CN116615238A - Antibody combinations for treating cancer and alleviating cytokine release syndrome - Google Patents

Abstract

The present disclosure provides methods for treating cancer and ameliorating cytokine release syndrome in a subject having a tumor. The method comprises administering to a subject in need thereof a bispecific anti-CD 20/anti-CD 3 antibody prior to administering to the subject an anti-PD-1 antibody. The disclosed methods represent an effective treatment for cancer, such as B-cell malignancies, and mitigate the potentially life-threatening effects of Cytokine Release Syndrome (CRS).

Description

Antibody combinations for treating cancer and alleviating cytokine release syndrome

Technical Field

The present disclosure relates to methods of treating cancer and methods of ameliorating cytokine release syndrome in a subject having a tumor or tumor cells. The method comprises administering to a subject in need thereof a bispecific antibody that binds to CD20 and CD3 prior to administering to the subject an antibody that binds to PD-1.

Background

B cell cancer is a heterogeneous group of cancers of the white blood cells called B lymphocytes and includes leukemia (located in the blood) and lymphoma (located in the lymph nodes). B-cell lymphomas include, but are not limited to, non-Hodgkin's lymphoma (NHL) and Hodgkin's Lymphoma (HL). Lymphomas are classified as indolent (slow growing) or invasive lymphomas. The common indolent lymphoma is follicular lymphoma, while the most common aggressive lymphoma is diffuse large B-cell lymphoma. B-cell leukemia includes, but is not limited to, acute lymphoblastic leukemia, hairy cell leukemia, and B-cell chronic lymphocytic leukemia.

Most B cell cancers express CD20 on the cell surface of mature B cells. Methods for treating cancer by targeting CD20 are known in the art. For example, the chimeric anti-CD 20 monoclonal antibody rituximab has been used or proposed for the treatment of cancers such as NHL, chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL) and small lymphocytic lymphoma (small lymphocytic lymphoma, SLL), or as monotherapy but more typically in combination with chemotherapy. While anti-CD 20 tumor targeting strategies have shown tremendous promise in clinical settings, not all patients respond to anti-CD 20 therapy and have shown that some patients either develop resistance to anti-CD 20 therapy or exhibit an incomplete response (e.g., partial depletion of peripheral B cells) for reasons that are not clear (but generally do not include loss of CD20 expression). Some patients relapse more aggressive phenotypically or chemotherapeutically resistant diseases. Many patients with invasive lymphomas have poor prognosis and have a chance of survival without recurrence of less than 50%. Prognosis for relapsed or treatment-refractory patients remains poor, with median survival after rescue treatment of 2 to 8 months. In addition, high dose chemotherapy leads to serious adverse side effects. Thus, there remains a great unmet need for treatments that are effective in preventing recurrence and have fewer side effects in patients with B cell cancer.

Activation of T cells occurs naturally through their T Cell Receptor (TCR) complexed with the CD3 subunit (cd3γε -cd3δε -cd3ζζ) by ligation with a cognate peptide displayed on a Major Histocompatibility Complex (MHC) molecule on an Antigen Presenting Cell (APC). anti-CD 3 antibodies have been shown to accumulate CD3 on T cells, resulting in T cell activation in a manner similar to the engagement of peptide-loaded MHC molecules with TCRs. Bispecific monoclonal antibodies that aim to target both CD20 and CD3 bridge CD20 expressing cells with cytotoxic T cells, resulting in polyclonal T cell killing directed against CD 20.

Programmed death-1 (PD-1) receptor signaling in the tumor microenvironment plays a key role in immune surveillance that allows tumor cells to evade the host immune system. Blockade of PD-1 signaling pathways has shown clinical activity in patients with a variety of tumor types, and antibody therapeutics 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 shows that PD-1 blocks clinical activity in patients with invasive NHL and Hodgkin's lymphoma (Lesokhin, et al 2014, abstract 291,56th ASH Annual Meeting and Exposition,San Francisco,Calif.; ansell et al 2015, N.Engl. J. Med.372 (4): 311-9).

However, toxicity management of cancer immunotherapy is a challenging clinical problem. Alleviation of cytokine release syndrome (cytokine release syndrome, CRS) or infusion-related reactions (IRR) is a hallmark of administration of certain therapeutic modalities, such as chimeric antigen receptor (chimeric antigen receptor, CAR) T cells and bispecific antibody-targeted T cells. T cell redirection therapies, including CAR T cell therapy and CD20 x CD3 bispecific antibodies, have been associated with increased serum cytokines in patients with B cell non-hodgkin lymphomas, which may lead to systemic inflammatory responses CRS (shimabakuro-Vornhagen et al JImmunother Cancer,2018,6 (1): 56). Low-grade CRS is commonly treated with antihistamines, antipyretics, and fluids for symptomatic treatment. Serious CRS may represent a life threatening adverse event requiring timely and aggressive treatment. Reducing tumor burden, limiting the dose of treatment administered, and the use of steroids prior to surgery reduces the occurrence of severe CRS, as does the use of anti-cytokine therapies. However, minimizing cytokine activity using dose limiting and treatment may have deleterious effects on the efficacy of immunotherapy. In addition, targeting PD-1/PD-L1 is likely to promote T cell activation and inhibit evasion of PD-1 mediated tumor immune surveillance, but also may increase cytokine release, resulting in higher incidence and severity of CRS.

Thus, there remains a strong need for effective cancer treatments, such as B-cell malignancies, while mitigating the potentially life threatening effects of CRS without negatively impacting the therapeutic benefit of the treatment.

Disclosure of Invention

The disclosed technology addresses the above-described needs in several respects. In one aspect, the present disclosure provides a method of treating a tumor or inhibiting tumor growth comprising: (a) selecting a subject with cancer; (b) Administering to the subject a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprising a first antigen-binding arm that specifically binds CD20 and a second antigen-binding arm that specifically binds CD 3; and (c) after step (b), administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1); wherein the method treats a tumor or inhibits tumor growth and improves Cytokine Release Syndrome (CRS) in the subject.

In another aspect, the present disclosure provides a method of improving Cytokine Release Syndrome (CRS) in a subject with a tumor, comprising: (a) selecting a subject with cancer; (b) Administering to the subject a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprising a first antigen-binding arm that specifically binds CD20 and a second antigen-binding arm that specifically binds CD 3; and (c) after step (b), administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1).

In some embodiments, the step of administering an anti-PD-1 antibody or antigen-binding fragment thereof to the subject further comprises administering an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific antibody or antigen-binding fragment thereof to the subject. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to the subject at least about 1 week prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, each of the bispecific antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject in one or more doses.

In some embodiments, the first dose of the bispecific antibody or antigen-binding fragment thereof is administered to the subject about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks prior to administration of the first dose of the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the first dose of the bispecific antibody or antigen-binding fragment thereof is administered to the subject about 5 weeks prior to administration of the first dose of the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to a subject at one or more doses of about 0.1mg/kg to about 15mg/kg of the subject's body weight. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to the subject in one or more doses of about 1mg to about 800 mg. In some embodiments, the bispecific antibody or antigen binding fragment thereof is administered to the subject once daily, once every two days, once every three days, once every five days, once weekly, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, each of the one or more doses of bispecific antibody or antigen binding fragment thereof is administered 0.5 to 12 weeks after the previous dose (immediately preceding dose).

In some embodiments, at least one of the one or more doses of bispecific antibody or antigen binding fragment thereof comprises a dose having a greater amount of bispecific antibody or antigen binding fragment thereof than its previous dose. In some embodiments, at least one of the one or more doses of bispecific antibody or antigen binding fragment thereof is administered in two or more separate doses. In some embodiments, at least one of the two or more separate doses comprises the same amount of bispecific antibody or antigen binding fragment thereof. In some embodiments, at least one of the two or more divided doses is administered at least about 0.5 days after the previous dose. In some embodiments, at least one of the two or more divided doses is administered about 1 day after the previous dose. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject at one or more doses of about 0.1mg/kg to about 20mg/kg of the subject's body weight. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject in one or more doses of about 1mg to about 1500 mg. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject once daily, once every two days, once every three days, once every five days, once weekly, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks. In some embodiments, at least one of the one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof is administered 0.5 to 12 weeks after the previous dose. In some embodiments, at least one of the one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof comprises a dose having a greater amount of the anti-PD-1 antibody or antigen-binding fragment thereof than its previous dose.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof or the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject intravenously, subcutaneously, or intraperitoneally.

In some embodiments, the subject has cytokine release syndrome. In some embodiments, the tumor comprises a B cell cancer. In some embodiments, the B cell cancer is selected from hodgkin's lymphoma, non-hodgkin's lymphoma, follicular lymphoma, small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, lymphomatoid granulomatosis, burkitt's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, and B cell chronic lymphocytic leukemia.

In some embodiments, the subject is resistant to, insufficiently responsive to, or relapses after a previous treatment. In some embodiments, the subject has been previously treated with an anti-CD 20 treatment. In some embodiments, the anti-CD 20 treatment comprises an anti-CD 20 antibody. In some embodiments, the treatment produces a therapeutic effect selected from the group consisting of: delay in tumor growth, reduced tumor cell number, tumor regression, increased survival, partial response, and complete response.

In some embodiments, the treatment produces an effect selected from the group consisting of: reduced cytokine release, reduced release of IL-2, IL-6, IL-10, TNF-alpha and/or IFN-gamma, reduced administration of dexamethasone, corticosteroids or analgesics, reduced number of immune related adverse events and reduced number of grade 3 or more adverse events.

In some embodiments, tumor growth is delayed by at least 10 days as compared to untreated subjects. In some embodiments, tumor growth is inhibited by at least 50% as compared to an untreated subject. In some embodiments, tumor growth is inhibited by at least 50% as compared to administration of a bispecific antibody or antigen-binding fragment thereof or an anti-PD-1 antibody or antigen-binding fragment thereof as a monotherapy to a subject.

In some embodiments, the method further comprises administering a third therapeutic agent or third treatment to the subject. In some embodiments, the third therapeutic agent or third treatment is selected from the group consisting of radiation, surgery, chemotherapeutic agents, cancer vaccines, PD-L1 inhibitors, LAG-3 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, CD28 activators, CD38 inhibitors, GITR agonists, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) antagonists, angiopoietin-2 (ang2) inhibitors, transforming growth factor beta (transforming growth factor beta, tgfβ) inhibitors, epidermal growth factor receptor (epidermal growth factor receptor, EGFR) inhibitors, antibodies to tumor-specific antigens, bcg (Bacillus Calmette-Guerin vaccinee), granulocyte-macrophage colony stimulating factor, cytotoxins, interleukin 6receptor (IL-6R) inhibitors, interleukin 4receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, oncolytic viruses, anti-inflammatory drugs, dietary supplements, and combinations thereof.

In some embodiments, the first antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (a-HCDR 1, a-HCDR2 and a-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3), and wherein a-HCDR1 comprises the amino acid sequence of SEQ ID NO: 14; A-HCDR2 comprises the amino acid sequence of SEQ ID NO. 15; A-HCDR3 comprises the amino acid sequence of SEQ ID NO. 16; LCDR1 comprises the amino acid sequence of SEQ ID NO. 17; LCDR2 comprises the amino acid sequence of SEQ ID NO. 18; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable region (heavy chain variable region, A-HCVR) having the amino acid sequence of SEQ ID NO. 11 and a light chain variable region (light chain variable region, LCVR) having the amino acid sequence of SEQ ID NO. 12.

In some embodiments, the second antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (B-HCDR 1, B-HCDR2 and B-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3), and wherein B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 20; B-HCDR2 comprises the amino acid sequence of SEQ ID NO. 21; B-HCDR3 comprises the amino acid sequence of SEQ ID NO. 22; LCDR1 comprises the amino acid sequence of SEQ ID NO. 17; LCDR2 comprises the amino acid sequence of SEQ ID NO. 18; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the second antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable region (B-HCVR) having the amino acid sequence of SEQ ID NO. 13 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO. 12.

In some embodiments, the bispecific antibody comprises: a first heavy chain comprising a HCVR of a first antigen binding domain; a second heavy chain comprising a HCVR of a second antigen binding domain; and a common light chain comprising a LCVR of a first antigen binding domain and a second antigen binding domain, wherein the first heavy chain comprises the amino acid sequence of SEQ ID No. 23. In some embodiments, the bispecific antibody comprises: a first heavy chain comprising a HCVR of a first antigen binding domain; a second heavy chain comprising a HCVR of a second antigen binding domain; and a common light chain comprising a LCVR of a first antigen binding domain and a second antigen binding domain, wherein the second heavy chain comprises the amino acid sequence of SEQ ID No. 25. In some embodiments, the bispecific antibody comprises: a first heavy chain comprising a HCVR of a first antigen binding domain; a second heavy chain comprising a HCVR of a second antigen binding domain; and a common light chain comprising a LCVR of a first antigen binding domain and a second antigen binding domain, wherein said light chain comprises the amino acid sequence of SEQ ID No. 24.

