Combination therapy with anti-CD40 antibodies

Abstract

The present invention relates to combination therapies using an anti-CD40 antibody. The combination therapy comprises (a) an antibody or an antigen-binding portion thereof that specifically binds to CD40, and (b) another immunotherapeutic agent having cancer treatment efficacy, which is not an anti-CD40 antibody or an antigen-binding fragment thereof. The invention also relates to a kit and a method of using the therapy.

Description

[0001] This application is a divisional application of the Chinese patent application filed on August 12, 2015 (application number 2021114200446, entitled “Combination therapy using anti-CD40 antibodies”). Technical Field

[0002] The present invention relates to a combination therapy for treating a solid tumor in a subject, and methods of using the combination therapy. The combination therapy comprises (a) an antibody or antigen-binding fragment thereof that specifically binds to CD40, and (b) another immunotherapeutic agent that specifically binds to an immune checkpoint molecule other than CD40. The present invention also relates to a kit and methods of using the combination therapy of the present invention. Background Art

[0003] Cancer is the leading cause of premature death in developed countries. The purpose of immunotherapy in cancer is to produce an effective immune response against tumors, particularly solid tumors, by the body. This can be achieved by, for example, destroying tolerance to tumor antigens, enhancing anti-tumor immune responses, and stimulating local cytokine responses at the tumor site. The key effector cells for long-lasting anti-tumor immune responses are activated tumor-specific effector T cells. Incomplete activation of effector T cells by, for example, dendritic cells can cause T cell anergy, which leads to ineffective anti-tumor responses, while sufficient induction of dendritic cells can produce effective amplification of activated effector T cells, thereby redirecting the immune response to the tumor.

[0004] The cell surface CD40 receptor molecule is a member of the tumor necrosis factor receptor superfamily (TNFR) and is a key regulator of innate and adaptive immune responses. It is expressed on human antigen presenting cells, especially B cells, dendritic cells and macrophages, as well as on normal cells such as fibroblasts, smooth muscle cells, endothelial cells and epithelial cells. In addition, it is expressed on a wide range of tumor cells (including all B lymphomas, 30%-70% of solid tumors, melanomas and carcinomas).

[0005] The natural ligand of CD40 (called CD154 or CD40L) is mainly expressed on mature T lymphocytes. CD40L-mediated signaling triggers several biological events, including immune cell activation, proliferation, and the production of cytokines and chemokines. Therefore, cellular and immune functions are enhanced by CD40 receptor stimulation. Its role in cell-mediated immune responses is well known. For example, activation of dendritic cells by CD40 stimulation can induce activation of effector T cells. Therefore, treatment with CD40 agonists can provide a means to redirect immune responses and amplify effector T cells against tumors.

[0006] Anti-tumor effects of some anti-CD40 antibodies have been reported, of which several mechanisms have been identified. Indirect effects have been observed for CD40-negative tumors, involving activation of antigen-presenting cells, in particular increased activity of tumor-specific cytotoxic T lymphocytes and natural killer cells (NK cells). Indirect and direct anti-tumor mechanisms have been observed for CD40-positive tumors, in which CD40 antibodies bound to tumor cells induce apoptosis. These mechanisms of anti-tumor activity can be supplemented by antibody-mediated cytotoxicity (ADCC). However, administration of anti-CD40 antibodies also produces adverse side effects, such as cytokine release syndrome.

[0007] Therefore, there remains a need for improved cancer therapies, particularly anti-CD40 antibodies and combination therapies thereof that are suitable for treating solid tumors. Summary of the invention

[0008] A first aspect of the invention provides a combination therapy for treating a solid tumor in a subject, comprising (a) an antibody or an antigen-binding portion thereof that specifically binds to CD40, and (b) another immunotherapeutic agent with cancer treatment efficacy, which is not an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO:7 and a heavy chain variable region of SEQ ID NO:8 for binding to human CD40, and wherein the additional immunotherapeutic agent specifically binds to an immune checkpoint molecule other than CD40.

[0009] In one embodiment, the solid tumor is selected from adenoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms tumor, lung tumor, colon tumor, lymphoma, breast tumor and melanoma. For example, the solid tumor can be a melanoma, such as a metastatic melanoma.

[0010] In one embodiment, the antibody or antigen binding portion thereof that specifically binds CD40 comprises or consists of an intact antibody.

[0011] In an alternative embodiment, the antibody or antigen-binding portion thereof that specifically binds to CD40 comprises or consists of an antigen-binding fragment selected from the group consisting of an Fv fragment (e.g., a single-chain Fv fragment or a disulfide-bonded Fv fragment) and a Fab-like fragment (e.g., a Fab fragment; a Fab' fragment or a F(ab)2 fragment).

