Methods of treating clear cell renal cell carcinoma with bispecific anti-PSMAx anti-CD28 antibodies

Abstract

The present disclosure provides methods for treating clear cell renal cell carcinoma (ccRCC) or metastatic ccRCC, reducing the severity of ccRCC or metastatic ccRCC, or inhibiting the growth of ccRCC or metastatic ccRCC. 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, the bispecific antibody or antigen-binding fragment thereof specifically binding to prostate specific membrane antigen (PSMA) and CD28.

Description

[0001] References to sequence lists

[0002] This application incorporates a computer-readable sequence list in ST.26 XML format, created on August 1, 2024, containing 58,834 bytes and titled 11742WO01_Sequence. Technical Field

[0003] This disclosure relates to a method for treating clear cell renal cell carcinoma (ccRCC), including metastatic ccRCC, the method comprising administering a therapeutically effective amount of a bispecific antibody to a subject in need, the bispecific antibody specifically binding to prostate-specific membrane antigen (PSMA) and CD28, and a method for treating ccRCC by administering a combination of the bispecific antibody and an antibody specifically binding to programmed death receptor-1 (PD-1). Background Technology

[0004] Clear cell renal cell carcinoma (ccRCC) represents the most common subtype of renal cell carcinoma, with a frequency of 75-80% at initial diagnosis (Makhov et al., *Molecular Cancer Ther.*, 17(7):1355-1364, 2018). Prostate-specific membrane antigen (PSMA), also known as FOLH1, glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADaseI), or N-acetyl-aspartylglutamate (NAAG) peptidase, is a homodimeric enzymatic type II transmembrane protein encoded by the folic acid hydrolase 1 (FOLH1) gene. PSMA is expressed in all subtypes of renal cell carcinoma, but is significantly more abundant in ccRCC (Baccala et al., *Urology*, 70(2):385-390, 2017). Internal immunohistochemistry (IHC) has confirmed that >90% of patients with ccRCC express PSMA, and this expression is generally confined to tumor angiogenesis, although it is not expressed in normal blood vessels (Chang et al., Cancer Research, 59(13):3192-3198, 1999). PSMA PET imaging studies have been conducted in patients with metastatic ccRCC and have shown to be more sensitive for detecting metastases than conventional imaging via CT / MRI (94.7% vs. 78.9%) (Rowe et al., Ann Nucl Med., 29(10):877-882, 2015), which may be of great significance for staging, patient management, and informed treatment decisions.

[0005] CD28 is a type I transmembrane protein with a single extracellular Ig-V-like domain that assembles into a homodimer and is expressed on the surface of T cells. CD28 is a receptor for both CD80 (B7.1) and CD86 (B7.2) proteins and is activated by CD80 or CD86 expressed on antigen-presenting cells (APCs). The binding of CD28 to CD80 or CD86 provides a co-stimulatory signal that is crucial for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 also provides a potent signal for the production of various interleukins. Following TCR activation, CD28 also enhances cellular signaling pathways, such as those controlled by the NFκB transcription factor. CD28 co-signaling is important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death. Anti-CD28 antibodies have been proposed for therapeutic purposes involving T cell activation. A 2006 clinical trial used a specific anti-CD28 antibody, TGN1412 (an anti-CD28 superagonist), in which TGN1412 was administered intravenously to six healthy volunteers at a dose of 0.1 mg / kg. Within two hours, all six patients developed a significant inflammatory response (cytokine storm), and within sixteen hours, all patients developed multiple organ failure. Treatment with corticosteroids resulted in the return of cytokine levels to normal within 2–3 days (Suntharalingam et al., Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412, New England Journal of Medicine (NEJM) 355:1018-1028 (2006)).

[0006] Programmed death receptor-1 (PD-1) signaling in the tumor microenvironment plays a crucial role in allowing tumor cells to evade immune surveillance by the host immune system. Blocking the PD-1 signaling pathway has demonstrated 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 (Lesokhin et al. 2014, Abstract 291, 56th ASH Annual Meeting and Exposition, San Francisco, California; Ansell et al. 2015, N. Engl. J. Med. 372(4):311-9).

[0007] Patients with metastatic ccRCC receive systemic therapy, including combinations of targeted and immunotherapies. Anti-PD-1 monotherapy and immune checkpoint inhibitors (ICIs) combined with nivolumab and pilimuumab both improved overall survival (OS) (Motzer et al., New England Journal of Medicine, 373(19):1803-1813, 2015). Standards of care for first-line therapy are stratified according to patient risk groups and include dual ICIs or combinations of anti-PD-1 therapy and anti-angiogenic agents (Powles et al., Ann Oncol., 32(12):1511-1519, 2021). There is no standard treatment in the second-line setting, and this remains an area of ​​high unmet need. Most commonly, patients receive tyrosine kinase inhibitor therapy in a second-line setting, which is tailored to the prior therapy received in the first-line setting (Tannir et al., Oncologist, 23(5):540-*555, 2018). Summary of the Invention

[0008] According to certain embodiments, this disclosure provides methods for treating, improving, or inhibiting the growth of at least one symptom or indication of PSMA-expressing clear cell renal cell carcinoma (e.g., metastatic clear cell renal cell carcinoma) in a subject. A method according to this aspect of the disclosure comprises administering to a subject in need a therapeutically effective amount of a bispecific antibody or an antigen-binding fragment thereof, said bispecific antibody or antigen-binding fragment thereof specifically binding to prostate-specific membrane antigen (PSMA) and CD28. In some embodiments, said bispecific antibody is administered in combination with an antibody or antigen-binding fragment thereof specifically binding to programmed death receptor-1 (PD-1).

[0009] On one hand, this disclosure provides a method for treating clear cell renal cell carcinoma in a subject of need, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain specifically binding to prostate-specific membrane antigen (PSMA); and a second antigen-binding domain specifically binding to human CD28.

[0010] In some embodiments, the bispecific antibody is administered to the subject at a dose of at least 30 mg once weekly (QW) for at least three weeks.

[0011] In some embodiments, the clear cell renal cell carcinoma is a metastatic clear cell renal cell carcinoma.

[0012] In some embodiments, the subject has received at least one prior therapy for clear cell renal cell carcinoma. In some cases, the subject has received anti-PD-1 therapy. In some cases, the subject has received anti-CTLA-4 therapy and / or a tyrosine kinase inhibitor. In some cases, the anti-CTLA-4 therapy is ipilimumab.

[0013] In some embodiments, the bispecific antibody or its antigen-binding fragment is administered as a monotherapy for at least three weeks, at least four weeks, at least five weeks, or at least six weeks.

[0014] In some embodiments, the methods discussed above or herein further comprise administering an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 100 mg. In some embodiments, the methods discussed above or herein further comprise administering an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 350 mg. In some embodiments, the methods discussed above or herein further comprise administering an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 1000 mg.

[0015] In some embodiments, the first antigen-binding domain of the bispecific antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), the three heavy chain complementarity-determining regions being contained within a heavy chain variable region (HCVR), the HCVR containing the amino acid sequence of SEQ ID NO: 1; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), the three light chain complementarity-determining regions being contained within a light chain variable region (LCVR), the LCVR containing the amino acid sequence of SEQ ID NO: 9; and the second antigen-binding domain of the bispecific antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), the three heavy chain complementarity-determining regions being contained within a heavy chain variable region (HCVR), the HCVR containing the amino acid sequence of SEQ ID NO: 5; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), the three light chain complementarity-determining regions being contained within a light chain variable region (LCVR), the LCVR containing the amino acid sequence of SEQ ID NO: 9.

[0016] In some cases, the first antigen-binding domain comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 2; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 3; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 4. In some cases, the second antigen-binding domain comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 6; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 7; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 8.

[0017] In some cases, the first antigen-binding domain and the second antigen-binding domain comprise: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 10; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 11; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 12.

[0018] In some cases, the first antigen-binding domain comprises: HCVR, which comprises the amino acid sequence of SEQ ID NO: 1; and LCVR, which comprises the amino acid sequence of SEQ ID NO: 9. In some cases, the second antigen-binding domain comprises: HCVR, which comprises the amino acid sequence of SEQ ID NO: 5; and LCVR, which comprises the amino acid sequence of SEQ ID NO: 9.

[0019] In some embodiments, the bispecific antibody comprises a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype IgG1. In some cases, the human IgG heavy chain constant region is isotype IgG4.

[0020] In some embodiments, the bispecific antibody comprises a chimeric hinge, the chimeric hinge being reduced relative to a wild-type hinge of the same isotype. Receptor binding.

[0021] In some embodiments, the first or second heavy chain of the bispecific antibody, but not both, contains a CH3 domain, which contains H435R (EU number) modification and Y436F (EU number) modification.

[0022] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the bispecific antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 13; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 14; and a common light chain comprising the amino acid sequence of SEQ ID NO: 15.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof binding to PD-1 comprises: (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 36; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 40.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof binding to PD-1 comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 37; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 38; and HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody or antigen-binding fragment thereof binding to PD-1 comprises: LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 41; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 42; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 43.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof that binds to PD-1 comprises: HCVR, the HCVR comprising the amino acid sequence of SEQ ID NO: 36; and LCVR, the LCVR comprising the amino acid sequence of SEQ ID NO: 40.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof that binds to PD-1 is an antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 44; and a light chain comprising the amino acid sequence of SEQ ID NO: 45.