In some embodiments, the bispecific antibody is ornitumumab (odronex mab).

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR 1, HCDR2, and HCDR 3) and three light chain complementarity determining regions (LCDR 1, LCDR2, and LCDR 3), and wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO. 4; HCDR3 comprises the amino acid sequence of SEQ ID NO. 5; LCDR1 comprises the amino acid sequence of SEQ ID NO. 6; LCDR2 comprises the amino acid sequence of SEQ ID NO. 7; and LCDR3 comprises the amino acid sequence of SEQ ID NO. 8. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID NO. 1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO. 2. In some embodiments, an anti-PD-1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO. 9 and a light chain having the amino acid sequence of SEQ ID NO. 10.

In some embodiments, the anti-PD-1 antibody is a cemipramiab (samipimab) Li Shan antibody.

The foregoing summary is not intended to limit each aspect of the disclosure, and additional aspects are described in other sections, such as in the detailed description below. The entire document is intended to be associated as a unified disclosure and it should be understood that all combinations of features described herein are contemplated even if such combinations of features are not present together in the same sentence, paragraph, or portion of the document. Other features and advantages of the disclosed subject matter will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Drawings

FIG. 1A shows an exemplary engineered reporter assay for evaluation of Ornituximab (REGN 1979) +cimiput Li Shan antibody (REGN 2810), wherein the titration range of Ornituximab is 500nM to 0.05pM.

FIG. 1B shows an exemplary engineered reporter assay for evaluation of Ornituximab (REGN 1979) +cimip Li Shan resistance (REGN 2810), wherein Ornituximab stimulates AP1-Luc activity from T cells incubated with WSU-DLCL2 cells.

FIG. 1C shows an exemplary engineered reporter assay for evaluation of Ornituximab (REGN 1979) +cimipran Li Shan antibody (REGN 2810), wherein stimulation of AP1-Luc in T cells by Ornituximab is significantly reduced in the presence of PD-L1 on WSU-DLCL2, which contributes to inhibition of T cell/AP 1-Luc activity and PD1 activation.

Fig. 2A is a first portion of the schematic (continuing to fig. 2B and 2C) showing a primary cd3+ T cell assay for evaluating the orituzumab + cimip Li Shan antibody.

Fig. 2B is a second portion of the schematic (starting from fig. 2A and continuing to fig. 2C) showing a primary cd3+ T cell assay for evaluating the orituzumab-cimetidine Li Shan antibody.

Fig. 2C is a third portion of the schematic (starting from fig. 2A and 2B) showing a primary cd3+ T cell assay for evaluating the orituzumab + cimip Li Shan antibody.

Figures 3A, 3B, 3C, 3D, 3E, 3F and 3G are graphs showing results from an ornitumumab + cimipran Li Shan anti-induction dosimetry (lead-in dosing assay) in which T cells exposed to an induction dose of ornitumumab exhibited cytokine rescue by cimipran Li Shan anti-treatment.

Fig. 3A is a graph showing results from an ornitumumab + cimetidine Li Shan anti-induction dosimetry, wherein T cells exposed to an induction dose of ornitumumab exhibit cytokine rescue by cimetidine Li Shan anti-treatment, wherein the graph shows IL-2 release in response to cimetidine Li Shan anti-titration in T cells not stimulated with WSU-DLCL2/PD-L1 (as measured by relative fluorescent units or RFU).

Fig. 3B is a graph showing the results from an ornitumumab + cimetidine Li Shan anti-induction dosimetry, wherein T cells exposed to an induction dose of ornitumumab exhibit cytokine rescue by treatment with cimitumumab Li Shan antibody, wherein the graph shows IL-2 release in response to a cimitumumab Li Shan anti-titration in T cells pre-stimulated with an initial dose of 1.3nM of ornitumumab.

Fig. 3C is an enhanced view of fig. 3B.

Fig. 3D is a graph showing ifnγ release in unstimulated T cells in response to a cimrpu Li Shan anti-titration.

Fig. 3E is a graph showing ifnγ release in response to cimiput Li Shan anti-titration in T cells pre-stimulated with 1.3nM of ornitumumab.

Fig. 3F is an enhanced view of fig. 3E.

FIG. 3G is a set of graphs showing PD-1 expression levels in unstimulated and prestimulated T cells.

FIG. 4 is a set of graphs showing cytokine responses from pre-stimulation in response to CTLA-4 and LAG-3 blockade.

FIG. 5 is a set of graphs showing T cell cytotoxicity of unstimulated T cells and T cells stimulated with 1.3nM ornitumumab.

Fig. 6A shows a quantitative system pharmacology (quantitative systems pharmacology, QSP) modeling framework that captures disease-lymphocyte-drug interactions associated with the mechanism of action of ornitumumab, which binds to CD20 on B cells and CD3 on T cells to form synapses.

Fig. 6B shows a QSP modeling framework that captures disease-lymphocyte-drug interactions associated with T cell activation, where ornitumumab binds to T cells and B cells and activates T cells, which enhances T cell mediated tumor cytotoxicity.

Fig. 6C shows a QSP modeling framework to capture disease-lymphocyte-drug interactions associated with the IL-6 dynamic model, where cimrpu Li Shan resists modulation of cytokine production by synapse formation and enhances release of IL-6 into the systemic circulation. [ IL, interleukin ]

Fig. 6D shows a QSP modeling framework that captures disease-lymphocyte-drug interactions associated with the mechanism of action of the cimiput Li Shan antibody, where the cimiput Li Shan antibody promotes T cell activation.

Fig. 6E shows a QSP modeling framework to capture disease-lymphocyte-drug interactions associated with the mechanism of combined action of ornitumumab/cimiput Li Shan anti-combination, where the ornitumumab/cimiput Li Shan anti-combination enhances T cell activation to kill tumor cells.

FIG. 6F shows a QSP modeling framework to capture disease-lymphocyte-drug interactions associated with PD-1 regulatory models, where PD-1 expression levels on activated T cells are a function of programmed cell death 1 (programmed cell death, PDCD 1) gene regulation. [ TCR, T cell receptor ]

FIG. 7 shows a representative example of an ornitumumab monotherapy regimen (1, 6, 12mg QW; weeks 1-36). Arrows indicate treatment with ornitumumab. [ QW, once a week ]

Fig. 8 shows a representative example of an ornitumumab/cimiput Li Shan anti-combination treatment regimen (ornitumumab 1, 6, 12mg QW/cimiput Li Shan anti 3mg/kg Q2W). The arrow in the upper row indicates treatment with ornitumumab; the arrow in the next row indicates treatment with the cimetidine Li Shan antibody. [ QW, once a week; Q2W, once every two weeks ]

Fig. 9A shows a comparison of simulated (line) and observed (dot) IL-6 concentrations over time in the QSP model of example 2 under both the aonitab monotherapy and the aonitab/cimiput Li Shan anti-combination therapy.

Fig. 9B shows another comparison of simulated (line) and observed (dot) IL-6 concentrations over time under both the aonitab monotherapy and the aonitab/cimip Li Shan anti-combination therapy in the QSP model of example 2. The model predicts that the IL-6 peak after the combined therapy of the ornitumumab/cimip Li Shan is higher than the peak after the single therapy of the ornitumumab.

FIG. 10 is a graph showing the effect of changing the onset time of cimrpu Li Shan antibody on IL-6 levels, as predicted by the QSP model in example 2, based on doses of ornitumumab of 1, 20 or 160mg QW and administration of cimrpu Li Shan antibody at different onset times (3 mg/kg Q2W). This model predicts that delaying the time of administration of the cimiput Li Shan antibody will attenuate peak levels of IL-6.

Figure 11 shows a simulated cytokine release (IL-6) profile based on an induction period of 4 weeks of ornitumumab monotherapy followed by a dose level of cimrpuzep Li Shan starting at 3, 30 and 350mg starting from week 5, as predicted by the QSP model in example 2. This model predicts that when the administration of the cimiput Li Shan antibody is initiated from week 5, the IL-6 level is similar to that of the ornitumumab monotherapy, regardless of the dose level of the cimiput Li Shan antibody.

Detailed Description

Targeted treatment of cancer has the potential to specifically recognize and destroy cancer cells while minimizing adverse side effects. However, these new treatments present unique challenges in determining a dosing regimen that is safe for the patient and avoids unwanted immune cell activation. The present disclosure is based, at least in part, on the unexpected discovery that: pretreatment of T cells with anti-CD 20/anti-CD 3 bispecific antibody (ornitumumab) and delayed exposure to anti-PD-1 antibody (cimetidine Li Shan antibody) resulted in a significant reduction in cytokine (e.g., IL-2) release when exposed to a combination of ornitude Shan Kangjia cimitude Li Shan antibodies compared to control T cells that did not receive the ornitumumab pretreatment. This reduction in cytokine levels seen after the pretreatment with ornitumumab supports the conclusion that priming ornitumumab prior to the addition of cimiput Li Shan antibody can help significantly reduce Cytokine Release Syndrome (CRS).

The disclosed methods generally comprise administering a bispecific anti-CD 20/anti-CD 3 antibody to a subject suffering from cancer for an induction period prior to administration of the anti-PD-1 antibody to the subject, which achieves dual objectives by effectively treating a tumor (e.g., B cell cancer) or inhibiting its growth while also avoiding or minimizing (i.e., achieving a lower incidence or severity of) immune-related adverse events CRS.

Therapeutic method

In one aspect, the present disclosure provides methods for treating, ameliorating or reducing the severity of at least one symptom or indication of cancer, or inhibiting the growth of cancer in a subject. The method comprises the following steps: (i) Administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprising a first antigen-binding arm that specifically binds CD20 and a second antigen-binding arm that specifically binds CD3, followed by (ii) administering to a subject in need thereof an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds PD-1. Furthermore, in this aspect, the method also improves CRS in the subject.

In another aspect, the present disclosure provides a method of ameliorating at least one symptom or indication of CRS or reducing the severity of at least one symptom or indication of CRS in a subject having a tumor. The method comprises the following steps: (i) Administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprising a first antigen-binding arm that specifically binds CD20 and a second antigen-binding arm that specifically binds CD3, followed by (ii) administering to a subject in need thereof an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds PD-1.

In some embodiments, the method comprises administering to a subject in need thereof one or more doses of an anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof prior to administering to the subject one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof. Upon administration of one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof may be administered in combination with one or more doses of the anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof.

As used herein, the term "treatment" and variations thereof means alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, delaying or inhibiting tumor growth, reducing tumor cell burden or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and/or disappearance, preventing tumor recurrence, and/or extending the survival duration of a subject.

As used herein, the expression "a subject in need thereof" means a human or non-human mammal that exhibits one or more symptoms and signs of cancer and/or has been diagnosed with cancer (including B cell cancer) and in need of treatment therefor. In some embodiments, the term "subject" is used interchangeably with the term "patient," e.g., 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: lymph node enlargement, abdominal swelling, chest pain/pressure, weight loss due to unknown reasons, fever, night sweat, sustained fatigue, anorexia, spleen enlargement, itching. The expression includes subjects suffering from primary or established B cell tumors. In some embodiments, the expression includes a human subject suffering from and in need of treatment for: b-cell malignancies, for example, hodgkin's lymphoma, non-hodgkin's lymphoma, follicular lymphoma, small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, lymphomatoid granulomatosis, burkitt's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, and B-cell chronic lymphocytic leukemia. In another embodiment, the expression includes a human having a pathological subtype of B cell cancer. In other specific embodiments, the expression includes subjects with a cd20+ tumor (e.g., a tumor with CD20 expression on ≡20% leukemia lymphoblastic cells as determined by flow cytometry). In certain embodiments, the expression "a subject in need thereof" includes patients suffering from B cell cancer that is resistant or refractory to prior treatment (e.g., treatment with conventional anti-cancer agents) or that is not adequately controlled by it. For example, the expression includes subjects that have been treated with a CD20 inhibitor (e.g., rituximab), a chemotherapeutic or immunomodulatory agent (e.g., a blocker of CTLA-4, 4-1BB, LAG3, or OX-40). The expression also includes subjects suffering from B cell malignancies for which conventional anti-cancer treatments are not desirable, for example due to toxic side effects. For example, the expression includes patients who have undergone one or more cycles of chemotherapy and have toxic side effects. In certain embodiments, the expression "a subject in need thereof" includes patients suffering from B cell malignancies, who have been treated but who subsequently relapse or metastasis. For example, such patients with B cell malignancies are treated with the methods of the present disclosure: the patient may have received treatment with one or more anti-cancer agents, resulting in tumor regression, however, subsequently relapsed cancer that is resistant to the one or more anti-cancer agents (e.g., a chemotherapy-resistant cancer).