[0012] In one embodiment, the antibody, or antigen-binding portion thereof, is human or humanized.

[0013] In one embodiment, the antibody or antigen binding portion thereof that specifically binds CD40 comprises at least one CDR selected from SEQ ID NOs 1, 2, 3, 4, 5 and 6, or a 'core' CDR sequence thereof (for sequence details, see Example 1 below).

[0014] Thus, the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the CDR sequences of SEQ ID NOs: 1, 2 and 3 and / or SEQ ID NOs: 4, 5 and 6, or 'core' CDR sequences thereof.

[0015] For example, the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain variable region of SEQ ID NO:7 and / or the heavy chain variable region of SEQ ID NO:8.

[0016] In one embodiment, the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain constant region of SEQ ID NO:11 and / or the heavy chain constant region of SEQ ID NO:12.

[0017] Thus, the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of SEQ ID NO:7 plus the light chain of SEQ ID NO:11, and / or SEQ ID NO:8 plus the heavy chain of SEQ ID NO:12.

[0018] The combination therapy of the present invention further comprises another immunotherapeutic agent that is effective in treating cancer and specifically binds to immune checkpoint molecules other than CD40. It should be understood that the therapeutic benefit of the additional immunotherapeutic agent can be mediated by weakening the function of inhibitory immune checkpoint molecules and / or by activating the function of stimulatory immune checkpoint molecules.

[0019] In another embodiment, the additional immunotherapeutic agent is selected from:

[0020] (a) Immunotherapeutic agents that bind to PD-1;

[0021] (b) immunotherapeutic agents that bind to CTLA-4;

[0022] (c) an immunotherapeutic agent that binds OX40; and

[0023] (d) Immunotherapeutic agents that bind CD137.

[0024] Therefore, the additional immunotherapeutic agent may be a PD1 inhibitor, such as an anti-PD1 antibody or an antigen-binding fragment thereof capable of inhibiting PD1 function (e.g., Nivolumab, Pembrolizumab, Lambrolizumab, Pidizumab and AMP-224). Alternatively, the PD1 inhibitor may comprise or consist of an anti-PD-L1 antibody or an antigen-binding fragment thereof capable of inhibiting PD1 function (e.g., MEDI-4736 and MPDL3280A).

[0025] In another embodiment, the additional immunotherapeutic agent is a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody or antigen-binding portion thereof.

[0026] In another embodiment, the additional immunotherapeutic agent activates OX40, such as an agonistic anti-OX40 antibody or antigen binding portion thereof.

[0027] In another embodiment, the additional immunotherapeutic agent activates CD137, such as an agonistic anti-CD137 antibody or antigen binding portion thereof.

[0028] It will be appreciated by those skilled in the art that the presence of two active agents (as described above) can provide a synergistic benefit in treating a solid tumor in a subject. "Synergy" includes a combined therapeutic effect of the two agents (e.g., as determined by reference to growth rate or tumor size) that is greater than the additive therapeutic effect of the two agents administered alone. Such synergy can be identified by testing the active agents alone and in combination in relevant cell line models of solid tumors.

[0029] Optionally, the combination therapy further comprises a third immunotherapeutic agent having cancer treating efficacy.

[0030] For example, the combination therapy may comprise (a) an antibody, or an antigen binding portion thereof, that specifically binds CD40, (b) an antibody, or an antigen binding portion thereof, that specifically binds PD1 or PD-L1, and (c) an antibody, or an antigen binding portion thereof, that specifically binds CTLA-4.

[0031] A second aspect of the present invention provides an antibody or antigen-binding portion thereof that specifically binds to CD40 for use in a method for treating a solid tumor, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO:7 and a heavy chain variable region of SEQ ID NO:8 for binding to human CD40, and wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 is used in combination with another immunotherapeutic agent having cancer treatment efficacy, which immunotherapeutic agent is not an anti-CD40 antibody or antigen-binding fragment thereof.

[0032] In one embodiment, the antibody, or antigen binding portion thereof, that specifically binds CD40 is as discussed above in relation to the first aspect of the invention.

[0033] In one embodiment, the additional immunotherapeutic agent having cancer treating efficacy is as discussed above in relation to the first aspect of the invention.

