first antigen binding domain comprises LCDR1 having the amino acid sequence of SEQ ID NO:29, LCDR2 having the amino acid sequence of SEQ ID NO:30, and LCDR3 having 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 a HCVR having the amino acid sequence of SEQ ID NO: 16 and a LCVR having the amino acid sequence of SEQ ID NO:

28.

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

28.

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

27.

35. The method of claim 33 or claim 34, wherein the second antigen binding domain comprises LCDR1 having the amino acid sequence of SEQ ID NO:29, LCDR2 having the amino acid sequence of SEQ ID NO:30, and LCDR3 having 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 a HCVR having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 24 and a LCVR having 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. The method of claim 37, wherein the human IgG heavy chain constant region is of isotype IgGl.

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

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

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 a 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 having 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 having 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 having the amino acid sequence of SEQ ID NO:

34.

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

35.

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

35.

47. The method of any one of claims 1 to 46, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 36; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

40.

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

39.

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

43.

50. The method of any one of claims 47 to 49, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a HCVR having the amino acid sequence of SEQ ID NO: 36 and a LCVR having 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 to PD-1 is an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 44 and a light chain having an amino acid sequence of SEQ ID NO:

45.

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 at a dose of 0.03 mg to 1000 mg per week.

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

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

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

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

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

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

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

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

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

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

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 salirumab or tocilizumab.

67. The method of any one of claims 1 to 66, wherein after 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: The prostate-specific antigen (PSA) level in the subject decreases by at least 50%; The PSA level in the subject decreases by at least 55%; The PSA level in the subject decreases by at least 60%; The PSA level in the subject decreases by at least 65%; The PSA level in the subject is reduced by at least 70%; The PSA level in the subject is reduced by at least 75%; The PSA level in the subject is reduced by at least 80%; The PSA level in the subject is reduced by at least 85%; The PSA level in the subject is reduced by at least 90%; The PSA level in the subject is reduced by at least 95%; The subject's PSA level is reduced by at least 96%; The PSA level in the subject is reduced by at least 97%; The PSA level in the subject is reduced by at least 98%; The PSA level in the subject is reduced by at least 99%; a decrease in size of at least one lesion in the subject, the PSMA PET signal of the lesion being less than the PSMA PET signal in the subject's liver; and / or The subject responded after a pseudoprogression.

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

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