The expression "a subject in need thereof" also includes subjects at risk of developing B cell cancer, for example, a person with a family history of lymphoma, a person with a past history of EB infection (e.g., infectious mononucleosis), or a person with an impaired immune system due to HIV infection or due to immunosuppressive drugs.

In certain embodiments, the methods of the present disclosure are useful for treating patients exhibiting elevated levels of one or more cancer-associated biomarkers (e.g., PD-L1, CD20, beta-2-microglobulin, lactate dehydrogenase, BCR-ABL fusion gene, ALK gene rearrangement). For example, the methods comprise administering to a patient having an elevated level of PD-L1 and/or CD20 a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the methods comprise administering a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof to a patient with elevated PD-L1 and/or CD20 levels, followed by administration of an anti-PD-1 antibody or antigen-binding fragment thereof to the patient.

In certain embodiments, the methods of the present disclosure may be used to ameliorate or reduce the severity of at least one symptom or indication of CRS in a subject with a tumor. CRS is a systemic inflammatory response that may be triggered by a variety of factors, including certain drugs. T cell activated cancer immunotherapy has a particularly high risk of CRS, typically due to the on-target effect induced by the binding of bispecific antibodies or chimeric antigen receptor CAR-T cells to their antigens, and subsequent activation of bystander immune cells and non-immune cells (e.g. endothelial cells). Activation of bystander cells results in a large release of a range of cytokines. IL-6, IL-10 and Interferon (IFN) -gamma are core cytokines that are continuously found to be elevated in the serum of patients with CRS. For treatment of T cell activation against tumor cells, CRS is triggered by the massive release of IFN- γ by the activated T cells or by the tumor cells themselves. Secreted IFN-gamma induces activation of other immune cells (most importantly macrophages), which in turn produce excess amounts of additional cytokines such as IL-6, TNF-alpha and IL-10.

In certain embodiments, the methods of the present disclosure are used in subjects with B cell cancer. The terms "tumor", "tumor cell", "cancer" and "malignant" are used interchangeably herein. The term "B cell cancer" as used herein refers to tumors of white blood cells called B lymphocytes and includes leukemia (located in the blood) and lymphoma (located in the lymph nodes). The present disclosure includes methods of treating both leukemia and lymphoma. In certain embodiments, B-cell cancers include, but are not limited to, hodgkin's lymphoma, non-hodgkin's lymphoma, follicular lymphoma, small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, lymphomatoid granulomatosis, burkitt's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and pathological subtypes thereof. B-cell lymphomas are generally classified into low and high grade, and generally correspond to indolent (slow growing) lymphomas and invasive lymphomas, respectively. The present disclosure includes methods of treating both indolent and aggressive lymphomas.

According to certain embodiments, the present disclosure includes methods for treating tumors, delaying or inhibiting tumor growth. In certain embodiments, the present disclosure includes methods of promoting tumor regression. In certain embodiments, the disclosure includes methods of reducing tumor cell burden or reducing tumor burden. In certain embodiments, the present disclosure includes methods of preventing tumor recurrence.

The method according to this aspect of the disclosure comprises sequentially administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof, wherein each antibody is administered to the subject in multiple doses, e.g., as part of a particular therapeutic dosing regimen. For example, a therapeutic dosing regimen may comprise administering one or more doses of a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof, wherein the one or more doses of the bispecific antibody or antigen-binding fragment thereof are administered to a subject at a frequency of about once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or less frequently. In certain embodiments, at least one dose of an anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof is administered in more than 1 fraction, e.g., in 2 to 5 fractions ("divided administration"), within a given administration period. The anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof may be administered in separate doses to reduce or eliminate cytokine "spikes" (spike) induced in response to antibody administration. Cytokine spikes refer to clinical symptoms of Cytokine Release Syndrome (CRS) or "cytokine storm" and infusion-related reactions, seen in patients administered anti-CD 20 antibodies.

In certain embodiments, one or more doses of an anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof are administered to a subject at the following frequency: about 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 every four weeks, once monthly, once every two months, once every three months, once every four months, or less frequently.

In certain embodiments, the methods of the present disclosure comprise administering one or more doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof to a subject in need thereof, wherein the dose of bispecific antibody is administered in divided doses or in more than 1 part, e.g., 2 parts, 3 parts, 4 parts, or 5 parts, within a given dosing period. In certain embodiments, the dose of bispecific antibody is divided into 2 or more fractions, wherein each fraction contains the same amount of antibody as the other fractions.

In some embodiments, one or more doses of the bispecific antibody or antigen-binding fragment thereof are administered to the subject at least about 1 week prior to administration of one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the first dose of bispecific antibody or antigen-binding fragment thereof is administered to the subject about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks prior to administration of the first dose of anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the first dose of bispecific antibody or antigen-binding fragment thereof is administered to the subject about 5 weeks (e.g., 5 weeks) prior to administration of the first dose of anti-PD-1 antibody or antigen-binding fragment thereof.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to the subject at one or more doses of about 0.1mg/kg to about 15mg/kg of the subject's body weight. In some embodiments, the bispecific antibody or antigen binding fragment thereof is administered to a subject in one or more doses of about 1mg to about 800 mg.

In some embodiments, at least one of the one or more doses of the bispecific antibody or antigen binding fragment thereof comprises a dose having a greater amount of bispecific antibody or antigen binding fragment thereof than its previous dose.

In some embodiments, at least one of the one or more doses of bispecific antibody or antigen binding fragment thereof is administered in two or more separate doses. In some embodiments, at least one of the two or more separate doses comprises the same amount of bispecific antibody or antigen binding fragment thereof. In some embodiments, at least one of the two or more divided doses is administered at least about 0.5 days after the previous dose. In some embodiments, each of the two or more divided doses is administered about 1 day after the previous dose.

For example, a dose of anti-CD 20/anti-CD 3 antibody comprising 1mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 parts, each part comprising 0.5mg, within a week. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 1mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions on two consecutive days, respectively, each portion comprising 0.5mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 4mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions within a week, each portion comprising 2mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 20mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions within a week, each portion comprising 10mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 20mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions on two consecutive days, respectively, each portion comprising 10mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 80mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions within a week, each portion comprising 40mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 80mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions on two consecutive days, respectively, each portion comprising 40mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 320mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions within a week, each portion comprising 160mg. In some embodiments, a dose of anti-CD 20/anti-CD 3 antibody comprising 320mg of anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof may be administered once a week, wherein the dose is administered in 2 portions on two consecutive days, respectively, each portion comprising 160mg.

In certain embodiments, the dose of bispecific antibody is administered in 2 or more portions, wherein the portions comprise unequal amounts of antibody, e.g., more or less than the first portion. For example, a dose comprising 360mg of an anti-CD 20/anti-CD 3 antibody may be administered once a week, wherein the dose is administered in 2 portions within a week, wherein the first portion comprises 200mg and the second portion comprises 160mg. As another example, a dose comprising 360mg of an anti-CD 20/anti-CD 3 antibody may be administered once within 2 weeks, wherein the dose is administered in 3 portions over a 2 week period, wherein the first portion comprises 120mg, the second portion comprises 120mg, and the third portion comprises 120mg.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject at one or more doses of about 0.1mg/kg to about 20mg/kg of the subject's body weight. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject in one or more doses of about 1mg to about 800 mg.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject once daily, once every two days, once every three days, once every five days, once weekly, once every two weeks, or once every three weeks. In some embodiments, each of the one or more doses of anti-PD-1 antibody or antigen-binding fragment thereof is administered 0.5 to 12 weeks after the previous dose.

In some embodiments, at least one of the one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof comprises a dose having a greater amount of the anti-PD-1 antibody or antigen-binding fragment thereof than its previous dose.

An example of a dosing regimen contemplated by the present disclosure is as follows:

in certain embodiments, the present disclosure includes methods of inhibiting, delaying or stopping tumor metastasis or tumor infiltration into peripheral organs. The method according to this aspect comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides methods for increasing anti-tumor efficacy or increasing tumor suppression.

The method according to this aspect of the disclosure comprises administering a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof to a subject having B cell cancer prior to administration of the therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof may be administered about 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, more than 8 days, more than 10 days, more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, or more than 8 weeks prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the methods provide increased tumor inhibition, e.g., by about 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80%, as compared to monotherapy with a bispecific antibody or an anti-PD-1 antibody.

In certain embodiments, the methods of the present disclosure comprise administering a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to a subject suffering from B cell cancer. For example, the method comprises administering a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof prior to administering the anti-PD-1 antibody or antigen-binding fragment thereof to a subject having B cell cancer. In some embodiments, the B cell cancer is hodgkin's lymphoma or non-hodgkin's lymphoma. In other embodiments, the B cell cancer is indolent or invasive. In certain embodiments, the subject does not respond to or relapse after prior treatment.

In certain embodiments, the methods of the present disclosure further comprise administering a bispecific anti-CD 20/anti-CD 3 antibody, or antigen-binding fragment thereof, in combination with an anti-PD-1 antibody to a subject suffering from cd20+ B cell cancer. In certain embodiments, the methods of the present disclosure comprise administering a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof prior to administering the anti-PD-1 antibody or antigen-binding fragment thereof to a subject having cd20+ B cell cancer. In some embodiments, the B cell cancer is acute lymphoblastic leukemia or chronic lymphoblastic leukemia. In other embodiments, the B cell cancer is indolent or invasive. In certain embodiments, the subject is unresponsive to prior treatment or relapses after prior treatment (e.g., with an anti-CD 20 inhibitor such as rituximab).

In certain embodiments, the methods of the present disclosure comprise administering a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to a subject in need thereof as a "first line" treatment (e.g., initial treatment). In other embodiments, the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with the anti-PD-1 antibody or antigen-binding fragment thereof is administered as a "second line" treatment (e.g., after a prior treatment). For example, a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof is administered as a "second line" treatment to a subject who relapses after prior treatment with, for example, chemotherapy or rituximab.

In certain embodiments, the methods of the present disclosure are used to treat patients with minimal residual disease (minimum residual disease, MRD) -positive disease. MRD refers to the small number of cancer cells that remain in a patient during or after the end of treatment, where the patient may or may not develop symptoms or signs of the disease. Such residual cancer cells, if not cleared, often lead to recurrence of the disease. The present disclosure includes methods of inhibiting and/or clearing residual cancer cells in a patient at the time of an MRD test. MRD may be determined according to methods known in the art (e.g., MRD flow cytometry). Methods according to this aspect of the disclosure include administering to a subject in need thereof a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof.

According to certain embodiments, the methods of the present disclosure comprise administering to the subject a therapeutically effective amount of each of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a third therapeutic agent or third therapy. The third therapeutic agent may be an agent selected from the group consisting of: such as radiation, chemotherapy, surgery, cancer vaccines, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), LAG3 inhibitors (e.g., anti-LAG 3 antibodies), CTLA-4 inhibitors, 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 beta (tgfβ) inhibitors, epidermal Growth Factor (EGFR) inhibitors, antibodies to tumor specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (melanoma-associated antigen, mage3), carcinoembryonic antigen (carcinoembryonic antigen, CEA), vimentin, tumor-M2-PK, prostate specific antigen (precursor-specific antigen, PSA), mucin-1, antibodies to MART-1 and CA19-9, oncolytic agents, vaccines (e.g., bacillus calmets), granulocyte-macrophage colony stimulating factors, cytotoxins, chemotherapeutic agents, IL-6R, IL-4, IL-21, IL-12, anti-inflammatory agents (e.g., anti-corticoids), and non-dietary supplements thereof).

In certain embodiments, the antibodies may be administered in combination with therapies including chemotherapeutic agents, radiation, and surgery. Herein, the phrase "in combination with … …" means that the antibody is administered to the subject simultaneously with, just prior to, or just after administration of the third therapeutic agent. In certain embodiments, the third therapeutic agent is administered as a co-formulation with the antibody. In a related embodiment, the disclosure includes a method comprising administering a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to a subject undergoing a background anti-cancer treatment regimen. Background anti-cancer treatment regimens may include, for example, the administration of a chemotherapeutic agent or radiation. Bispecific anti-CD 20/anti-CD 3 antibodies or antigen-binding fragments thereof in combination with anti-PD-1 antibodies or antigen-binding fragments thereof may be added above background anti-cancer treatment regimens. In some embodiments, the antibody is added as part of a "step-down" regimen, wherein the subject gradually withdraws (e.g., in a stepwise manner) from the background anti-cancer treatment over time, while the antibody is administered to the subject at a constant dose, or at an elevated dose, or at a reduced dose over time.