[0027] In some embodiments, the bispecific antibody or its antigen-binding fragment is administered to the subject once weekly at a dose of 30 mg to 900 mg for at least three weeks. In some embodiments, the bispecific antibody or its antigen-binding fragment is administered to the subject once weekly at a dose of 100 mg to 900 mg for at least three weeks. In some embodiments, the bispecific antibody or its antigen-binding fragment is administered to the subject once weekly at a dose of 300 mg to 900 mg for at least three weeks. In some embodiments, the bispecific antibody or its antigen-binding fragment is administered to the subject once weekly at a dose of 30 mg, 100 mg, 300 mg, 600 mg, or 900 mg for at least three weeks.

[0028] This disclosure also covers the use of bispecific antibodies and / or anti-PD-1 antibodies (and antigen-binding fragments of either) in the manufacture of medicaments for treating cancers expressing PSMA, as illustrated in any of the embodiments of the methods discussed above or herein. This disclosure also covers bispecific antibodies and / or anti-PD-1 antibodies (and antigen-binding fragments of either) in any of the embodiments of the methods discussed above or herein. This disclosure also covers pharmaceutical compositions comprising bispecific antibodies and / or anti-PD-1 antibodies (and antigen-binding fragments of either) in any of the embodiments of the methods discussed above or herein.

[0029] In various embodiments, any feature or component of the embodiments discussed above or herein may be combined, and such combinations are covered within the scope of this disclosure. Any particular value discussed above or herein may be combined with another related value discussed above or herein to enumerate a range of values ​​having an upper and lower end representing a range, and such ranges are covered within the scope of this disclosure.

[0030] Other embodiments of this disclosure will become apparent from the following detailed description. Attached Figure Description

[0031] Figure 1 An example of a study flowchart illustrating QW dosing of REGN5678 monotherapy as discussed in Example 2 is presented. Participants with evidence of progressive disease may receive a low dose of cimiprimab (3 mg IV Q3W) after receiving REGN5678 monotherapy for at least 6 weeks. Participants may also switch to REGN5678 Q3W dosing after 12 months of treatment. Detailed Implementation

[0032] It should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims. Any embodiment or feature of an embodiment may be combined with each other, and such combinations are expressly covered within the scope of this disclosure. Any particular value discussed above or herein may be combined with another related value discussed above or herein to enumerate a range of values ​​having an upper and lower end indicating a range, and such ranges are covered within the scope of this disclosure.

[0033] 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 this disclosure pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value may differ from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0034] Although any methods and materials similar to or equivalent to those described and used herein may be used in practice or testing of this disclosure, preferred methods and materials are now described. All patents, applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety.

[0035] Methods used to treat or inhibit cancer growth

[0036] This disclosure includes methods for treating, improving, or reducing the severity of at least one symptom or indication of cancer (e.g., metastatic clear cell renal cell carcinoma) in a subject, or inhibiting the growth of said at least one symptom or indication. A method according to this aspect of the disclosure comprises administering a therapeutically effective amount of a bispecific antibody against PSMA and CD28 to a subject in need. As used herein, the terms “treat,” “treating,” etc., mean improving symptoms, temporarily or permanently eliminating the triggers of symptoms, delaying or inhibiting tumor growth, reducing tumor cell burden or tumor load, promoting tumor regression, shrinking, necrosis, and / or disappearing of the tumor, preventing tumor recurrence, and / or prolonging the survival of the subject. In some cases, the method further comprises administering a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that binds to human PD-1.

[0037] As used herein, "subject" or "subject requiring treatment" means a human or non-human mammal exhibiting one or more symptoms or indications of cancer, and / or having been diagnosed with cancer including clear cell renal cell carcinoma (ccRCC) (e.g., metastatic ccRCC) and requiring treatment for said cancer. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may be diagnosed with a primary or metastatic tumor and / or have one or more symptoms or indications, including but not limited to lymphadenopathy, abdominal swelling, unexplained pain, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, and / or splenomegaly. Expression includes subjects with a primary or confirmed renal cell carcinoma tumor having a clear cell component. In a specific embodiment, expression includes human subjects with ccRCC expressing PSMA and requiring treatment. In other specific embodiments, expression includes subjects with a PSMA+ tumor (e.g., a tumor expressing PSMA as determined by flow cytometry). In some embodiments, the expression "subject in need" includes patients with ccRCC that is resistant, refractory, or poorly controlled to prior therapies (e.g., treatment with conventional anticancer agents, including antiPD-1 therapy, antiCTLA-4 therapy, or treatment with tyrosine kinase inhibitors). For example, the expression includes subjects who have been treated with chemotherapy, or antiPD-1 therapy, or antiPD-1 therapy and antiCTLA-4 therapy (e.g., ipilimumab), or antiPD-1 therapy and tyrosine kinase inhibitors, or antiPD-1 therapy, antiCTLA-4 therapy, and tyrosine kinase inhibitors. The expression also includes subjects with ccRCC tumors that are unsuitable for conventional anticancer therapy, 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 therapies and experienced toxic side effects. In some embodiments, the expression "subject in need" includes patients with ccRCC tumors who have received treatment but subsequently relapsed or metastasized. For example, a patient with ccRCC tumors may have received treatment with one or more anticancer agents, resulting in tumor regression; however, the cancer, which subsequently recurs and is resistant to one or more anticancer agents, is treated using the methods disclosed herein.

[0038] In some embodiments, the methods of this disclosure can be used to treat patients with histologically or cytologically confirmed renal cell carcinoma having a clear cell component. The terms “tumor,” “cancer,” and “malignant tumor” are used interchangeably herein. As used herein, the term “clear cell renal cell carcinoma” refers to a tumor of the kidney, including metastatic tumors originating from the kidney having a clear cell component.

[0039] According to some embodiments, this disclosure includes methods for treating, delaying, or inhibiting tumor growth. In some embodiments, this disclosure includes methods for promoting tumor regression. In some embodiments, this disclosure includes methods for reducing tumor cell burden or reducing tumor load. In some embodiments, this disclosure includes methods for preventing tumor recurrence. According to this aspect of the disclosure, the method comprises administering to a subject in need a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof, optionally 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 treatment dosing regimen. For example, the treatment dosing regimen may comprise administering one or more doses of the anti-PSMA x CD28 antibody or an antigen-binding fragment thereof to the subject at a frequency of approximately once daily, every two days, every three days, every four days, every five days, every six days, once weekly, every two weeks, every three weeks, every four weeks, once monthly, every two months, every three months, every four months, or lower. In some embodiments, the anti-PSMA x anti-CD28 antibody or an antigen-binding fragment thereof is administered once weekly. In some embodiments, the anti-PSMA x anti-CD28 antibody or its antigen-binding fragment is administered to the subject once every three weeks. In some embodiments, one or more doses of the anti-PD-1 antibody or its antigen-binding fragment are administered to the subject at a frequency of approximately once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, once every four months, or lower. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is administered to the subject once every three weeks.

[0040] In some embodiments, this disclosure includes methods for inhibiting, delaying, or stopping tumor metastasis or tumor invasion into peripheral organs. According to this aspect, the method comprises administering to a subject in need a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof, optionally in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0041] In specific embodiments of combination therapy, the anti-PSMA / CD28 bispecific antibody or its antibody-binding fragment is administered to the subject prior to the anti-PD-1 antibody or its antigen-binding fragment. In some cases, the anti-PSMA / CD28 antibody or its antigen-binding fragment may be administered approximately 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 longer prior to the anti-PD-1 antibody or its antigen-binding fragment. In some cases, the anti-PSMA / CD28 antibody or its antigen-binding fragment is administered as a monotherapy for three, four, five, or six weeks prior to the administration of the anti-PD-1 antibody or its antigen-binding fragment.

[0042] In some embodiments, the methods of this disclosure are used to treat patients with MRD-positive disease. Minimal residual disease (MRD) refers to a small number of cancer cells remaining in a patient's body during or after treatment, where the patient may or may not exhibit symptoms or signs of the disease. Such residual cancer cells often lead to disease recurrence if not eliminated. This disclosure includes methods for inhibiting and / or eliminating residual cancer cells in a patient during an MRD test. MRD can be determined according to methods known in the art, such as MRD flow cytometry. According to this aspect of the disclosure, the method comprises administering a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof, optionally in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, to a subject in need.

[0043] According to certain embodiments, the method of this disclosure includes administering to a subject a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof, optionally in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, and optionally a third therapeutic agent or therapy. The third therapeutic agent or therapy can be a drug or therapy selected from the group consisting of: for example, reflex, 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 (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, cytotoxins, chemotherapy 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 nonsteroidal anti-inflammatory drugs, and dietary supplements such as antioxidants. In some embodiments, antibodies may be administered in combination with therapies including chemotherapy, radiation, and surgery. As used herein, the phrase “in combination with” means administering one or more antibodies to a subject concurrently with, before, or after administration of a third therapeutic agent. In some embodiments, one or more antibodies and a third therapeutic agent are administered in separate formulations.