In certain embodiments, the methods of the present disclosure comprise administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof, wherein administration of the antibody results in increased inhibition of tumor growth. In certain embodiments, tumor growth is inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% as compared to an untreated subject or a subject administered either antibody as monotherapy. In certain embodiments, administration of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and/or the anti-PD-1 antibody or antigen-binding fragment thereof results in increased tumor regression, tumor shrinkage, and/or disappearance. In certain embodiments, administration of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and/or the anti-PD-1 antibody or antigen-binding fragment thereof results in a delay in tumor growth and onset compared to an untreated subject or a subject treated with either antibody as monotherapy, e.g., tumor growth may be delayed for about 3 days, more than 3 days, about 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years. In certain embodiments, administration of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof can prevent tumor recurrence and/or increase survival duration of a subject, e.g., 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, as compared to an untreated subject or a subject administered either antibody as a monotherapy. In certain embodiments, the combined administration of antibodies increases progression free survival or overall survival.

In certain embodiments, administration of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof increases response and duration of response in a subject, e.g., 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% as compared to an untreated subject or a subject that has received either antibody as a monotherapy. In certain embodiments, administration of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to a subject suffering from B cell cancer results in complete disappearance of all signs of tumor cells ("complete response"). In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and/or a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof to a subject having B cell cancer results in a reduction in tumor cell or tumor size of at least 30% or more ("partial response"). In certain embodiments, administration of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and/or the anti-PD-1 antibody or antigen-binding fragment thereof to a subject having B cell cancer results in complete or partial disappearance of tumor cells/lesions (including new measurable lesions). Tumor reduction may be measured by any method known in the art, for example, X-ray, positron emission tomography (positron emission tomography, PET), computed tomography (computed tomography, CT), magnetic resonance imaging (magnetic resonance imaging, MRI), cytology, histology, or molecular genetic analysis.

In certain embodiments, the combination of administered antibodies is safe and well tolerated by the patient, wherein there is no increase in adverse side effects (e.g., increased CRS or increased T cell activation) compared to a patient administered a bispecific antibody as monotherapy.

anti-PD-1 antibodies and antigen binding fragments thereof

According to certain exemplary embodiments of the present disclosure, the methods comprise administering a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof. The term "antibody" as used herein includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains, and two light (L) chains, as well as multimers thereof (e.g., igM), that are interconnected by disulfide bonds. In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL 1). The VH and VL regions can be further subdivided into regions of hypervariability termed complementarity determining regions (complementarity determining region, CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FR of the anti-IL-4R antibody (or antigen binding portion thereof) may be identical to the human germline sequence, or may be naturally or artificially modified. Amino acid consensus sequences can be defined based on parallel analysis of two or more CDRs.

The term "antibody" as used herein also includes antigen binding fragments of whole antibody molecules. The terms "antigen binding portion" of an antibody, an "antigen binding fragment" of an antibody, and the like as used herein, encompass any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The antigen binding fragment of an antibody may be derived, e.g., from an intact antibody molecule, using any suitable standard technique involving manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody, e.g., proteolytic digestion or recombinant genetic engineering techniques. Such DNA is known and/or may be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthesized. The DNA can be sequenced and manipulated by chemical means or by using molecular biological techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, to create cysteine residues, to modify, add or delete amino acids, and the like.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) a dAb fragment; and (vii) a minimal identifying unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated Complementarity Determining Region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (small modular immunopharmaceutical, SMIP), and shark variable IgNAR domains are also included in the expression "antigen-binding fragments" as used herein.

The antigen binding fragment of an antibody typically comprises at least one variable domain. The variable domain may have any size or amino acid composition and typically comprises at least one CDR adjacent to or within one or more framework sequences. In the presence of V _L Domain related V _H In the antigen binding fragment of the domain, V _H Domain and V _L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and comprise V _H -V _H 、V _H -V _L Or V _L -V _L A dimer. Alternatively, the antigen-binding fragment of the antibody may comprise monomer V _H Or V _L A domain.

In certain embodiments, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be present in antigen binding fragments of antibodies of the present disclosure include: (i) V (V) _H -C _H 1；(ii)V _H -C _H 2；(iii)V _H -C _H 3；(iv)V _H -C _H 1-C _H 2；(v)V _H -C _H 1-C _H 2-C _H 3；(vi)V _H -C _H 2-C _H 3；(vii)V _H -C _L ；(viii)V _L -C _H 1；(ix)V _L -C _H 2；(x)V _L -C _H 3；(xi)V _L -C _H 1-C _H 2；(xii)V _L -C _H 1-C _H 2-C _H 3；(xiii)V _L -C _H 2-C _H 3, a step of; and (xiv) V _L -C _L . In any configuration of variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly linked to each other, or may be linked by whole or part of a hinge or linker region. The hinge region can be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Furthermore, antigen binding fragments of antibodies of the present disclosure may comprise one or more monomers V with each other and/or with one or more monomers V _H Or V _L The domains are non-covalently associated (e.g., via disulfide bonds) with homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above.

The term "antibody" as used herein also includes multispecific (e.g., bispecific) antibodies. The multispecific antibody or antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format may be suitable in the context of an antibody or antigen-binding fragment of an antibody of the present disclosure using conventional techniques available in the art. For example, the disclosure includes methods comprising using bispecific antibodies, wherein one arm of the immunoglobulin is specific for PD-1 or a fragment thereof and the other arm of the immunoglobulin is specific for a second therapeutic target or conjugated to a therapeutic moiety. Exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, scFv-based formats or diabody bispecific formats, igG-scFv fusions, double Variable Domain (DVD) -Ig, tetravalent body tumors (Quadroma), knob-in-hole (knobs-intro-holes), common light chains (e.g., a long Common light chain under the projection-in-pocket, etc.), crossMab, crossFab, (SEED) body, leucine zipper, duobody, igG1/IgG2, double Acting Fab (DAF) -IgG and Mab ² Bispecific formats (for reviews of the foregoing formats, see, e.g., klein et al 2012, mAbs 4:6,1-11 and references cited therein). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugates, for example, wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes of defined composition, valency, and geometry. (see, e.g., kazane et al, J.am. Chem. Soc. Epub: dec.4, 2012).

The antibodies used in the methods of the present disclosure may be human antibodies. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nonetheless, the human antibodies of the present disclosure may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in CDRs, and in particular in CDR 3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

The antibodies used in the methods of the present disclosure may be recombinant human antibodies. The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinant combinatorial human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., taylor et al (1992) nucleic acids res.20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means that involves splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have a human originVariable and constant regions of germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus, the V of the recombinant antibodies _H And V _L The amino acid sequence of the region is such that: although derived from human germline V _H And V _L Sequences and related thereto, but may not naturally occur in human antibody germline libraries in vivo.

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

According to certain exemplary embodiments of the present disclosure, an anti-PD-1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR), a Light Chain Variable Region (LCVR), and/or a Complementarity Determining Region (CDR) comprising any amino acid sequence of an anti-PD-1 antibody as shown in U.S. patent publication No. 20150203579. In certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof useful in the context of the methods of the present disclosure comprises: heavy chain complementarity determining region (HCDR) of Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID NO. 1 and amino acid comprising SEQ ID NO.2Light chain complementarity determining regions (LCDR) of the Light Chain Variable Region (LCVR) of the sequence. According to certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR 1, HCDR2, and HCDR 3) and three LCDRs (LCDR 1, LCDR2, and LCDR 3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO. 4; HCDR3 comprises the amino acid sequence of SEQ ID NO. 5; LCDR1 comprises the amino acid sequence of SEQ ID NO. 6; LCDR2 comprises the amino acid sequence of SEQ ID NO. 7; and LCDR3 comprises the amino acid sequence of SEQ ID NO. 8. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO. 1 and the LCVR of SEQ ID NO. 2. In certain embodiments, the methods of the present disclosure comprise the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 9. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 10. An exemplary antibody comprising a HCVR (which comprises the amino acid sequence of SEQ ID NO: 1) and a LCVR (which comprises the amino acid sequence of SEQ ID NO: 2) is known as REGN2810 (also known as a Zealand Li Shan antibody, ) Is a fully human anti-PD-1 antibody. According to certain exemplary embodiments, the methods of the present disclosure include the use of a cimrpu Li Shan antibody or a biological equivalent thereof.

The term "biological equivalent" as used herein refers to an anti-PD-1 antibody or PD-1 binding protein or fragment thereof that is a drug equivalent or drug replacement that does not exhibit a significant difference in its absorbance and/or extent of absorption from the absorbance and/or extent of absorption of the cimipran Li Shan antibody when administered in the same molar dose (single or multiple doses) under similar experimental conditions. In the context of the present disclosure, the term refers to an antigen binding protein that binds to PD-1 without clinically significant differences from cimetidine Li Shan in terms of its safety, purity and/or potency.

Other anti-PD-1 antibodies that may be used in the context of the methods of the present disclosure include, for example, antibodies known and known in the art as nivorumab (U.S. patent No.8,008,449), pembrolizuumab (U.S. patent No.8,354,509), MEDI0608 (U.S. patent No.8,609,089), pidilizuumab (U.S. patent No.8,686,119), or any anti-PD-1 antibody as described in U.S. patent nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8900587.

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

In some cases, "reduced binding to PD-1 at acidic pH compared to neutral pH" is based on K of an antibody that binds to PD-1 at acidic pH _D Value and K of antibodies binding to PD-1 at neutral pH _D The ratio of values (and vice versa). For example, if the antibody or antigen binding fragment thereof exhibits an acidic/neutral K of about 3.0 or greater _D For purposes of this disclosure, an antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to PD-1 at acidic pH compared to neutral pH". In certain exemplary embodiments, the acid/neutral K of the antibodies or antigen binding fragments of the disclosure _D The ratio may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies that have reduced (or enhanced) binding to a particular antigen at an acidic pH as compared to a neutral pH. Furthermore, modification of the antigen binding domain at the amino acid level may result in antibodies with pH dependent properties. For example, by replacing one or more amino acids of an antigen binding domain (e.g., within a CDR) with histidine residues, antibodies can be obtained that have reduced antigen binding at acidic pH relative to neutral pH. As used herein, the expression "acidic pH" means a pH of 6.0 or less.

Bispecific anti-CD 20/anti-CD 3 antibodies

According to certain exemplary embodiments of the present disclosure, the method comprises administering a therapeutically effective amount of a bispecific antibody that specifically binds CD3 and CD 20. Such antibodies may be referred to herein as, for example, "anti-CD 20/anti-CD 3" or "anti-CD 20 xcd 3" or "CD20 xcd 3" bispecific antibodies, or other similar terms.

As used herein, the expression "bispecific antibody" refers to an immunoglobulin comprising at least a first antigen binding domain and a second antigen binding domain. In the context of the present disclosure, a first antigen binding domain specifically binds a first antigen (e.g., CD 20), and a second antigen binding domain specifically binds a second, different antigen (e.g., CD 3). Each antigen binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each domain comprising three CDRs. In the context of bispecific antibodies, the CDRs of a first antigen binding domain can be represented by the prefix "a" and the CDRs of a second antigen binding domain can be represented by 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.

The first antigen binding domain and the second antigen binding domain are each associated with a separate multimeric domain. As used herein, a "multimeric domain" is any macromolecule, protein, having the ability to associate with a second multimeric domain of the same or similar structure or composition A polypeptide, peptide or amino acid. In the context of the present disclosure, the multimerizing component is the Fc portion of an immunoglobulin (comprising C _H 2-C _H 3 domain), for example an Fc domain of IgG selected from isotypes IgG1, igG2, igG3 and IgG4, and any isotype within each isotype group.

Bispecific antibodies of the present disclosure generally comprise two multimeric domains, e.g., two Fc domains, each independently being part of a separate antibody heavy chain. The first and second multimeric domains may have the same IgG isotype, e.g., igG1/IgG1, igG2/IgG2, igG4/IgG4. Alternatively, the first and second multimeric domains may have different IgG isotypes, e.g., igG1/IgG2, igG1/IgG4, igG2/IgG4, etc.

Any bispecific antibody format or technique can be used to prepare bispecific antigen binding molecules of the disclosure. For example, an antibody or fragment thereof having a first antigen binding specificity may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen binding specificity, to produce a bispecific antigen binding molecule. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, scFv-based formats or diabody bispecific formats, igG-scFv fusions, double Variable Domains (DVD) -Ig, tetravalent body tumors, carina-in-pockets, common light chains (e.g., common light chains with carina-in-pockets, etc.), crossMab, crossFab, (SEED) bodies, leucine zippers, duobody, igG1/IgG2, double Acting Fab (DAF) -IgG, and Mab, for example ² Bispecific formats (for reviews of the foregoing formats, see, e.g., klein et al 2012, mAbs 4:6,1-11 and references cited therein).

In the context of bispecific antibodies of the present disclosure, an Fc domain may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) as compared to a wild-type, naturally occurring form of the Fc domain. For example, the disclosure includes bispecific antigen binding molecules comprising one or more modifications in the Fc domainDecoration, which results in a modified Fc domain having altered binding interactions (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule comprises C _H 2 or C _H 3, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH ranging from about 5.5 to about 6.0). Some non-limiting examples of such Fc modifications are disclosed in U.S. patent publication No.20150266966, which is incorporated herein in its entirety.

The present disclosure also includes a method comprising a first C _H 3 domain and second Ig C _H 3 domain bispecific antibody wherein the first and second Ig C _H The 3 domains differ from each other by at least one amino acid, and wherein the difference in at least one amino acid reduces binding of the bispecific antibody to protein a as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C _H 3 domain binding protein A, and a second Ig C _H The 3 domain comprises mutations that reduce or eliminate protein A binding, such as H95R modifications (IMGT exon numbering; H435R (EU numbering)). Second C _H 3 may also comprise a Y96F modification (IMGT; Y436F (EU)). Second C _H Other modifications that may be present in 3 include: in the case of the IgG1 antibody, D16E, L, 18M, N, S, K, N, V M, and V821 (IMGT; D356E, L358M, N384S, K, 392, N, V, 397M, and V4221 (EU)); in the case of IgG2 antibodies, N44S, K N and V821 (IMGT; N384S, K392N and V4221 (EU)); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E Q, and V821 (IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 (EU)).

In certain embodiments, the Fc domains may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, the chimeric Fc domain may comprise part or all of a polypeptide derived from human IgG1, human IgG2, or human IgG 4C _H C of zone 2 _H 2, and a part or all of C derived from human IgG1, human IgG2, or human IgG4 _H 3 sequence. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise and be derived from The "lower hinge" sequence of a human IgG1, human IgG2 or human IgG4 hinge region combines the "upper hinge" sequences derived from a human IgG1, human IgG2 or human IgG4 hinge region. One specific example of a chimeric Fc domain that may be included in any of the antigen binding molecules shown herein comprises from N-terminus to C-terminus: [ IgG4C _H1 ]- [ IgG4 upper hinge]- [ IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen binding molecules shown herein comprises from N-terminus to C-terminus: [ IgG 1C _H1 ]- [ IgG1 upper hinge]- [ IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen binding molecules of the present disclosure are described in U.S. patent publication No.20140243504, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements may have altered Fc receptor binding, which in turn affects Fc effector function.

According to certain exemplary embodiments of the present disclosure, the bispecific anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof comprises a heavy chain variable region (a-HCVR and B-HCVR), a Light Chain Variable Region (LCVR), and/or a Complementarity Determining Region (CDR) comprising any amino acid sequence of a bispecific anti-CD 20/anti-CD 3 antibody as shown in U.S. patent publication No. 20150266966. In certain exemplary embodiments, bispecific anti-CD 20/anti-CD 3 antibodies, or antigen-binding fragments thereof, useful in the context of the methods of the present disclosure comprise: (a) a first antigen binding arm that binds CD20, comprising: a heavy chain complementarity determining region (a-HCDR 1, a-HCDR2, and a-HCDR 3) comprising a heavy chain variable region (a-HCVR) of the amino acid sequence of SEQ ID No. 11 and a light chain complementarity determining region (LCDR) comprising a Light Chain Variable Region (LCVR) of the amino acid sequence of SEQ ID No. 12, and (b) a second antigen binding arm binding to CD3 comprising: heavy chain CDRs (B-HCDR 1, B-HCDR2 and B-HCDR 3) of a HCVR (B-HCVR) comprising the amino acid sequence of SEQ ID NO. 13 and light chain CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO. 12. According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO. 14; A-HCDR2 comprises the amino acid sequence of SEQ ID NO. 15; A-HCDR3 comprises the amino acid sequence of SEQ ID NO. 16; LCDR1 comprises the amino acid sequence of SEQ ID NO. 17; LCDR2 comprises the amino acid sequence of SEQ ID NO. 18; LCDR3 comprises the amino acid sequence of SEQ ID NO. 19; B-HCDR1 comprises the amino acid sequence of SEQ ID NO. 20; B-HCDR2 comprises the amino acid sequence of SEQ ID NO. 21; and B-HCDR3 comprises the amino acid sequence of SEQ ID NO. 22. In other embodiments, the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprises: (a) A first antigen binding arm comprising a HCVR (A-HCVR) comprising SEQ ID NO. 11 and a LCVR comprising SEQ ID NO. 12; and (B) a second antigen binding arm comprising a HCVR (B-HCVR) comprising SEQ ID NO. 13 and a LCVR comprising SEQ ID NO. 12.

In one embodiment, the bispecific antibody comprises: a first heavy chain of an HCVR comprising a first antigen binding domain, a second heavy chain of an HCVR comprising a second antigen binding domain, and a common light chain of an LCVR comprising first and second antigen binding domains, wherein the first heavy chain comprises the amino acid sequence of SEQ ID No. 23. In one embodiment, the bispecific antibody comprises: a first heavy chain of the HCVR comprising a first antigen binding domain, a second heavy chain of the HCVR comprising a second antigen binding domain, and a common light chain of the LCVR comprising the first and second antigen binding domains, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 25. In one embodiment, the bispecific antibody comprises: a first heavy chain of an HCVR comprising a first antigen binding domain, a second heavy chain of an HCVR comprising a second antigen binding domain, and a common light chain of an LCVR comprising both the first and second antigen binding domains, wherein the light chain comprises the amino acid sequence of SEQ ID No. 24. In one embodiment, the bispecific antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO. 23, a second heavy chain comprising the amino acid sequence of SEQ ID NO. 25, and a common light chain comprising the amino acid sequence of SEQ ID NO. 24.

Other bispecific anti-CD 20/anti-CD 3 antibodies that can be used in the context of the methods of the present disclosure include, for example, any of the antibodies shown in U.S. patent publication nos. 20140088295 and 20150166661. In some embodiments, the bispecific anti-CD 20/anti-CD 3 antibody may be REGN1979 (also known as ornitumumab), as shown in PCT publication WO2020047389 A1.

Combination therapy

According to certain embodiments, the methods of the present disclosure comprise administering to a subject in need thereof an anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the method comprises administering one or more doses of an anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof to the subject prior to administering one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof to the subject. Upon administration of one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof, the anti-PD-1 antibody or antigen-binding fragment thereof may be administered in combination with one or more doses of the anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof.

In certain embodiments, the methods of the present disclosure comprise administering antibodies with additive or synergistic activity to treat cancer, preferably heme cancer, more preferably B cell cancer (e.g., non-hodgkin's lymphoma or acute lymphoblastic leukemia). As used herein, the expression "in combination with … …" means that the anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof is administered before, after or simultaneously with the anti-PD-1 antibody or antigen-binding fragment thereof. The term "in combination with … …" also includes sequential or simultaneous administration of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof. For example, when administered "before" the anti-PD-1 antibody, one or more doses of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof may be administered more than about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week before the administration of one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof. By "concurrently" with an anti-PD-1 antibody is meant that the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof is administered to a subject in a separate dosage form, or in a single combined dosage formulation comprising both the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof, within less than 5 minutes of administration of the anti-PD-1 antibody or antigen-binding fragment thereof (before, after, or simultaneously).

In certain embodiments, the methods of the present disclosure comprise administering a third therapeutic agent, wherein the third therapeutic agent is an anti-cancer drug. As used herein, "anticancer drug" means any agent used to treat cancer, including but not limited to cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P' - (DDD)), biologicals (e.g., antibodies and interferons), and radiopharmaceuticals. As used herein, "cytotoxin or cytotoxic agent" also refers to a chemotherapeutic agent and refers to any agent that is detrimental to cells. Examples include TAXOL (paclitaxel), temozolomide, cytochalasin B (cytochalasin B), poncirin D (gramicidin D), ethidium bromide, emetidine, cisplatin, mitomycin, etoposide (etoposide), teniposide (teniposide), vincristine (vincristine), vinblastine (vinblastine), colchicine, doxorubicin (doxorubicin), daunorubicin (daunorubicin), dihydroxyanthrax dione, mitoxantrone (mitoxantrone), mithramycin (mithramycin), actinomycin D (actinomycin D), 1-dehydrogenine, glucocorticoid, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranol (procaine), and puromycin (puromycin), and homologs thereof.

In certain embodiments, the methods of the present disclosure include administering a third therapeutic agent selected from the group consisting of radiation, surgery, cancer vaccine, PD-L1 inhibitor (e.g., anti-PD-L1 antibody), LAG-3 inhibitor, CTLA-4 inhibitor (e.g., ipilimumab)), TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, CD38 inhibitor, CD28 activator, another antagonist of a T cell co-inhibitor or ligand (e.g., an antibody directed against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2, 3-dioxygenase (IDO) inhibitor, vascular Endothelial Growth Factor (VEGF) antagonist [ e.g., "VEGF-Trap" or other VEGF inhibitory fusion proteins as shown in U.S. patent No.7,087,411, or anti-VEGF antibody or antigen binding fragment thereof (e.g., bevacizumab) or ranibizumab (ranib)), a receptor (e.g., a small molecule of the like, e.g., panitumumab), an agonist (e.g., panitumumab), a receptor (e.g., panaxadib), a receptor (e.g., panaxan agonist (37), a receptor (e.g., panaxadiab), a receptor (p), a receptor (e.g., panaxazetidine), a receptor (p) or a receptor (p-35, a receptor (panaxatrix), a receptor (e.g., panaxbib), a receptor (panaxa receptor (e.g., panaxbib-9) CA125, melanoma-associated antigen 3 (MAGE 3), carcinoembryonic antigen (CEA), vimentin, tumor M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and antibodies to CA 19-9), vaccines (e.g., BCG, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony stimulating factor), cytotoxins, oncolytic viruses, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), radiation therapy, IL-6R inhibitors (e.g., sha Lilu mab (sarilumab)), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-12, IL-21, and IL-15), antibody-drug conjugates (ADCs) (e.g., anti-CD 19-DM4 ADC and anti-DS 6-DM4 ADC), chimeric antigen receptor T cells (e.g., CD 19-targeted T cells), anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), dietary supplements (e.g., antioxidants), and combinations thereof.

Pharmaceutical composition and administration

The present disclosure includes methods comprising administering to a subject an anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antibodies are contained in separate or combined (single) pharmaceutical compositions. The pharmaceutical compositions of the present disclosure may be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerability, and the like. A number of suitable formulations can be found in the prescription set known to all pharmaceutical chemists: remington's Pharmaceutical Sciences, mack Publishing Company, easton, pa. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (e.g., LIPOFECTIN), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al PDA (1998) J Pharm Sci Technol 52:52:238-311.

A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., wu et al, 1987, j. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (mucocutaneous lining) (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents.

The pharmaceutical compositions of the present disclosure may be delivered subcutaneously or intravenously with standard needles and syringes. Furthermore, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically use a replaceable cartridge containing a pharmaceutical composition. Once all of the pharmaceutical composition within 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 may then be reused. In disposable pen delivery devices, there is no replaceable cartridge. In contrast, disposable pen delivery devices are pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is free of the pharmaceutical composition, the entire device is discarded.

In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used; see, e.g., medical Applications of Controlled Release, langer and Wise (eds.), 1974, crc Pres., boca Raton, fla. In another embodiment, the controlled release system may be placed in proximity to the target of the composition, thus requiring only a portion of the systemic dose (see, e.g., goodson,1984, in Medical Applications of Controlled Release (supra), vol.2, pp.115-138). Other controlled release systems are discussed in Langer's review (1990,Science 249:1527-1533).

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, instillation, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibodies or salts thereof in a sterile aqueous or oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with suitable solubilizing agents such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [ e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adducts of hydrogenated castor oil) ] and the like. As the oily medium, for example, sesame oil, soybean oil, or the like is used, which may be used in combination with a solubilizing agent (for example, benzyl benzoate, benzyl alcohol, or the like). The injection thus prepared is preferably filled in a suitable ampoule.

Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared in unit dosage forms suitable for the dosage of the active ingredient. Such dosage forms in unit dosage form include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like.