[0044] In some embodiments, the method of this disclosure includes administering a therapeutically effective amount of a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof, optionally in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, to a subject in need. In some embodiments, administration of the bispecific antibody or optional combination results in tumor growth inhibition of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, compared to an untreated subject. In some embodiments, administration of the bispecific antibody or optional combination results in increased tumor regression, tumor shrinkage, and / or disappearance. In some embodiments, administration of the bispecific antibody or optional combination results in delayed tumor growth and development, for example, tumor growth may be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years, compared to an untreated subject. In some embodiments, administration of bispecific antibodies or optional combinations prevents tumor recurrence and / or prolongs the survival of subjects, for example, by extending the duration of survival by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months compared to untreated subjects. In some embodiments, administration of bispecific antibodies or optional combinations prolongs progression-free survival or overall survival. In some embodiments, administration of bispecific antibodies or optional combinations increases the subject's response and the duration of the response, for example, 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% compared to untreated subjects. In some embodiments, administration of bispecific antibodies or optional combinations to patients with ccRCC results in the complete disappearance of all evidence of tumor cells (“complete response”). In some embodiments, administration of bispecific antibodies or optionally combinations thereof to a patient with ccRCC results in a reduction of tumor cells or tumor size by at least 30% or more (“partial response”). In some embodiments, administration of bispecific antibodies or optionally combinations thereof to a patient with ccRCC results in the complete or partial disappearance of tumor cells / lesions, including new measurable lesions. Tumor shrinkage 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), cytological, histological, or molecular genetic analysis.

[0045] In some cases, a subject's response to treatment 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 < 10 mm (< 1 cm). PR is defined as a reduction of at least 30% in the sum of the diameters of the target lesions, relative to the baseline sum. PD is defined as an increase of at least 20% in the sum of the diameters of the target lesions, relative to the minimum sum in the study (including the baseline sum if it is the minimum in the study). In addition to a relative increase of 20%, the sum must also indicate an absolute increase of at least 5 mm (0.5 cm). (Note: The appearance of one or more new lesions is also considered progression). SD is defined as a contraction that is neither sufficient to qualify as PR nor sufficient to qualify as PD, relative to the minimum sum of the diameters in the study.

[0046] Bispecific anti-PSMA x anti-CD28 antibody

[0047] According to certain exemplary embodiments of this disclosure, the method includes administering a therapeutically effective amount of a bispecific antibody that specifically binds to CD28 and PSMA or their antigen-binding fragments. Such antibodies and fragments may be referred to herein as, for example, "anti-PSMA / anti-CD28," or "anti-PSMA x CD28," or "PSMA x CD28" bispecific antibody or its antigen-binding fragment, or other similar terms.

[0048] As used herein, the expression "bispecific antibody" refers to an immunoglobulin containing at least a first antigen-binding domain and a second antigen-binding domain. In the context of this 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 unique antigen (e.g., CD28). Each antigen-binding domain of a bispecific antibody contains a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), and each domain contains three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen-binding domain may be designated with the prefix "A," and the CDRs of the second antigen-binding domain may be designated with the prefix "B." Thus, the CDRs of the first antigen-binding domain may be referred to herein as A-HCDR1, A-HCDR2, and A-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as B-HCDR1, B-HCDR2, and B-HCDR3.

[0049] The first antigen-binding domain and the second antigen-binding domain are each linked to a separate multimerizing domain. As used herein, a "multimerizing domain" means any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to bind to a second multimerizing domain of the same or similar structure or composition. In the context of this disclosure, the multimerizing component is the Fc portion of an immunoglobulin (containing C... H2 -C H3 (domains), for example, Fc domains selected from isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype of IgG within each isotype group.

[0050] The bispecific antibodies disclosed herein typically comprise two multimerizing domains, for example, two Fc domains that are each independently part of a separate antibody heavy chain. The first and second multimerizing domains can be the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerizing domains can be different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0051] Any bispecific antibody format or technique can be used to prepare the bispecific antibodies of this disclosure. For example, an antibody or antibody fragment having first antigen binding specificity can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to another molecular entity, such as an antibody or antibody fragment having second antigen binding specificity, to produce a bispecific antibody. Specific exemplary bispecific formats that may be used in the context of this disclosure include, but are not limited to, the following: for example, scFv-based or biantibody bispecific formats, IgG-scFv fusion, dual variable domain (DVD)-Ig, quadroma, knocks-into-holes, common light chains (e.g., common light chains with knocks-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab2 bispecific formats (see, for example, Klein et al. 2012 Monoclonal Antibodies (mAbs) 4:6, 1-11, and the references cited therein for a review of the aforementioned formats).

[0052] In the context of bispecific antibodies disclosed herein, the Fc domain may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version. For example, this disclosure includes bispecific antibodies that contain one or more modifications to the Fc domain, resulting in a modified Fc domain having a modified binding interaction (e.g., enhanced or weakened) between Fc and FcRn. In one embodiment, the bispecific antibody comprises C H2 Or C H3 Modifications to the Fc domain that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with a pH range of 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 by reference in its entirety.

[0053] This disclosure also includes bispecific antibodies, said bispecific antibodies comprising a first C H 3 structural domains and second Ig C H 3 structural domains, where the first and second IgC H The three domains differ from each other by at least one amino acid, and wherein, compared to a bispecific antibody lacking amino acid differences, at least one amino acid difference reduces the binding of the bispecific antibody to protein A. In one embodiment, the first Ig C H The 3-domain binds to protein A, and the second IgC... H Domain 3 contains mutations that reduce or eliminate protein A binding, such as H95R modification (via IMGT exon numbering; H435R, via EU numbering). Second C H 3 may further include the Y96F modification (via IMGT; Y436F, via EU). See, for example, U.S. Patent No. 8,586,713. It may be in the second C H The additional modifications found in 3 include: in the case of IgG1 antibody, D16E, L18M, N44S, K52N, V57M and V82I (via IMGT; D356E, L358M, N384S, K392N, V397M and V422I, via EU); in the case of IgG2 antibody, N44S, K52N and V82I (IMGT; N384S, K392N and V422I, via EU); and in the case of IgG4 antibody, Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (via IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I, via EU).

[0054] In some embodiments, the Fc domain may be chimeric, with combinations derived from Fc sequences of more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise sequences derived from human IgG1, human IgG2, or human IgG4 C. H C in Zone 2 H 2. Part or all of the sequence, and C derived from human IgG1, human IgG2 or human IgG4 H 3. Part or all of the sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may contain an “upper hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, which combines with a “lower hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. Specific examples of chimeric Fc domains that may be included in any antibody described herein contain, from the N-terminus to the C-terminus: [IgG4 C 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 antibody described herein comprises, from the N-terminus to the C-terminus: [IgG1C 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 may be included in any antibody of the present disclosure are described in U.S. Patent Publication No. 20140243504, which is incorporated herein by reference in its entirety. Chimeric Fc domains and chimeric heavy chain constant regions having these general structural arrangements, and their variants, may have altered Fc receptor binding, which in turn affects Fc effector function.

[0055] According to certain exemplary embodiments of this disclosure, a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment 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) containing any amino acid sequence of the amino acid sequence of a bispecific anti-PSMA / anti-CD28 antibody as set forth in WO 2019 / 246514. In some exemplary embodiments, a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment that can be used in the context of the methods of this disclosure comprises: (a) a first antigen-binding arm that specifically binds to PSMA, the first antigen-binding arm comprising a heavy chain complementarity-determining region (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) and a light chain complementarity-determining region (A-LCVR) of a light chain variable region (A-LCVR) (A-LCDR1, A-LCDR2, and A-LCDR3), wherein the A-HCVR comprises the amino acid sequence of SEQ ID NO: 1, and the A-LCVR comprises the amino acid sequence of SEQ ID NO: 1. (a) the amino acid sequence of SEQ ID NO: 9; and (b) a second antigen-binding arm that specifically binds to CD28, the second antigen-binding arm comprising the heavy chain CDRs (B-HCDR1, B-HCDR2 and B-HCDR3) of HCVR (B-HCVR) and the light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of LCVR (B-LCVR), wherein the HCVR (B-HCVR) comprises the amino acid sequence of SEQ ID NO: 5 and the LCVR (B-LCVR) comprises 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 another embodiment, the bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment comprises: (a) a first antigen-binding arm comprising HCVR (A-HCVR) and LCVR (A-LCVR), wherein HCVR (A-HCVR) comprises SEQ ID NO: 1 and LCVR (A-LCVR) comprises SEQ ID NO: 9; and (b) a second antigen-binding arm comprising HCVR (B-HCVR) and LCVR (B-LCVR), wherein HCVR (B-HCVR) comprises SEQ ID NO: 5 and HCVR (B-HCVR) comprises SEQ ID NO: 9. In some exemplary embodiments, the bispecific anti-PSMA x CD28 antibody comprises: a PSMA binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and the light chain comprising the amino acid sequence of SEQ ID NO: 15; and a CD28 binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and the light chain comprising the amino acid sequence of SEQ ID NO: 15.