Administration protocol

The present disclosure includes methods comprising administering a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof to a subject at a dosing frequency of about four times per week, twice per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per eight weeks, once per twelve weeks, or less frequently, so long as a therapeutic response is achieved. In certain embodiments, the disclosure includes methods comprising administering an anti-PD-1 antibody, or antigen-binding fragment thereof, to a subject at a dosing frequency of about four times per week, twice per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per eight weeks, once per twelve weeks, or less frequently, so long as a therapeutic response is achieved. In certain embodiments, the methods involve administering an anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof at a dosing frequency of about four times a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently, so long as a therapeutic response is achieved.

According to certain embodiments of the present disclosure, multiple doses of an anti-CD 20/anti-CD 3 bispecific antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof may be administered to a subject over a defined period of time. Methods according to this aspect of the disclosure include sequentially administering one or more doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof to a subject.

As used herein, "sequentially administering" means that each dose of antibody is administered to a subject at different time points, e.g., on different days, at predetermined intervals (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof, followed by one or more second doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof, and optionally followed by one or more third doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof. In certain embodiments, the method further comprises sequentially administering a single initial dose of an anti-PD-1 antibody or antigen-binding fragment thereof to the patient, followed by one or more second doses of an anti-PD-1 antibody or antigen-binding fragment thereof, and optionally followed by one or more third doses of an anti-PD-1 antibody or antigen-binding fragment thereof.

According to certain embodiments of the present disclosure, multiple doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof may be administered to a subject over a defined period of time. The method according to this aspect of the disclosure comprises sequentially administering to a subject multiple doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof. As used herein, "sequentially administering" means that each dose of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with the anti-PD-1 antibody or antigen-binding fragment thereof is administered to a subject at different time points separated by a predetermined interval (e.g., hours, days, weeks, or months), e.g., on different days.

The terms "initial dose", "second dose" and "third dose" refer to the temporal sequence of administration. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "baseline dose"); a "second dose" is a dose administered after the initial dose; and "third dose" is the dose administered after the second dose. The initial, second and third doses may all comprise the same amount of antibody (bispecific antibody or anti-PD-1 antibody). However, in certain embodiments, during the course of treatment, the amounts contained in the initial, second, and/or third doses are different from each other (e.g., adjusted upward or downward as the case may be). In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen in a "loading dose" followed by subsequent doses (e.g., a "maintenance dose") that are administered based on a lower frequency. For example, bispecific antibodies can be administered to a patient suffering from B cell carcinoma at a loading dose of about 1 to 3mg/kg, followed by one or more maintenance doses of about 0.1mg/kg to about 15mg/kg of patient body weight.

In one exemplary embodiment of the present disclosure, each second dose and/or third dose is 1/2 to 14 weeks after the previous dose (e.g., 1/2, 1 ¹ / ₂ 、2、2 ¹ / ₂ 、3、3 ¹ / ₂ 、4、4 ¹ / ₂ 、5、5 ¹ / ₂ 、6、6 ¹ / ₂ 、7、7 ¹ / ₂ 、8、8 ¹ / ₂ 、9、9 ¹ / ₂ 、10、10 ¹ / ₂ 、11、11 ¹ / ₂ 、12、12 ¹ / ₂ 、13、13 ¹ / ₂ 、14、14 ¹ / ₂ Or more weeks). The phrase "previous dose" as used herein means a dose of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof (and/or an anti-PD-1 antibody or antigen-binding fragment thereof) administered to a patient in a sequence of multiple administrations without intervening doses immediately prior to the administration of the next dose in the sequence.

Methods according to this aspect of the disclosure may include administering to the patient any number of second and/or third doses of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof (and/or the anti-PD-1 antibody or antigen-binding fragment thereof). For example, in certain 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. Also, in certain 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.

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 1 to 2 weeks after 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 2 to 4 weeks after the previous dose. Alternatively, the frequency of the second and/or third doses administered to the patient may vary over the course of the treatment regimen. The doctor can also adjust the frequency of administration during the course of treatment according to the needs of the individual patient after the clinical examination.

In certain embodiments, one or more doses of the bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and/or the anti-PD-1 antibody or antigen-binding fragment thereof are administered at the beginning of the treatment regimen as an "induction dose" based on a higher frequency (twice weekly, once weekly, or once 2 weeks), followed by a subsequent dose ("consolidation dose" or "maintenance dose") based on a lower frequency (e.g., once 4 to 12 weeks) of administration.

The present disclosure includes methods comprising sequentially administering to a patient a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof to treat B cell cancer (e.g., non-hodgkin's lymphoma, acute lymphoblastic leukemia). In some embodiments, the methods comprise administering one or more doses of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof, followed by one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the methods comprise administering a single dose of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof, followed by one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, one or more bispecific antibodies may be administered at a dose of about 0.1mg/kg to about 15mg/kg, followed by one or more anti-PD-1 antibodies or antigen-binding fragments thereof at a dose of about 0.1mg/kg to about 20mg/kg, to inhibit tumor growth and/or prevent tumor recurrence in a subject with B cell cancer. In some embodiments, administration of a bispecific antibody at one or more doses followed by administration of one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof results in increased anti-tumor efficacy (e.g., greater tumor growth inhibition, increased prevention of tumor recurrence compared to untreated subjects or subjects administered either antibody as monotherapy).

Dosage of

The amount of bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof and/or anti-PD-1 antibody or antigen-binding fragment thereof administered to a subject according to the methods of the present disclosure is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount of an antibody (bispecific anti-CD 20/anti-CD 3 or antigen binding fragment thereof, or an antibody anti-PD-1 antibody or antigen binding fragment thereof) that results in one or more of the following compared to an untreated subject or a subject administered either antibody as monotherapy: (a) Reducing the severity or duration of B cell cancer symptoms; (b) Inhibit tumor growth, or increase tumor necrosis, tumor shrinkage, and/or tumor disappearance; (c) tumor growth and occurrence delay; (d) inhibiting or delaying or preventing tumor metastasis; (e) preventing recurrence of tumor growth; (f) increasing survival of a subject having B cell cancer; (g) Reduced use or need for conventional anti-cancer therapies (e.g., reduced or eliminated use of chemotherapeutic or cytotoxic agents); and/or (h) reduced cytokine release or reduced immune related adverse events.

In the case of bispecific anti-CD 20/anti-CD 3 antibodies or antigen-binding fragments thereof, the therapeutically effective amount may be about 0.02mg, about 0.05mg, about 0.1mg, about 0.5mg, about 1mg, about 5mg, about 10mg, about 20mg, about 40mg, about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg, about 500mg, about 520mg, about 540mg, about 560mg, about 580mg about 660mg, about 680mg, about 700mg, 720mg, about 740mg, about 760mg, about 780mg, about 800mg, 820mg, about 840mg, about 860mg, about 880mg, about 900mg, about 920mg, about 940mg, about 960mg, about 980mg, about 1000mg, about 1020mg, about 1040mg, about 1060mg, about 1080mg, about 1100mg, about 1120mg, about 1140mg, about 1160mg, about 1180mg, about 1200mg of the bispecific anti-CD 20/anti-CD 3 antibody or antigen binding fragment thereof.

In the case of an anti-PD-1 antibody, or antigen-binding fragment thereof, a therapeutically effective amount may be from about 0.05mg to about 1500mg, for example, about 0.05mg, about 0.1mg, about 1.0mg, about 1.5mg, about 2.0mg, about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 620mg, about 640mg, about 660mg, about 680mg, about 700mg, about 720mg, about 740mg, about 760mg, about 780mg, about 800mg, about 900mg, about 1000mg, about 1050mg, about 1100mg, or about 1200mg of the anti-PD-1 antibody. In certain embodiments, 250mg of the anti-PD-1 antibody is administered. In certain embodiments, 350mg of an anti-PD-1 antibody or antigen-binding fragment thereof is administered.

The amount of bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof or anti-PD-1 antibody or antigen-binding fragment thereof contained in a single dose may be expressed in terms of milligrams of antibody per kilogram of subject body weight (i.e., mg/kg or mpk). In certain embodiments, the anti-PD-1 antibodies or bispecific anti-CD 20/anti-CD 3 antibodies used in the methods of the present disclosure may be administered to a subject at a dose of about 0.0001 to about 100mg/kg of subject body weight. For example, an anti-PD-1 antibody or antigen-binding fragment thereof may be administered at a dose of about 0.1mg/kg to about 20mg/kg of patient body weight. The bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof may be administered at a dose of about 0.1mg/kg to about 15mg/kg of patient body weight.

Additional definitions

To facilitate an understanding of specific embodiments of compositions and methods according to the present disclosure, some express definitions are provided to facilitate an express disclosure of aspects of the present disclosure. Unless defined otherwise, 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 belongs.

As used herein, "pharmaceutical agent" means a chemical compound, a mixture of chemical compounds, a biological macromolecule (e.g., a nucleic acid, an antibody, a protein, or a portion thereof (e.g., a peptide)), or an extract made from biological material such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may make them suitable as "therapeutic agents", which are biologically, physiologically or pharmacologically active substances that act locally or systemically in a subject.

As used herein, "therapeutic agent," "therapeutically effective agent," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that imparts some beneficial effect upon administration to a subject. Beneficial effects include enabling diagnostic determinations; improvement of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally combat diseases, symptoms, disorders or pathological conditions.

As used herein, the term "disease" is intended to be generally synonymous and used interchangeably with the terms "disorder" and "condition" (as in a medical condition), as they both reflect an abnormal condition (e.g., inflammatory disorder) of one of the human or animal body or its parts that impair normal function, the term "disease" is generally manifested by distinguishing between signs and symptoms, and results in a reduction in the duration or quality of life of the human or animal.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not destroy the biological activity or characteristics of the composition and is relatively non-toxic, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, a "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, that participate in carrying or transporting the compounds of the present disclosure within or to a subject so that they can perform their intended function. Typically, such compounds are carried or transported from one organ or body part to another organ or body part. Each salt and carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of materials that may be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; phosphate buffer solution; a diluent; granulating agent; a lubricant; an adhesive; a disintegrant; a wetting agent; an emulsifying agent; a colorant; a release agent; a coating agent; a sweetener; a flavoring agent; a flavoring agent; a preservative; an antioxidant; a plasticizer; a gelling agent; a thickener; a hardening agent; a setting agent; a suspending agent; a surfactant; a humectant; a carrier; a stabilizer; and other non-toxic compatible substances for pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like that are compatible with the activity of one or more components of the present disclosure and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

The term "in vitro" as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, etc., rather than within a multicellular organism.

The term "in vivo" as used herein refers to events occurring within multicellular organisms such as non-human animals.

Unless the context clearly indicates otherwise, nouns without quantitative word modifications as used herein mean one and more.

The terms "comprising," "including," "containing," or "having," and variations thereof, as used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter, unless otherwise specified.

As used herein, the phrases "in one embodiment," "in embodiments," "in some embodiments," and the like are reused. Such phrases are not necessarily referring to the same embodiment, but they may refer to the same embodiment unless the context indicates otherwise.

As used herein, the term "and/or"/"means any item, any combination of items, or all items associated with the term.

As used herein, the word "substantially" does not exclude "complete", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the present disclosure, if necessary.

As used herein, the term "each" when used in relation to a collection of items is intended to identify a single item in the collection, but does not necessarily refer to each item in the collection. An exception may occur if an explicit disclosure or context clearly dictates otherwise.

As used herein, the term "about" or "approximately" when applied to one or more destination values refers to values similar to the reference value. In some embodiments, unless otherwise indicated or apparent from the context, the term "about" or "approximately" refers to a value that falls within any direction (greater than or less than) of the reference value(s) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (except where such number would exceed 100% of the possible values). Unless otherwise indicated herein, the term "about" is intended to include values approaching the stated range, e.g., weight percentages, which are equivalent in terms of the function of the individual ingredients, compositions or embodiments.

As disclosed herein, a plurality of value ranges are provided. It is to be understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Every smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where neither, nor both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. With respect to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, they may occur in any order unless otherwise indicated. Where a method includes a combination of steps, each and every combination or sub-combination of steps is encompassed within the scope of the present disclosure unless otherwise indicated herein.

Each of the publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety to the extent they do not contradict the present disclosure. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior application. Furthermore, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Examples

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

Example 1

Targeted treatment of cancer has the potential to specifically recognize and destroy cancer cells while minimizing adverse side effects. However, these new treatments present unique challenges in determining the initial dose that is safe for the patient and avoids unwanted immune cell activation. The present disclosure demonstrates how to use human immune cell-based assays to assess the predicted level of immune cell activation over a range of drug concentrations, and to address these challenges in a way that supports the lowest expected biological effect level (minimum anticipated biological effect level, MABEL) and facilitates clinical study design. Although the objective of combination therapy is to enhance the activity of immune cells against cancer cells, it is desirable to assess the potential risk associated with immune cell over-activation. The assays disclosed herein are directed to solving the safety issue and provide basis for the suggested dosing regimen involving cimip Li Shan anti + ornitumumab.