[0056] In some exemplary embodiments, a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment that can be used in the context of the methods of this disclosure comprises: (a) a first antigen-binding arm that specifically binds to PSMA, the first antigen-binding arm comprising a heavy chain complementarity-determining region (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) and a light chain complementarity-determining region (A-LCVR) of a light chain variable region (A-LCVR), wherein the A-HCVR comprises the amino acid sequence of SEQ ID NO: 16, and the A-LCVR comprises the amino acid sequence of SEQ ID NO: 16. (a) the amino acid sequence of CD28; and (b) a second antigen-binding arm that specifically binds to CD28, the second antigen-binding arm comprising the heavy chain CDRs (B-HCDR1, B-HCDR2 and B-HCDR3) of HCVR (B-HCVR) and the light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of LCVR (B-LCVR), wherein the HCVR (B-HCVR) comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 24, and the LCVR (B-LCVR) comprises the amino acid sequence of SEQ ID NO: 28. According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 17; A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 18; A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 19; A-LCDR1 comprises the amino acid sequence of SEQ ID NO: 29; A-LCDR2 comprises the amino acid sequence of SEQ ID NO: 30; A-LCDR3 comprises the amino acid sequence of SEQ ID NO: 31; B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25; B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 26; 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 another embodiment, the bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment comprises: (a) a first antigen-binding arm comprising HCVR (A-HCVR) and LCVR (A-LCVR), wherein HCVR (A-HCVR) comprises SEQ ID NO: 16 and LCVR (A-LCVR) comprises SEQ ID NO: 28; and (b) a second antigen-binding arm comprising HCVR (B-HCVR) and LCVR (B-LCVR), wherein HCVR (B-HCVR) comprises SEQ ID NO: 20 or SEQ ID NO: 24 and HCVR (B-HCVR) comprises SEQ ID NO: 28. In some exemplary embodiments, the bispecific anti-PSMA x CD28 antibody comprises: a PSMA-binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and the light chain comprising the amino acid sequence of SEQ ID NO: 35; and a CD28-binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and the light chain comprising the amino acid sequence of SEQ ID NO: 35. In some exemplary embodiments, the bispecific anti-PSMA x CD28 antibody comprises: a PSMA-binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and the light chain comprising the amino acid sequence of SEQ ID NO: 35; and a CD28-binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and the light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0057] The methods disclosed herein also cover bioequivalents of the bispecific antibodies discussed herein. As used herein, the term "bioequivalent" refers to an antibody or fragment thereof that, under similar experimental conditions, whether administered as a single or multiple dose at the same molar dose, shows no significant difference in absorption rate and / or absorption extent compared to the bispecific antibodies discussed above. In the context of this disclosure, the term refers to an antigen-binding protein that binds to PSMA and CD28 and has no clinically significant differences in safety, purity, and / or potency compared to the bispecific antibodies discussed herein.

[0058] Anti-PD-1 antibody and its antigen-binding fragment

[0059] According to certain exemplary embodiments of this disclosure, the method comprises administering a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to PD-1. As used herein, the term "antibody" includes an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, and a polymer thereof (e.g., IgM). In typical antibodies, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy-chain constant region contains three structural domains C. H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L ) and the light chain constant region. The light chain constant region contains a structural domain (C L 1). V can be... H District and V L The region is further subdivided into highly variable regions known as complementary determinant regions (CDRs), interspersed with more conservative regions known as frame regions (FRs). Each V H and V L It consists 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 different embodiments of this disclosure, the FRs of the anti-IL-4R antibody (or its antigen-binding portion) may be identical to the human germline sequence or may be natural or artificially modified. The common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0060] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody can be derived from the whole antibody molecule, for example, using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering, which involves the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into suitable conformations, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0061] 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) the smallest recognition unit consisting of amino acid residues of a hypervariable region of a mimic antibody (e.g., a separated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variant IgNAR domains, are also included in the term "antigen-binding fragments" as used herein.

[0062] Antibody antigen-binding fragments will typically contain at least one variable domain. Variable domains can have any size or amino acid composition and will typically contain at least one CDR adjacent to or within one or more frame sequences. L V associated with the structural domain H In the antigen-binding fragment of the domain, V H and V L Domains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimers and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0063] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of the variable and constant domains that may be found in the antigen-binding fragments of the antibodies of this 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 H2-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 the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or connected via full-length or partial hinge or connector regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, which create flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may comprise the domains listed above and / or one or more monomers V. H or V L Homodimers or heterodimers (or other polymers) of any variable and constant domain configurations in which the domains (e.g., via disulfide bonds) are non-covalently associated.

[0064] The antibodies used in the methods of this disclosure may be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, the disclosed human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in the CDR and specifically in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence.

[0065] The antibodies used in the methods of this disclosure may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells (further described below), antibodies isolated from a recombinant combined human antibody library (further described below), antibodies isolated from animals (e.g., mice) that are transgenic for the human immunoglobulin gene (see, for example, Taylor et al. (1992) Nucleic Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence onto another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody is... H District and V L The amino acid sequence of the region is as follows: Although it originates from human lineage V H Sequence and V L The sequence is related to it, but it may not be naturally present in human antibody germline libraries.

[0066] According to certain embodiments, the antibodies used in the methods of this disclosure specifically bind to PD-1. The term "specific binding," etc., means that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used in the context of this disclosure, antibodies that specifically bind to "PD-1" include those with Kc concentrations 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 Antibodies that bind to PD-1 or a portion thereof, as measured in surface plasmon resonance assays. However, isolated antibodies that specifically bind to human PD-1 may be cross-reactive with other antigens, such as PD-1 molecules from other (non-human) species.

[0067] According to certain exemplary embodiments of this disclosure, an anti-PD-1 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR), which comprises any amino acid sequence as set forth in the amino acid sequence of an anti-PD-1 antibody as described in U.S. Patent No. 9,987,500. In certain exemplary embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof that may be used in the context of the methods of this disclosure comprises: a heavy chain complementarity-determining region (HCDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 36; and a light chain complementarity-determining region (LCDR) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 40. According to some embodiments, an anti-PD-1 antibody or its antigen-binding fragment comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 37; HCDR2 comprises the amino acid sequence of SEQ ID NO: 38; HCDR3 comprises the amino acid sequence of SEQ ID NO: 39; LCDR1 comprises the amino acid sequence of SEQ ID NO: 41; LCDR2 comprises the amino acid sequence of SEQ ID NO: 42; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 43. In yet other embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises: HCVR, which comprises SEQ ID NO: 36; and LCVR, which comprises SEQ ID NO: 40. In some embodiments, the method of this disclosure comprises using an anti-PD-1 antibody, wherein the antibody comprises a heavy chain, which comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-PD-1 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 45. An exemplary antibody comprising an HCVR including the amino acid sequence of SEQ ID NO: 36 and an LCVR including the amino acid sequence of SEQ ID NO: 40 is a fully human anti-PD-1 antibody known as REGN2810 (also known as cimiprimab, LIBTAYO). ®According to certain exemplary embodiments, the methods of this disclosure include the use of REGN2810 or its bioequivalence. As used herein, the term "bioequivalence" refers to a pharmaceutical equivalent or substitute for an anti-PD-1 antibody or PD-1 binding protein or fragment thereof, which, when administered at the same molar dose under similar experimental conditions, whether as a single or multiple dose, does not show a significant difference in absorption rate and / or absorption rate compared to REGN2810. In the context of this disclosure, the term refers to an antigen-binding protein that binds to PD-1 and has no clinically significant difference from REGN2810 in terms of safety, purity, and / or potency.

[0068] Other anti-PD-1 antibodies that may be used in the context of the methods disclosed herein include, for example, antibodies mentioned in the art and referred to as nivolumab (US Patent No. 8,008,449), pembrolizumab (US Patent No. 8,354,509), MEDI0608 (US Patent No. 8,609,089), pidilizumab (US Patent No. 8,686,119), or any anti-PD-1 antibody as set forth in US Patent Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0069] Anti-PD-1 antibodies used in the context of the methods of this disclosure may have pH-dependent binding properties. For example, PD-1 antibodies used in the methods of this disclosure may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, anti-PD-1 antibodies of this disclosure may exhibit enhanced binding to their antigen 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 smaller. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0070] In some cases, "the binding of antibody to PD-1 is reduced at acidic pH compared to neutral pH" refers to the fact that antibody binding to PD-1 is reduced at acidic pH. D Value and K value of antibody binding to PD-1 at neutral pH DThis is expressed in terms of the ratio of values ​​(or vice versa). For example, if the antibody or its antigen-binding fragment exhibits an acidic / neutral K value of approximately 3.0 or greater. D The ratio, for the purposes of this disclosure, can be considered as the antibody or its antigen-binding fragment exhibiting "reduced binding to PD-1 at acidic pH compared to neutral pH." In some exemplary embodiments, the acidic / neutral K of the antibody or antigen-binding fragment of this disclosure... D The ratio can be approximately 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 greater.

[0071] Antibodies exhibiting pH-dependent binding properties can be obtained, for example, by screening for antibody populations that show reduced (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. Alternatively, modifications to the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids of the antigen-binding domain (e.g., within the CDR) with histidine residues, antibodies exhibiting reduced binding to antigens at acidic pH relative to neutral pH can be obtained. As used herein, the expression "acidic pH" means a pH of 6.0 or less.