Current immunotherapeutic molecules for cancer treatment aim to enhance T cell mediated tumor clearance by one or more of the following: (1) TCR/CD3: t cells are activated by T cell receptor complex recognition of MHC peptide complexes (termed "signal 1") or bypassed by the use of bispecific antibody activation-related CD3 signaling molecules targeting tumor-associated antigen (tamor-associated antigen, TAA) and CD 3; (2) co-stimulatory signals: the involvement of co-stimulatory receptors such as CD28 serves as "signal 2" for T cell activation, which is necessary for enhancement of T cell mediated immune responses. CD28 may be activated by the natural ligands CD80 and CD86 expressed on antigen-presenting cells (APC), or bypassed by bispecific antibodies targeting tumor-associated antigens (TAA) and CD 28; and (3) inhibit signal: inhibitory receptors whose expression on activated T cells is significantly enhanced, such as PD-1, CTLA-4 (competing with CD28 for binding to CD80 and CD 86) and LAG-3, reduce T cell activation upon binding to their corresponding ligands and thus need to be blocked to release inhibition (braking) of T cell activation.

The possibility that the combination of ornitumumab and PD-1 block was more effective, especially when tumors express multiple TAAs and/or are PD-l1+. For this reason, primary human T cell assays have been developed to address these issues and to assist in the development of drug delivery strategies.

FIGS. 1A, 1B and 1C show engineered reporter assays for evaluation of the resistance to ornitumumab (REGN 1979) +cimiput Li Shan. Engineered reporter T cells (Jurkat/AP 1-Luc/PD-1) were incubated with WSU-DLCL2 cells or WSU-DLCL2/PD-L1 cells and the onditumumab and cimiput Li Shan anti-titrations, the cimiput Li Shan anti-concentration was plotted on the x-axis and the onditumumab titration was represented by a different colored line. FIG. 1A shows that the titration range of the ornitumumab was 500nM to 0.05pM. FIG. 1B shows that ornitumumab stimulated AP1-Luc activity from T cells incubated with WSU-DLCL2 cells, with little to no effect observed from titration of cimiput Li Shan antibody, as expected by the lack of PD-L1 in the system. FIG. 1C shows that in the presence of PD-L1 on WSU-DLCL2, the stimulation of AP1-Luc in T cells by ornitumumab is significantly reduced, which contributes to inhibition of T cell/AP 1-Luc activity and PD-1 activation. The use of cimetidine Li Shan resistance restored activity.

FIGS. 2A, 2B and 2C show primary CD3+ T cell assays for evaluating the resistance of Ornituximab (REGN 1979) +cimetidine Li Shan. Cd3+ T cells were isolated from healthy donor PBMCs and frozen down (assay condition 1) or incubated with WSU-DLCL2 cells in the presence of 200pM (condition 2) or 1.3nM (condition 3) of ornitumumab at 37 ℃ and 5% CO2 for 72 hours. T cells were then re-isolated from WSU-DLCL2 (note that the ornitumumab molecule was still on T cells) and used in bioassays in the presence of WSU-DLCL2 or WSUDLCL 2/PD-L1. At this time, unstimulated and prestimulated T cells were also stained for CD25, CTLA-4, PD-1 and LAG 3. The ornitumumab titration range was 243nM to 0.11nM. For T cells obtained from condition 1, this is the first dose of ornitumumab. For T cells obtained from conditions 2 and 3, this is its second dose of ornitumumab. The anti-titration range for cimiput Li Shan was 300nM to 0.78pM. All T cells received only one dose of cimetidine Li Shan antibody. In addition, T cells obtained from condition 3 were also subjected to +/-anti-CTLA-4 and anti-LAG 3 combinations (50 nM each). Assay plates were incubated at 37℃for 72 hours at 5% CO 2. Supernatants were collected from all plates and subjected to ALPHALISA to detect IL-2 and IFNγ release. Only assay data from WSU/PD-L1 conditions are shown. WSU-DLCL2 wild-type data do not show any response to cimetidine Li Shan resistance, as determined to be PD-L1 negative. The results of 0.2nM pre-stimulation were similar to those of 1.3 nM.

Figures 3A, 3B, 3C, 3D, 3E, 3F and 3G show the results of an ornitumumab (REGN 1979) +cimiput Li Shan anti-induction dosimetry. T cells exposed to the introduced dose of ornitumumab underwent cytokine rescue by the cimetidine Li Shan anti-treatment. T cells isolated, frozen and receiving only one dose of ornitumumab responded minimally to treatment with cimiput Li Shan in the presence of WSU/PD-L1 cells (fig. 3A). FIG. 3B shows IL-2 levels in T cells pre-stimulated with an initial dose of 1.3nM of ornitumumab without combination. In the absence of cimetidine Li Shan antibody, 1.3nM pretreated T cells were detectable with increasing concentration of ornitumumab. An enhanced view of 1.3nM pre-stimulated T cells revealed a high sensitivity to the treatment with cimetidine Li Shan in combination with ornitumumab (fig. 3C). Release of ifnγ followed a similar trend to IL-2, cytokine levels decreased significantly with pretreatment and became more sensitive to the cimetidine Li Shan anti-combination (fig. 3D, 3E and 3F). Single treatment with ornitumumab in pre-stimulated cells resulted in increased ifnγ release with increased doses of ornitumumab. Unstimulated and prestimulated T cell staining showed higher PD1 surface expression on prestimulated T cells (fig. 3G).

FIG. 4 shows that cytokine responses from pre-stimulation can be further enhanced by blocking CTLA-4 and LAG-3. 1.3nM pre-stimulated T cells were treated with ornitumumab (REGN 1979) and cimipne Li Shan anti-titration (FIG. 3B). IL-2 response was significantly reduced compared to unstimulated T cells, but was more sensitive to the cimiput Li Shan antibody (FIG. 3A). IL-2 release was significantly improved by adding 50nM anti-CTLA-4 and anti-LAG 3 antibodies and titrating the onditumumab and cimetidine Li Shan antibodies. Ifnγ release also underwent similar rescue by treatment. According to staining data, expression of PD-1 (FIG. 3G), CTLA-4 and LAG-3 were all increased when T cells were stimulated with the Ornituximab+WSU cells, as compared to unstimulated cells.

Figure 5 shows that T cell cytotoxicity was not impaired by the pretreatment with ornitumumab (REGN 1979) and was enhanced by the cimetidine Li Shan antibody. Untreated and preactivated human T cells as well as Vybrant CFDA-SE labeled WSU/PD-L1 cells were plated with 10-fold serial dilutions of ornitumumab and cimetidine Li Shan antibody (starting from 300nM and 100nM, respectively) in complete medium at 5% CO2 at 37℃at an E:1T ratio for 3 days. After the end of the culture, surviving WSU/PDL1 cells and T cells were assessed for activation and proliferation using flow cytometry. Unstimulated T cells kill target cells in a dose-dependent manner under the treatment of ornitumumab. The treatment with cimiput Li Shan did not affect killing. At all doses tested, pre-stimulated T cells were sensitive to cimetidine Li Shan anti-treatment and combined with ornitumumab enhanced target cell killing.

By bioassays as described above, T cell mediated cytokine responses of test molecules from both single and combined treatments can be detected. For the ornitumumab + cimiput Li Shan anti-program, data can be generated rapidly by Jurkat/AP1-luc reporter assay, which provides in vitro evidence for the rationale behind this combination. From the data generated, these molecules were tested using primary human T cells in accordance with an "in" dosing model that generated a dose titration curve for cimiput Li Shan antibody in the presence of ornitumumab in vitro. This bioassay resulted in the following key findings: (i) Subsequent doses of ornitumumab produced a significantly weaker IL-2 response; (ii) T cells treated with ornitumumab are increasingly susceptible to treatment with cimiput Li Shan; and (iii) T cells significantly up-regulate inhibitory checkpoint receptors when stimulated with ornitumumab.

Example 2

Quantitative System Pharmacology (QSP) modeling framework for assessing cytokine release mediated by an Ornituximab (REGN 1979)/cimiput Li Shan anti-REGN 2810 combination therapy in patients with B-NHL

QSP is an emerging mathematical modeling approach that integrates current understanding of disease biology, drug mechanisms of action, and in vivo/in vitro/clinical data to generate hypotheses and predictions that address specific problems in both preclinical and clinical studies. This example describes a study using the QSP modeling method to evaluate clinical cytokine profiles following both the single therapy with ornitumumab and the combination therapy with cimiput Li Shan antibody (PD-1 inhibitor).

The object is: ornituximab (REGN 1979) is a bispecific antibody based entirely on human IgG4, which is associated with CD3 ⁺ T cells and CD20 ⁺ B cell binding, targeting CD20 by T cell mediated cytotoxicity ⁺ Tumor cells. The engagement of CD3 on T cells and CD20 on B cells activates T cells. During T cell activation, inflammatory cytokines are secreted which can lead to significant but temporary increases in circulating cytokine concentrations and can lead to systemic inflammatory responses, known as CRS. The study uses QSP modeling to evaluate cytokine (as represented by IL-6) profiles after both the single therapy with ornitumumab and the combination therapy with ornitumumab and cimip Li Shan antibody (REGN 2810; PD-1 inhibitor) in patients with B-NHL. One goal of this work is to predict cytokine profiles for different dosing regimens.

The method comprises the following steps: a QSP model was developed that integrates the pharmacokinetics of both ornitumumab and cimiput Li Shan antibodies, kinetics of T and B cells, disease characteristics of NHL, and cytokine data from both ornitumumab monotherapy study (NCT 02290951) and ornitumumab/cimiput Li Shan anti-combination study (NCT 02651662). In the QSP model, the mechanism of cytokine release is described as: (1) A three-molecule synapse is formed when ornitumumab binds to both CD3 on T cells and target CD20 on B cells; and (2) the PD-1 pathway regulates T cell activation. To describe the cytokine profile under combined therapy with ornitumumab/cimiput Li Shan, it was assumed that cimiput Li Shan antibody increased stimulation of IL-6 production by activating more T cells and decreased inhibition of the immune system to attenuate cytokine release. The model was calibrated using the concentrations of IL-6, ornitumumab, and cimiput Li Shan antibodies from NHL patients under monotherapy and in combination, as well as in vivo/in vitro data (e.g., tumor growth rate, CD20/CD3 expression levels, T/B cell baseline levels, and drug affinity data).

The QSP model integrates several sources of data including data describing the mechanism and pharmacokinetics of the orituzumab and ciminopril Li Shan antibodies, T cell and B cell kinetics, CD20/CD3/PD-1 expression levels, disease characteristics of NHL, and clinical cytokine data from phase 1 studies of the orituzumab monotherapy (NCT 02990951) and the orituzumab/ciminopril Li Shan anti-combination therapy (NCT 02651662). The data sources used to provide information for this QSP model are shown in table 1.

Table 1: data sources for QSP models

FIH, human first; PK, pharmacokinetics; SPD, sum of diameter products.

Conceptually, a QSP model was constructed using different modules to capture the interaction of both each drug alone and in combination with malignant lymphocytes. The cytokine release mechanism is described in two parts: (i) Cytokine release due to the formation of a three-molecule synapse when ornitumumab binds to both CD3 on T cells and CD20 on B cells after monotherapy (fig. 6A-6B); and (ii) a cytokine profile following treatment with an ornitumumab/cimiput Li Shan antibody combination; cimrpu Li Shan antibodies affected the modulation of T cell activation by interaction with the PD-1 pathway (fig. 6B-6F).

Various cytokines (e.g., IL-6, IL-10, IFN-gamma, TNF-alpha) are released upon administration of the ornitumumab. To simplify the model, IL-6 was chosen as a representative cytokine, since most cytokine time curves are similar, and IL-6 was considered a key cytokine associated with CRS (Cheng et al, J Biol Chem,288 (17): 11771-78 (2013)). The model was calibrated using IL-6 concentration data from patients with B-NHL treated in phase 1 studies of both the ornitumumab monotherapy and the ornitumumab/cimiput Li Shan anti-combination therapy. Representative schemes are depicted in fig. 7 and 8. Other in vivo/in vitro data include tumor growth rate, CD20/CD3 expression levels, T cell and B cell baseline levels, and drug affinity assays. (Cheng et al, J Biol Chem 288 (17): 11771-78 (2013)).

In this simulation, 300 virtual patients with aggressive NHL were created using the monte carlo sampling method (Monte Carlo sampling approach).