[0072] Combination therapy

[0073] According to some embodiments, the methods of this disclosure include administering to a subject a combination of an anti-PSMA / anti-CD28 bispecific antibody or an antigen-binding fragment thereof with an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, the methods of this disclosure include administering an antibody for additive or synergistic activity to treat PSMA-expressing cancers, preferably ccRCC. In some embodiments, the combination of an anti-PSMA x CD28 bispecific antibody (e.g., mAb1) and an anti-PD-1 antibody (e.g., cimiprimab) produces a synergistic therapeutic effect in the treatment of metastatic clear cell renal cell carcinoma. As used herein, the expression “bind to” means that the anti-PSMA / anti-CD28 bispecific antibody or an antigen-binding fragment thereof is administered before, after, or simultaneously with the anti-PD-1 antibody or an antigen-binding fragment thereof. The term “bind to” also includes sequentially or simultaneously administering 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" a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment may be administered more than 72 hours, approximately 72 hours, approximately 60 hours, approximately 48 hours, approximately 36 hours, approximately 24 hours, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, approximately 1 hour, or approximately 30 minutes before administration of the bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment. When administered "after" a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment may be administered approximately 30 minutes, approximately 1 hour, approximately 2 hours, approximately 4 hours, approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 60 hours, approximately 72 hours, or more than 72 hours after administration of the bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment. "Simultaneous" administration with bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment means that within 30 minutes of (before, after, or simultaneously with) administration of bispecific anti-PSMA / anti-CD28 antibody or its antibody-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment is administered to the subject in a single dosage form or as a single combined dose formulation comprising both the anti-PD-1 antibody or its antigen-binding fragment and the bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment.

[0074] In some embodiments, the methods of this disclosure include administration of a second therapeutic agent (i.e., when a bispecific antibody is administered without the administration of an anti-PD-1 antibody) or a third therapeutic agent (i.e., when a combination of a bispecific antibody and an anti-PD-1 antibody is administered), wherein the second or third therapeutic agent is an anticancer drug or anticancer therapy. In some embodiments, the methods of this disclosure include administration of an anti-PSMA / anti-CD28 bispecific antibody or an antigen-binding fragment thereof, and optionally an anti-PD-1 antibody or an antigen-binding fragment thereof, in combination with radiotherapy, surgery, or other anticancer therapies to produce a long-lasting antitumor response and / or prolong the survival of patients with PSMA-expressing cancers.

[0075] In some embodiments, the methods disclosed herein include administering radiotherapy before, simultaneously with, or after administering a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment thereof and optionally an anti-PD-1 antibody or its antibody-binding fragment thereof to a cancer patient. For example, radiotherapy may be administered to the tumor lesion at one or more doses after administering one or more doses of the antibody. In some embodiments, local radiotherapy may be administered to the tumor lesion after systemic administration of a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment thereof and optionally an anti-PD-1 antibody or its antigen-binding fragment thereof to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablation radiation).

[0076] Efficacy and monitoring

[0077] The methods discussed in this disclosure may further include tumor biopsy, imaging, and cytokine release syndrome (CRS) monitoring and management to assess efficacy and safety in individual subjects or subject populations.

[0078] Tumor biopsy

[0079] As discussed in this article, patients with soft tissue disease may undergo core or excisional biopsies of the soft tissue lesion if clinically accessible during screening and / or treatment. For patients without clinically accessible soft tissue disease, bone biopsies may be performed if feasible. In addition to clinical diagnostic uses, any available tissue from samples collected at different time points (e.g., formalin-fixed paraffin-embedded or preserved in blocks for molecular extraction), as well as archival specimens from previous treatments, can be used for biomarker assays. Specifically, these samples can be evaluated using in situ imaging employing probes targeting gene targets associated with REGN5678 (PSMA, CD28) and optionally decimiprimab (PD-L1), as well as biomarkers of immune activation, suppression, and function, and tumor cell phenotype.

[0080] Tumor tissue biopsies, if available, can also be used for gene expression profiling (using RNA sequencing or other methods) as a measure of complex tumor microenvironment phenotypes, whole-exome sequencing, or other mutation profiling, as well as for targeted studies of gene variants (tumor mutations), such as those affecting DNA repair pathways. They can also be analyzed using next-generation T-cell receptor library sequencing as a measure of tumor-associated T-cell clonal proliferation.

[0081] Imaging

[0082] Prostate-specific membrane antigen (PSMA) PET / CT has been shown to provide a sensitive measure of PSMA expression and tumor burden in cancer patients expressing PSMA. It allows for the detection of tumor lesions and improves the effectiveness of tumor response assessment and treatment strategy decision-making.

[0083] CRS monitoring and management

[0084] Cytokine release from superagonist anti-CD28 bivalent antibodies, bispecific antibodies, and similar molecules has been observed. Cytokine release syndrome (CRS) typically presents clinically during or within hours to days after infusion. In a clinical study of six patients treated with a bivalent anti-CD28 superagonist antibody (TGN1412), life-threatening acute CRS occurred, with patients becoming critically ill within 12 to 16 hours. Previous experience with bispecific antibodies targeting tumor antigens and CD3 has shown that when CRS occurs, these events are most prominent after the first 1 or 2 weeks of treatment and are generally transient, even with higher doses administered in subsequent weeks. This has also been observed in combination with cimipril. CRS generally occurs more frequently during the first 2 weeks of treatment in any given patient and decreases in frequency with subsequent exposures. Based on these findings, the risk of initial CRS onset at the third dose or later is considered low.

[0085] 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 well-tolerated in B-cell tumors, with no serious CRS events (no grade ≥ 3 CRS). In cynomolgus monkeys, subcutaneous administration of the same bispecific antibody produced lower CRS rates compared to intraperitoneal administration. max Delayed T max and lower plasma cytokine levels. The methods discussed herein may include measures to address potential safety concerns arising from cytokine release, including:

[0086] (1) Cytokine monitoring;

[0087] (2) Management of CRS using anti-IL-6 pathway therapy (e.g., thalidomide or tocilizumab) and corticosteroids; and

[0088] (3) In the event of observed CRS, the prescribed administration of pre-drug administration and the use of a lower dose at the initial dosing visit shall be made before the dose level (DL) is increased to the full dose.

[0089] Patients presenting with symptoms consistent with severe CRS, including but not limited to persistent fever, neurological disturbances (including altered mental status, lethargy, and seizures), signs of clinical toxicity (hypotension, requiring at least one IV vasopressor, or hypoxia [PO2 < 90%]), may be considered for pharmacological intervention using anti-IL-6 pathway therapy (e.g., thalidomide or tocilizumab) and / or high-dose steroids. This disclosure considers such additions to the methods discussed herein.

[0090] Corticosteroids can also be used to manage CRS, specifically in cases with neurological symptoms. Generally, corticosteroids should be used when: 1) IRR / CRS does not respond adequately to anti-IL-6 pathway therapy (e.g., thalidomide or tocilizumab), or 2) anti-IL-6 pathway therapy is not in the best interest of the patient.

[0091] Pharmaceutical composition and administration

[0092] This disclosure includes methods comprising administering to a subject a bispecific anti-PSMA / anti-CD28 antibody or an antigen-binding fragment thereof, optionally in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein one or more antibodies (or fragments) are contained in a single or combined (single) pharmaceutical composition. The pharmaceutical compositions of this disclosure can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerability, etc. Many suitable formulations can be found in all formularies known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and vesicle-containing lipids (cationic or anionic) (such as LIPOFECTIN). TMDNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbon wax emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon wax. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm Sci Technol 52:238-311.

[0093] Various delivery systems are known and can be used to administer the pharmaceutical compositions disclosed herein, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus injection, or by injection, and can be administered in combination with other bioactive agents.

[0094] The disclosed pharmaceutical compositions can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, pen delivery devices are readily applicable for delivering the disclosed pharmaceutical compositions. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition in the cartridge has been administered and the cartridge is 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 disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0095] Many reusable pen delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN™ (OwenMumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, and OPTIPEN. STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany) are just a few examples. Examples of disposable pen delivery devices used for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), KWIKPEN™ (Eli Lilly), and SURECLICK. TM Automatic injectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA) TM The pen (Abbott Labs, Abbott Park IL) is just a few examples.

[0096] In some cases, drug compositions can be delivered in controlled-release systems. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used; see *Medical Applications of Controlled Release*, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, *Medical Applications of Controlled Release*, ibid., Vol. 2, pp. 115–138). Other controlled-release systems are discussed in the comments of Langer, 1990, *Science* 249:1527–1533.

[0097] Injectable formulations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying, for example, the antibody described above or its salts in a sterile aqueous or oily medium conventionally used for injection. As an aqueous medium for injection, examples include physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with a suitable solubilizer. The injection prepared in this way is preferably contained in a suitable ampoule.

[0098] Advantageously, the pharmaceutical compositions described above for use are prepared into dosage forms in unit doses suitable for containing a specific amount of the active ingredient. Such unit-dose dosage forms include, for example, vials or pre-filled syringes.

[0099] Application plan

[0100] This disclosure includes a method comprising administering to a subject a bispecific anti-PSMA x CD28 antibody or an 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 as low as possible to achieve a therapeutic response, optionally in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0101] According to certain embodiments of this disclosure, multiple doses of a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment thereof, optionally in combination with an anti-PD-1 antibody or its antigen-binding fragment, can be administered to a subject over a defined time period. A method according to this aspect of the disclosure comprises administering one or more doses of a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment thereof to a subject, optionally sequentially together with one or more doses of an anti-PD-1 antibody or its antigen-binding fragment thereof. As used herein, “sequentially administer” means administering each dose of antibody to the subject at different time points, such as on different dates spaced apart by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes a method comprising sequentially administering a single initial dose of antibody (or fragment) to a patient, followed by one or more second doses of antibody (or fragment), and optionally then administering one or more third doses of antibody (or fragment).

[0102] The terms "initial dose," "second dose," and "third dose" refer to the order in which they are administered. Thus, the "initial dose" is the dose administered at the start of the treatment regimen (also known as the "baseline dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of antibody or its antigen-binding fragment (e.g., a bispecific antibody). However, in some embodiments, the amounts contained in the initial, second, and / or third doses differ from each other during the treatment process (e.g., appropriately increased or decreased). In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of the treatment regimen, followed by subsequent doses administered at a lower frequency (e.g., a "maintenance dose").

[0103] In one exemplary embodiment of this disclosure, each second and / or third dose is administered 1 / 2 week to 14 weeks or longer immediately following the previous dose (e.g., 1 / 2 week, 1 week, 1 1 / 2 weeks, 2 weeks, 2 1 / 2 weeks, 3 weeks, 3 1 / 2 weeks, 4 weeks, 4 1 / 2 weeks, 5 weeks, 5 1 / 2 weeks, 6 weeks, 6 1 / 2 weeks, 7 weeks, 7 1 / 2 weeks, 8 weeks, 8 1 / 2 weeks, 9 weeks, 9 1 / 2 weeks, 10 weeks, 10 weeks, 11 weeks, 11 weeks, 12 weeks, 12 weeks, 13 weeks, 13 weeks, 14 weeks, 14 weeks or longer). As used herein, the phrase “immediately following the previous dose” means, in a sequence of multiple administrations, the dose of bispecific anti-PSMA / anti-CD28 or its antigen-binding fragment (and optionally anti-PD-1 antibody or its antigen-binding fragment) administered to the patient before the administration of the next immediately following dose in the sequence, without intermediate doses.

[0104] The method according to this aspect of the disclosure may include administering any number of second and / or third doses of a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment thereof to a patient, and optionally an anti-PD-1 antibody or its antigen-binding fragment thereof. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient.

[0105] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient one, two, or three weeks (e.g., one or three weeks) immediately following the previous dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient one to four weeks (e.g., one or three weeks) immediately following the previous dose. Alternatively, the frequency of administration of the second and / or third doses to the patient may vary during the treatment regimen. The physician may also adjust the administration frequency during the treatment process, depending on the individual patient's needs following a clinical examination.

[0106] In some embodiments, at the start of the treatment regimen, one or more doses of a bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment, along with optionally an anti-PD-1 antibody or its antigen-binding fragment, are administered at a higher frequency (twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks) as an “induction dose,” followed by subsequent doses (“consolidation dose” or “maintenance dose”) at the same or lower frequency (e.g., once every 4–12 weeks).

[0107] This disclosure includes a method comprising administering a bispecific antibody or its antigen-binding fragment at a dose of at least 30 mg once weekly (QW) to an eligible subject for at least three weeks. In various embodiments, the monotherapy regimen may last from three weeks to 36 weeks or longer. In some cases, the duration of the monotherapy regimen is at least four weeks, at least five weeks, or at least six weeks. In some cases, the monotherapy regimen is for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 weeks. In some cases, the method further comprises administering an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 100 mg every three weeks (Q3W). In some cases, the method further comprises administering an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 350 mg every three weeks (Q3W). In some cases, the method further comprises administering an antibody or an antigen-binding fragment thereof that specifically binds to programmed death receptor-1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 1000 mg (e.g., Q3W, Q4W, Q5W, or Q6W). In some cases, administration of the anti-PD-1 antibody or its antigen-binding fragment is performed after a single-therapy regimen.

[0108] This disclosure also includes a method comprising administering to a 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), wherein the bispecific antibody is administered to the subject at a dose of at least 30 mg once weekly (QW) for at least three weeks. In some cases, the bispecific antibody is administered as a monotherapy during a lead regimen lasting at least three, four, five, or six weeks. In some cases, the lead regimen is for or for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 weeks.

[0109] dose

[0110] The amount of bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment thereof, and optionally anti-PD-1 antibody or its antigen-binding fragment thereof, administered to a subject according to the methods of this disclosure is generally a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means an amount of antibody (bispecific anti-PSMA / anti-CD28 antibody and optionally anti-PD-1 antibody) or its antigen-binding fragment thereof that causes one or more of the following: (a) reducing the severity or duration of symptoms of cancer (e.g., ccRCC); (b) inhibiting tumor growth or increasing tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delaying tumor growth and development; (d) inhibiting, delaying, or stopping tumor metastasis; (e) preventing tumor growth and recurrence; (f) prolonging the survival of patients with cancer (e.g., ccRCC); and / or (g) reducing the use or need for conventional anticancer therapies (e.g., reducing or eliminating the use of chemotherapy agents or cytotoxic agents) compared to untreated subjects.

[0111] In the case of bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment, the therapeutically effective dose can be from about 0.3 mg to about 2000 mg, for example, 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 bispecific anti-PSMA / anti-CD28 antibody or its antigen-binding fragment. In some embodiments, a subject is given 30 mg, 100 mg, 300 mg, 600 mg, or 900 mg of a bispecific anti-PSMA x anti-CD28 antibody or its antigen-binding fragment (e.g., once weekly or once every three weeks) to treat PSMA-expressing cancers or PSMA-expressing clear cell renal cell carcinoma (e.g., metastatic ccRCC).

[0112] In the case of an anti-PD-1 antibody or its antigen-binding fragment, the effective therapeutic dose can be from about 3 mg to about 100 mg (when following a monotherapy regimen of a bispecific antibody). Therefore, in some embodiments, the effective dose of the anti-PD-1 antibody (or fragment) can be about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg. In some embodiments, a combination of 3 mg to 99 mg of an anti-PD-1 antibody or its antigen-binding fragment (e.g., every three weeks) with a bispecific antibody or its antigen-binding fragment is administered to the subject to treat PSMA-expressing cancers or PSMA-expressing clear cell renal cell carcinoma (e.g., metastatic ccRCC). In some embodiments, a subject is given 300 mg to 400 mg or 650 mg to 750 mg of an anti-D-1 antibody or its antigen-binding fragment (e.g., every three weeks or every six weeks) to treat PSMA-expressing cancers or PSMA-expressing clear cell renal cell carcinoma (e.g., metastatic ccRCC). In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment may be administered at a dose of about 3 mg to about 1000 mg.Therefore, in some embodiments, the effective amount of the anti-PD-1 antibody (or fragment) can be approximately 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, etc. mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg or 1000 mg (e.g., administered once every three weeks or once every six weeks).

[0113] Table 1 below shows a summary of the sequences cited in this article and their corresponding SEQ ID NOs.

[0114] Table 1: Summary of Sequences

[0115]

[0116]

[0117] Example

[0118] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the methods and compositions of this disclosure, 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 figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should still be taken into account. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0119] Example 1: Generation of bispecific antibodies binding to prostate-specific membrane antigen (PSMA) and CD28

[0120] Bispecific antibodies comprising an anti-PSMA-specific binding domain and an anti-CD28-specific binding domain are constructed using standard methods, wherein each of the anti-PSMA antigen-binding domain and the anti-CD28 antigen-binding domain contains a different unique HCVR that pairs with a common LCVR. In some cases, bispecific antibodies are constructed using the heavy chain from the anti-CD28 antibody, the heavy chain from the anti-PSMA antibody, and the common light chain (see Table 2).

[0121] The bispecific antibody generated according to an example of the invention comprises two separate antigen-binding domains (i.e., binding arms). The first antigen-binding domain comprises a heavy chain variable region (“CD28-VH”) derived from the anti-CD28 antibody, and the second antigen-binding domain comprises a heavy chain variable region (“PSMA-VH”) derived from the anti-PSMA antibody. Both anti-PSMA and anti-CD28 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.

[0122] Table 2 summarizes the components of the antigen-binding domains of the various constructed anti-PSMAxCD28 bispecific antibodies. The corresponding CDR sequences and full-length heavy and light chain sequences are identified in Table 1 (refer to the "-001", "-002", and "-003" bispecific antibodies in Table 2).

[0123] Table 2: Summary of the components of the anti-PSMAxCD28 bispecific antibody

[0124]

[0125] Example 2: A Phase 1 / 2 Study of Bispecific Anti-PSMA x Anti-CD28 Antibody in Patients with Clear Cell Renal Cell Carcinoma

[0126] This is an open-label, phase 1 / 2, first-in-human study evaluating the safety, tolerability, pharmacokinetics (PK), and antitumor activity of mAb1 (REGN5678) in therapeutically experienced clear cell renal cell carcinoma (ccRCC).

[0127] Research Objectives

[0128] The main objective of the research is:

[0129] ●Evaluate the safety, tolerability, and pharmacokinetics (PK) of mAb1 alone and in combination with cimiprimab (dose escalation).

[0130] ●According to the RESIST criteria 1.1 for evaluating response to solid tumors, the efficacy of mAb1 alone and in combination with cimipril (dose extension) was assessed using objective response rate (ORR) measurements.