Results: in a simulated population of 300 patients with aggressive NHL, the model predicts: (i) When the ornitumumab and the cimiput Li Shan antibody were administered in combination, the IL-6 concentration was higher on week 1, day 1, as compared to the ornitumumab monotherapy, consistent with the observations of phase 1 trials (fig. 9A-9B); (ii) Delayed introduction of the cimiput Li Shan anti-co-administration attenuated IL-6 release, with progressive decrease in IL-6 release with increased delay in actuation of the cimiput Li Shan anti-actuation (fig. 10); and (iii) when cimiput Li Shan anti-administration (after the administration of ornitumumab monotherapy) was initiated from week 5, the effects of cimiput Li Shan anti-dose levels (3, 30 and 350 mg) on IL-6 concentration were expected to be minimal (fig. 11).

Model-based simulations indicate that IL-6 concentration peaks mostly on day 1 after an initial split dose of 1mg at week 1 under ornitumumab monotherapy. Cytokine concentrations decreased over time, even though the doses of ornitumumab increased to 10-fold and 100-fold at weeks 2 and 3, respectively. When both ornitumumab and cimiput Li Shan antibody were administered on day 1, the average peak of IL-6 concentration was higher than Yu Aoni for the tuzumab monotherapy due to the effect of cimiput Li Shan antibody on the PD-1/PD-L1 signaling pathway, consistent with the observed data.

Conclusion: this exploratory model-based assessment provides insight into IL-6 kinetics following both ornitumumab and cimiput Li Shan anti-treatment. Based on model simulations, the risk of IL6 release is higher if both ornitumumab and cimiput Li Shan antibody are administered simultaneously starting on day 1 of week 1. The introduction of the phase of introduction of the ornitumumab prior to the addition of the cimiput Li Shan antibody can limit the cytokine concentration to a level similar to that of the ornitumumab monotherapy.

Reference to the literature

1.Smith EJ，et al.Sci Rep.2015；5：17943.

2.Choi BD，et al.Expert Opin Biol Ther.2011；11(7)：843-53.

3.Shimabukuro-Vornhagen A，et al.J Immunother Cancer.2018；6(1)：56.

4.Cheng X，et al.J Biol Chem.2013；288(17)：11771-78.

5.Betts A，et al.AAPS J.2019；21：66.

6.Huh YO，et al.Am J Clin Pathol.2001；116(3)：437-43.

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<110> Regeneron Pharmaceuticals Inc.

HERMANN, Aynur

ULLMAN, Erica

ZHU, Min

KHAKSAR TOROGHI, Masood

<120> antibody combinations for treating cancer and alleviating cytokine release syndrome

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Claims (47)

1. A method of treating a tumor or inhibiting tumor growth comprising:

(a) Selecting a subject having cancer;

(b) Administering to the subject a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprising a first antigen-binding arm that specifically binds CD20 and a second antigen-binding arm that specifically binds CD 3; and

(c) After step (b), administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1);

wherein the method treats a tumor or inhibits tumor growth and improves Cytokine Release Syndrome (CRS) in the subject.

2. A method of ameliorating Cytokine Release Syndrome (CRS) in a subject having a tumor, comprising:

(a) Selecting a subject having cancer;

(b) Administering to the subject a therapeutically effective amount of a bispecific anti-CD 20/anti-CD 3 antibody or antigen-binding fragment thereof comprising a first antigen-binding arm that specifically binds CD20 and a second antigen-binding arm that specifically binds CD 3; and

(c) After step (b), administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1).

3. The method of claim 1 or 2, wherein step (c) further comprises administering to the subject an anti-PD-1 antibody or antigen-binding fragment thereof in combination with the bispecific antibody or antigen-binding fragment thereof.

4. The method of any one of claims 1 to 3, wherein the bispecific antibody or antigen-binding fragment thereof is administered to the subject at least about 1 week prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof.

5. The method of any one of claims 1 to 4, wherein each of the bispecific antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject in one or more doses.

6. The method of claim 5, wherein the first dose of the bispecific antibody or antigen-binding fragment thereof is administered to the subject about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks prior to administration of the first dose of the anti-PD-1 antibody or antigen-binding fragment thereof.

7. The method of claim 5, wherein the first dose of the bispecific antibody or antigen-binding fragment thereof is administered to the subject about 5 weeks prior to administration of the first dose of the anti-PD-1 antibody or antigen-binding fragment thereof.

8. The method of any one of claims 1 to 7, wherein the bispecific antibody or antigen binding fragment thereof is administered to the subject at one or more doses of about 0.1mg/kg to about 15mg/kg of body weight of the subject.

9. The method of any one of claims 1 to 7, wherein the bispecific antibody or antigen binding fragment thereof is administered to the subject in one or more doses of about 1mg to about 800 mg.

10. The method of claim 8 or 9, wherein the bispecific antibody or antigen binding fragment thereof is administered to the subject once daily, once every two days, once every three days, once every five days, once weekly, once every two weeks, once every three weeks, or once every four weeks.

11. The method of claim 8 or 9, wherein each of the one or more doses of the bispecific antibody or antigen binding fragment thereof is administered 0.5 to 12 weeks after the previous dose.

12. The method of any one of claims 8 to 11, wherein at least one of the one or more doses of the bispecific antibody or antigen binding fragment thereof comprises a dose having a greater amount of the bispecific antibody or antigen binding fragment thereof than its previous dose.

13. The method of any one of claims 8 to 12, wherein at least one of the one or more doses of the bispecific antibody or antigen binding fragment thereof is administered in two or more separate doses.

14. The method of claim 13, wherein at least one of the two or more separate doses comprises the same amount of the bispecific antibody or antigen-binding fragment thereof.

15. The method of claim 13, wherein at least one of the two or more divided doses is administered at least about 0.5 days after the previous dose.

16. The method of claim 15, wherein at least one of the two or more separate doses is administered about 1 day after the previous dose.

17. The method of any one of claims 1 to 16, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject at one or more doses of about 0.1mg/kg to about 20mg/kg of subject body weight.

18. The method of any one of claims 1 to 16, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject in one or more doses of about 1mg to about 1500 mg.

19. The method of claim 17 or 18, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject once daily, once every two days, once every three days, once every five days, once weekly, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks.

20. The method of claim 17 or 18, wherein at least one of the one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof is administered 0.5 to 12 weeks after the previous dose.

21. The method of any one of claims 17 to 20, wherein at least one of the one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof comprises a dose having a greater amount of the anti-PD-1 antibody or antigen-binding fragment thereof than its previous dose.

22. The method of any one of claims 1 to 21, wherein the bispecific antibody or antigen-binding fragment thereof or the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject intravenously, subcutaneously, or intraperitoneally.

23. The method of claim 1, wherein the subject has a cytokine release syndrome.

24. The method of any one of claims 1 to 23, wherein the tumor comprises a B cell carcinoma.

25. The method of claim 24, wherein the B-cell cancer is selected from hodgkin's lymphoma, non-hodgkin's lymphoma, follicular lymphoma, small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, lymphomatoid granulomatosis, burkitt's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, and B-cell chronic lymphocytic leukemia.

26. The method of any one of claims 1 to 25, wherein the subject is resistant to, insufficiently responsive to, or relapsed after a previous treatment.

27. The method of any one of claims 1 to 26, wherein the subject has been previously treated with an anti-CD 20 treatment.

28. The method of claim 27, wherein the anti-CD 20 treatment comprises an anti-CD 20 antibody.

29. The method of any one of claims 1 to 28, wherein the treatment produces a therapeutic effect selected from the group consisting of: delay in tumor growth, reduced tumor cell number, tumor regression, increased survival, partial response, and complete response.

30. The method of any one of claims 1 to 29, wherein the treatment produces an effect selected from the group consisting of: reduced cytokine release, reduced release of IL-2, IL-6, IL-10, TNF-alpha and/or IFN-gamma, reduced administration of dexamethasone, corticosteroids or analgesics, reduced number of immune related adverse events and reduced number of grade 3 or more adverse events.

31. The method of claim 29 or 30, wherein tumor growth is delayed by at least 10 days compared to untreated subjects.

32. The method of any one of claims 1 to 31, wherein tumor growth is inhibited by at least 50% compared to an untreated subject.

33. The method of claim 32, wherein tumor growth is inhibited by at least 50% as compared to administration of the bispecific antibody or antigen-binding fragment thereof or the anti-PD-1 antibody or antigen-binding fragment thereof as a monotherapy to a subject.

34. The method of any one of claims 1 to 33, further comprising administering a third therapeutic agent or third treatment to the subject.

35. The method of claim 34, wherein the third therapeutic agent or third treatment is selected from the group consisting of radiation, surgery, chemotherapeutic agents, cancer vaccines, PD-L1 inhibitors, LAG-3 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, CD28 activators, CD38 inhibitors, GITR agonists, indoleamine-2, 3-dioxygenase (IDO) inhibitors, vascular Endothelial Growth Factor (VEGF) antagonists, angiopoietin-2 (Ang 2) inhibitors, transforming growth factor beta (tgfβ) inhibitors, epidermal Growth Factor Receptor (EGFR) inhibitors, antibodies to tumor-specific antigens, bcg, granulocyte-macrophage colony stimulating factor, cytotoxins, interleukin 6 receptor (IL-6R) inhibitors, interleukin 4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, oncolytic viruses, anti-inflammatory drugs, dietary supplements, and combinations thereof.

36. The method of any one of claims 1 to 35, wherein the first antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (a-HCDR 1, a-HCDR2 and a-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3), and wherein a-HCDR1 comprises the amino acid sequence of SEQ ID NO 14; A-HCDR2 comprises the amino acid sequence of SEQ ID NO. 15; A-HCDR3 comprises the amino acid sequence of SEQ ID NO. 16; LCDR1 comprises the amino acid sequence of SEQ ID NO. 17; LCDR2 comprises the amino acid sequence of SEQ ID NO. 18; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19.

37. The method of any one of claims 1 to 36, wherein the first antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable region (a-HCVR) having the amino acid sequence of SEQ ID No. 11 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID No. 12.

38. The method of any one of claims 1 to 37, wherein the second antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (B-HCDR 1, B-HCDR2 and B-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3), and wherein B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 20; B-HCDR2 comprises the amino acid sequence of SEQ ID NO. 21; B-HCDR3 comprises the amino acid sequence of SEQ ID NO. 22; LCDR1 comprises the amino acid sequence of SEQ ID NO. 17; LCDR2 comprises the amino acid sequence of SEQ ID NO. 18; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19.

39. The method of any one of claims 1 to 38, wherein the second antigen-binding arm of the bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable region (B-HCVR) having the amino acid sequence of SEQ ID No. 13 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID No. 12.

40. The method of any one of claims 1 to 39, wherein the bispecific antibody comprises: a first heavy chain comprising a HCVR of a first antigen binding domain; a second heavy chain comprising a HCVR of a second antigen binding domain; and a common light chain comprising a LCVR of the first antigen-binding domain and the second antigen-binding domain, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO. 23.

41. The method of any one of claims 1 to 40, wherein the bispecific antibody comprises: a first heavy chain comprising a HCVR of a first antigen binding domain; a second heavy chain comprising a HCVR of a second antigen binding domain; and a common light chain comprising a LCVR of the first antigen-binding domain and the second antigen-binding domain, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO. 25.

42. The method of any one of claims 1 to 41, wherein the bispecific antibody comprises: a first heavy chain comprising a HCVR of a first antigen binding domain; a second heavy chain comprising a HCVR of a second antigen binding domain; and a common light chain comprising a LCVR of said first antigen binding domain and said second antigen binding domain, wherein said light chain comprises the amino acid sequence of SEQ id No. 24.

43. The method of any one of claims 1 to 35, wherein the bispecific antibody is ornitumumab.

44. The method of any one of claims 1 to 43, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR 1, HCDR2, and HCDR 3) and three light chain complementarity determining regions (LCDR 1, LCDR2, and LCDR 3), and wherein HCDR1 comprises the amino acid sequence of SEQ ID NO 3; HCDR2 comprises the amino acid sequence of SEQ ID NO. 4; HCDR3 comprises the amino acid sequence of SEQ ID NO. 5; LCDR1 comprises the amino acid sequence of SEQ ID NO. 6; LCDR2 comprises the amino acid sequence of SEQ ID NO. 7; and LCDR3 comprises the amino acid sequence of SEQ ID NO. 8.

45. The method of any one of claims 1 to 44, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID No. 1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID No. 2.

46. The method of any one of claims 1 to 43, wherein the anti-PD-1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 9 and a light chain having the amino acid sequence of SEQ ID No. 10.

47. The method of any one of claims 1 to 43, wherein the anti-PD-1 antibody is a cimrpox Li Shan antibody.