[0131] The secondary objective of the research is:

[0132] ●Efficacy of mAb1 alone and in combination with cimiprimab (dose escalation and dose extension) was evaluated according to RESIST 1.1 criteria, such as by ORR measurement.

[0133] ● Characterize the safety features of each extended group (dose extension).

[0134] ●Characteristics of mAb1 alone and PK in combination with cimiprimab (dose extension)

[0135] ●Assess the immunogenicity of mAb1 (dose escalation and dose expansion).

[0136] Research Design

[0137] This is an open-label, phase 1 / 2, first-in-human study evaluating the safety, tolerability, pharmacokinetic (PK), and antitumor activity of mAb1 (anti-PSMAxCD28) in treated, clear cell renal cell carcinoma (ccRCC). Once disease progression occurs, a low-dose cimiprimab (3 mg IV Q3W) can be added after at least 6 weeks of mAb1 monotherapy in both the dose-escalation and dose-extension groups. The study is divided into two parts: dose-escalation and dose-extension.

[0138] During the dose escalation period, patients will receive up to 900 mg intravenous (IV) doses of monotherapy mAb1 once a week (QW).

[0139] During dose extension, participants will receive mAb1 with MTD / estimated RP2D.

[0140] Study duration

[0141] The total duration of each patient's participation in the study will vary depending on the occurrence of one or more of the following: disease progression, intolerable adverse events (AEs), withdrawal of consent, or meeting of study withdrawal criteria. For participants in the dose escalation and expansion cohorts, the study consists of four time periods: a screening period of up to 28 days; a treatment period of mAb1 monotherapy consisting of a series of 6-week (42-day) treatment cycles; a combination therapy period of low-dose cimiprimab (3 mg IV Q3W) consisting of a series of 6-week (42-day) treatment cycles for participants with progressive disease after receiving at least 6 weeks of mAb1 monotherapy; and a follow-up period (90 days) for participants who discontinue therapy, followed by survival follow-up.

[0142] Increasing the dose of mAb1 will increase the QW from 30 mg to 900 mg (dose of 30 mg, 100 mg, 300 mg, 600 mg and 900 mg).

[0143] research group

[0144] The study population included men and women with ccRCC who had progressed during or after ≥1 line of prior systemic therapy approved in a metastatic setting. Prior therapy must have included anti-PD-1 / PD-L1 therapy and ipilimumab and / or a tyrosine kinase inhibitor.

[0145] Inclusion criteria: Patients must meet the following criteria to be eligible for inclusion in the study:

[0146] 1. Males and females aged 18 years and older.

[0147] 2. Renal cell carcinoma with clear cell components confirmed by histology or cytology.

[0148] 3. Metastatic clear cell renal cell carcinoma (ccRCC) with at least one measurable lesion diagnosed according to the Response Evaluation Criteria for Solid Tumors (RESIST) 1.1 criteria.

[0149] 4. Progression during or following ≥ first-line approved systemic therapy in a metastatic setting. Prior treatment must include anti-PD-1 / PD-L1 therapy and ipilimumab and / or a tyrosine kinase inhibitor.

[0150] 5. Able and willing to provide medical records or newly acquired tumor tissue. If medical records or fresh tissue are unavailable, a pathology report confirming the diagnosis may be submitted.

[0151] 6. Eastern Cooperative Oncology Group (ECOG) performance status is 0 or 1.

[0152] 7. Sufficient organ and bone marrow function as recorded below:

[0153] a. Hemoglobin ≥ 8.5 g / dL

[0154] b. Absolute neutrophil count ≥ 1.5 x 10⁻⁶ 9 / L

[0155] c. Platelet count ≥ 100 x 10 9 / L

[0156] 8. Serum creatinine ≤ 1.5 x ULN or estimated glomerular filtration rate > 50 mL / min / 1.73 m 2 24-hour urinary creatinine collection can replace the calculated creatinine clearance rate to meet eligibility criteria.

[0157] 9. Sufficient liver function:

[0158] a. Total bilirubin ≤ 1.5 x ULN (≤ 3 x ULN if the tumor involves the liver)

[0159] b. AST ≤ 2.5 x ULN (≤ 5 x ULN if the tumor involves the liver)

[0160] c. ALT ≤ 2.5 x ULN (≤ 5 x ULN if the tumor involves the liver)

[0161] d. Alkaline phosphatase (ALP) ≤ 2.5 x ULN (≤ 5 x ULN if the tumor involves the liver or bone)

[0162] Notice:

[0163] ● For patients with tumor-related liver involvement, if AST level ≥ 3 x ULN or ALT ≥ 3 x ULN and bilirubin level ≥ 2 x ULN, they will be excluded regardless of the above criteria.

[0164] ● Individuals with Gilbert's syndrome do not need to meet total bilirubin requirements, provided their total bilirubin levels are not higher than their historical levels. Gilbert's syndrome must be properly documented as part of their medical history.

[0165] 10. Willing and able to comply with outpatient visit and research procedures.

[0166] 11. Provide informed consent forms signed by the study patients.

[0167] 12. Able to understand and complete research-related questionnaires.

[0168] Exclusion criteria: Patients meeting any of the following criteria will be excluded from the study:

[0169] 1. Currently undergoing treatment under other research programs.

[0170] 2. Has participated in the study of the investigational drug or device within 4 weeks of receiving the first dose of the investigational therapy.

[0171] 3. Patients who have received approved systemic therapy (including sipuleucel-T) within 3 weeks of administration or have not yet recovered from any acute toxicity (i.e., grade ≤ 1 or baseline), except for laboratory changes as described in the inclusion criteria, and as follows:

[0172] a. Patients with neuropathy of grade 2 or lower

[0173] b. Hair loss

[0174] 4. Has received radiation therapy or surgery within 14 days of the first administration of the study drug, or has not recovered from an AE (i.e., grade ≤ 1 or baseline), in addition to laboratory changes as described in the inclusion criteria, and as follows:

[0175] a. Patients with neuropathy grade ≤ 2

[0176] 5. Patients who have received any prior systemic biological therapy within 5 half-lives of the first dose of the study therapy, except for anti-PD-1 therapy requiring clearance within 3 half-lives.

[0177] Exceptions: Patients who have previously received bevacizumab or other patients who have received non-immunomodulatory antibody therapy with a half-life of more than 7 days may be treated after discussion with the organizer.

[0178] 6. Has already received previous PSMA-targeted therapy

[0179] 7. Dose Escalation: Dose escalation is indicated for patients who have received prior anticancer immunotherapy (except ciproxetine-T) within 5 half-lives prior to study treatment. Examples of immunomodulatory agents include blockers of CTLA-4, 4-1BB (CD137), or OX-40, therapeutic vaccines, anti-PD-1 / PD-L1, phospholipase 3-kinase (PI3K)δ inhibitors, or cytokine-based anticancer therapies. Note: This excludes patients who have received investigational cell-based therapies (e.g., CAR-T cells). An exception is the approval of anti-PD-1 therapy after 3 half-lives of clearance.

[0180] 8. Dosage Expansion: Prior anticancer immunotherapy has been received. Examples of immunomodulatory agents include blockers of CTLA-4, 4-1BB (CD137), or OX-40, therapeutic vaccines, anti-PD-1 / PD-L1, PI3Kδ inhibitors, CAR-T cell or cytokine anticancer therapy. Note: Prior treatment with ciprofloxacin-T is permitted.

[0181] 9. Participants who had not recovered from immune-mediated adverse events (i.e., grade ≤1 or baseline) 3 months prior to starting investigational drug therapy, except for endocrine disorders adequately managed with hormone replacement.

[0182] 10. Participants who permanently discontinued anticancer immunomodulatory therapy due to immune-related adverse events (AEs).

[0183] 11. Another malignant tumor that is developing or requires active treatment, except in the following cases:

[0184] a. Non-melanoma skin cancer that has already received potentially curable treatment

[0185] b. Any tumor that has been proven effective with clear local control (with or without continuous adjuvant hormone therapy).

[0186] 12. Any symptoms requiring continuous / consecutive corticosteroid therapy (>10 mg prednisone / day or an anti-inflammatory equivalent) within 1 week prior to the first dose of study therapy. Patients requiring a short course of steroids (maximum 2 days, 1 week prior to enrollment) or physiological replacement are not excluded.

[0187] 13. There is current or recent (within the last 5 years) evidence of a significant autoimmune disease requiring systemic immunosuppressive therapy. This does not exclude the following conditions: vitiligo, remitted childhood asthma, endocrine disorders requiring only hormone replacement therapy (such as hypothyroidism or type 1 diabetes), or psoriasis not requiring systemic treatment.

[0188] 14. History of CNS metastases, including previously treated metastases.

[0189] 15. The patient experienced encephalitis, meningitis, neurodegenerative disease (excluding mild dementia that does not affect activities of daily living [ADL]), or uncontrolled seizures within one year prior to the first dose of study therapy.

[0190] 16. A known history or any evidence of interstitial lung disease or active non-infectious pneumonia (within the past 5 years). A history of radiation-induced pneumonia in the field of radiation is permissible.

[0191] 17. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B, or hepatitis C; or diagnosis of immunodeficiency.

[0192] Notice:

[0193] ● Patients will be tested for hepatitis C virus (HCV) and hepatitis B virus (HBV) during screening.

[0194] ● Allow HIV patients whose infection is under control (viral load is undetectable spontaneously or with a stable antiviral regimen (HIV RNA polymerase chain reaction [PCR]), and CD4 count is above 350).

[0195] ● Patients who are positive for hepatitis B surface antigen (HepBsAg+) and whose infection is under control (serum hepatitis B virus DNA PCR below AND detection limit and who are receiving hepatitis B antiviral therapy) are eligible.

[0196] ● Participants who are HBsAg negative but positive for total HBV core antibody (HBcAb+) are eligible if they meet the following requirements: If serum HBV DNA PCR is above the detection limit at screening, HBV antiviral therapy must be started before entering the study. If serum HBV DNA PCR is below the detection limit, HBsAg must be monitored regularly.

[0197] ● Allow patients who are positive for hepatitis C antibodies (HCV Ab+) and whose infection has been controlled (either spontaneously or in response to a previously successful anti-HCV therapy process, and whose HCV RNA is undetectable by PCR).

[0198] 18. Any infection requiring hospitalization or treatment with IV antibiotics within 2 weeks of the first dose of the study therapy.

[0199] 19. Receive the live vaccine within 4 weeks of the planned start date of the investigational drug.

[0200] 20. Has previously received an allogeneic stem cell transplant or an organ transplant at any time, or has received an autologous stem cell transplant within 12 weeks of the start of the study treatment.

[0201] 21. Known allergy or hypersensitivity to cimiprimab or any component of the investigational drug.

[0202] 22. Known mental illnesses or substance abuse disorders that may interfere with participation in the study requirements.

[0203] 23. Any medical condition, complication, physical examination results, metabolic dysfunction, or clinical laboratory abnormality that the investigator believes makes the patient unsuitable for participation in the clinical trial due to high safety risk and / or potential impact on the interpretation of study results, including but not limited to severe cardiovascular disease (e.g., New York Heart Association Class III or IV heart disease, myocardial infarction within the previous 6 months, unstable arrhythmia, or unstable angina) and / or severe pulmonary disease (e.g., history of obstructive pulmonary disease and symptomatic bronchospasm).

[0204] 24. Active infection, including

[0205] a. Infections requiring hospitalization or treatment with IV antibiotics within 2 weeks of the start of the study therapy.

[0206] b. Known active pulmonary tuberculosis or a history of incompletely treated active or latent pulmonary tuberculosis. An acceptable treatment for latent pulmonary tuberculosis is 9 months of oral isoniazid 300 mg daily or an equivalent proven regimen.

[0207] 25. History of invasive opportunistic infections, including but not limited to histoplasmosis, coccidioidomycosis, Pneumocystis jirovecii, or aspergillosis or John Cunningham virus (progressive multifocal leukoencephalopathy).

[0208] Research on treatment

[0209] The specified dose level of mAb1 will be administered via IV infusion or SC injection over 30 minutes to 2 hours, either QW or Q3W. Cimiprimab (for the specified group) will be administered via IV infusion over 30 minutes, Q3W. Low-dose cimiprimab (3 mg) will be administered as a bolus Q3W via IV infusion or IV injection to eligible participants. If both mAb1 and cimiprimab are administered on the same day, mAb1 will be administered first.

[0210] Study endpoints

[0211] The primary endpoint of the study was to evaluate mAb1 alone and in combination with cimipril:

[0212] Dosage escalation:

[0213] ● Safety is measured by the incidence and severity of treatment-emergent adverse events (TEAEs) / adverse events of particular concern (AESIs) / serious adverse events (SAEs) and grade ≥ 3 laboratory abnormalities during the treatment period and up to 90 days after the last dose of study or until the start of a new therapy for the patient’s cancer (whichever occurs first).

[0214] ● Tolerability, measured by the incidence of dose-limiting toxicities (DLTs) from the first dose to the end of the DLT observation period, using mAb1 alone and in combination with cimipril (if applicable).

[0215] ● Changes in serum mAb1 concentration over time

[0216] Dose expansion:

[0217] ●According to the RESIST 1.1 standard, ORR is based on verified complete response (CR) or partial response (PR).

[0218] Results - Administration of mAb1 or a combination of mAb1 and cimiprimab to patients with ccRCC was well-tolerated and effective in treating ccRCC.

[0219] This disclosure is not limited to the specific embodiments described herein. In fact, various modifications to this disclosure, 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.

[0220] sequence

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] *******

Claims

1. A method of treating clear cell renal cell carcinoma in a subject in need thereof, the method comprising administering to the subject a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to prostate-specific membrane antigen (PSMA); and a second antigen-binding domain that specifically binds to human CD28.

2. The method of claim 1, wherein the bispecific antibody is administered to the subject at a dose of at least 30 mg once a week (QW) for at least three weeks.

3. The method of claim 1 or 2, wherein the clear cell renal cell carcinoma is metastatic clear cell renal cell carcinoma.

4. The method of any one of claims 1 to 3, wherein the subject has received at least one prior therapy for clear cell renal cell carcinoma.

5. The method of claim 4, wherein the subject has received an anti-PD-1 therapy.

6. The method of claim 4 or 5, wherein the subject has received an anti-CTLA-4 therapy and / or a tyrosine kinase inhibitor.

7. The method of claim 6, wherein the anti-CTLA-4 therapy is ipilimumab.

8. The method of any one of claims 2 to 7, wherein the bispecific antibody or antigen-binding fragment thereof is administered as a monotherapy for at least three weeks, at least four weeks, at least five weeks, or at least six weeks.

9. The method of any one of claims 1 to 8, further comprising administering an antibody or antigen-binding fragment thereof that specifically binds to Programmed Death Receptor- 1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 100 mg.

10. The method of any one of claims 1 to 8, further comprising administering an antibody or antigen-binding fragment thereof that specifically binds to Programmed Death Receptor- 1 (PD-1), wherein the antibody or antigen-binding fragment is administered at a dose of 3 mg to 350 mg.

11. The method of any one of claims 1 to 10: (a) wherein the first antigen-binding domain comprises: three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR), the HCVR comprising the amino acid sequence of SEQ ID NO: 1; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR), the LCVR comprising the amino acid sequence of SEQ ID NO: 9, and (b) wherein the second antigen-binding domain comprises: three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR), the HCVR comprising the amino acid sequence of SEQ ID NO: 2; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR), the LCVR comprising the amino acid sequence of SEQ ID NO:

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

9.

12. The method of claim 11, wherein the first antigen binding domain comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 3; and a HCDR3 comprising the amino acid sequence of SEQ ID NO:

4.

13. The method of claim 11 or 12, wherein the second antigen binding domain comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 6; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a HCDR3 comprising the amino acid sequence of SEQ ID NO:

8.

14. The method of any one of claims 11 to 13, wherein the first antigen binding domain and the second antigen binding domain comprise: a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 11; and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

12.

15. The method of any one of claims 11 to 14, wherein the first antigen binding domain comprises: a HCVR comprising the amino acid sequence of SEQ ID NO: 1; and a LCVR comprising the amino acid sequence of SEQ ID NO:

9.

16. The method of any one of claims 11 to 15, wherein the second antigen binding domain comprises: a HCVR comprising the amino acid sequence of SEQ ID NO: 5; and a LCVR comprising the amino acid sequence of SEQ ID NO:

9.

17. The method of any one of claims 1 to 16, wherein the bispecific antibody comprises a human IgG heavy chain constant region.

18. The method of claim 17, wherein the human IgG heavy chain constant region is of isotype IgG1.

19. The method of claim 17, wherein the human IgG heavy chain constant region is of isotype IgG4.

20. The method of claim 18 or 19, wherein the bispecific antibody comprises a chimeric hinge that is reduced relative to a wild-type hinge of the same isotype. receptor binding.

21. The method of any one of claims 17-20, 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.

22. The method of any one of claims 1-16, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:

13.

23. The method of any one of claims 1-16, wherein the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:

14.

24. The method of any one of claims 1-16, wherein the bispecific antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 13; and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 14; and a common light chain comprising the amino acid sequence of SEQ ID NO:

15.

25. The method of any one of claims 9-24, 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) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 36; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

40.

26. The method of claim 25, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 37; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 38; and a HCDR3 comprising the amino acid sequence of SEQ ID NO:

39.

27. The method of claim 25 or 26, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises: a LCDR1 comprising the amino acid sequence of SEQ ID NO: 41; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 42; and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

43.

28. The method of any one of claims 25-27, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises: an HCVR comprising the amino acid sequence of SEQ ID NO: 36; and an LCVR comprising the amino acid sequence of SEQ ID NO:

40.

29. The method of claim 28, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 is an antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 44; and a light chain comprising the amino acid sequence of SEQ ID NO:

45.

30. The method of any one of claims 1-29, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 30 mg to 900 mg once weekly for at least three weeks.

31. The method of claim 30, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 100 mg to 900 mg once weekly for at least three weeks.

32. The method of claim 30, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 300 mg to 900 mg once weekly for at least three weeks.

33. The method of claim 30, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of 30 mg, 100 mg, 300 mg, 600 mg, or 900 mg once weekly for at least three weeks.

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