[0034] A related third aspect of the present invention provides use of an antibody or an antigen-binding portion thereof that specifically binds to CD40 in the preparation of a medicament for treating solid tumors, wherein the antibody or the antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO:7 and a heavy chain variable region of SEQ ID NO:8 for binding to human CD40, and wherein the antibody or the antigen-binding portion thereof that specifically binds to CD40 is used in combination with another immunotherapeutic agent having cancer treatment efficacy, which immunotherapeutic agent is not an anti-CD40 antibody or antigen-binding fragment thereof.

[0035] In one embodiment, the antibody, or antigen binding portion thereof, that specifically binds CD40 is as discussed above in relation to the first aspect of the invention.

[0036] In one embodiment, the additional immunotherapeutic agent having cancer treating efficacy is as discussed above in relation to the first aspect of the invention.

[0037] The fourth aspect of the present invention provides a pharmaceutical composition comprising (a) an antibody or an antigen-binding portion thereof that specifically binds to CD40, and (b) another immunotherapeutic agent with cancer treatment efficacy, which is not an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8 for binding to human CD40.

[0038] In one embodiment, the antibody, or antigen binding portion thereof, that specifically binds CD40 is as discussed above in relation to the first aspect of the invention.

[0039] In one embodiment, the additional immunotherapeutic agent having cancer treating efficacy is as discussed above in relation to the first aspect of the invention.

[0040] The fifth aspect of the present invention provides a kit for treating solid tumors, comprising (a) an antibody or an antigen-binding portion thereof that specifically binds to CD40, and (b) another immunotherapeutic agent with cancer treatment efficacy, wherein the immunotherapeutic agent is not an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8 for binding to human CD40.

[0041] In one embodiment, the antibody, or antigen binding portion thereof, that specifically binds CD40 is as discussed above in relation to the first aspect of the invention.

[0042] In one embodiment, the additional immunotherapeutic agent having cancer treating efficacy is as discussed above in relation to the first aspect of the invention.

[0043] A sixth aspect of the present invention provides a method for treating a solid tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of: (a) administering to the subject a therapeutically effective amount of an antibody or an antigen-binding portion thereof that specifically binds to CD40, and (b) administering to the subject a therapeutically effective amount of another immunotherapeutic agent with cancer treatment efficacy, the immunotherapeutic agent being not an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8 for binding to human CD40.

[0044] In one embodiment, the antibody, or antigen binding portion thereof, that specifically binds CD40 is as discussed above in relation to the first aspect of the invention.

[0045] In one embodiment, the additional immunotherapeutic agent having cancer treating efficacy is as discussed above in relation to the first aspect of the invention.

[0046] In another embodiment, steps (a) and (b) are performed simultaneously or wherein step (b) is performed from 24 hours to two weeks after step (a), from 24 hours to one week after step (a), from 24 hours to 72 hours after step (a), or from 24 hours to 48 hours after step (a).

[0047] In one embodiment, step (a) comprises locally administering the antibody to the tumor site.

[0048] In one embodiment, at least 30% of the amount of antibody administered in step (a) remains at the tumor site 4 hours after administration, preferably wherein at least 40% of said amount remains at the tumor site 4 hours after administration.

[0049] In one embodiment, the additional therapeutic agent of step (b) is formulated with at least one pharmaceutically acceptable diluent or carrier into a composition suitable for systemic administration.

[0050] In one embodiment, step (a) is performed on multiple separate occasions and step (b) is performed such that exposure of the subject to the additional therapeutic agent is continuous for the duration of the method.

[0051] In one embodiment, the subject is a human.

[0052] The inventors have also surprisingly demonstrated that certain anti-CD40 antibodies remain at the site of a solid tumor after administration to a subject. Thus, only a small fraction of the antibody escapes from the tumor site into the vascular or lymphatic circulation of the subject.

[0053] This results in very effective treatments, as well as reduced side effects, enabling the use of lower doses of the antibody. These advantages are particularly evident when such antibodies are administered locally to a tumor site in a subject, but may also be evident when the antibody is administered systemically.

[0054] However, one skilled in the art will appreciate that the anti-CD40 antibody may also be administered systemically (eg, intravenously or subcutaneously) to a subject.

[0055] The inventors have also surprisingly demonstrated that administration of an anti-CD40 antibody that is retained at the tumor site in combination with systemic administration of an additional therapeutic agent to a subject results in an improved therapeutic effect relative to administration of the anti-CD40 antibody alone.

[0056] Thus, the present invention provides a method of treating a solid tumor in a subject, the method comprising: (a) administering to the subject a therapeutically effective amount of an antibody that specifically binds to CD40 and remains at the tumor site after administration, and optionally (b) systemically administering to the subject a therapeutically effective amount of an additional therapeutic agent. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, as long as step (a) precedes step (b). In step (a), the anti-CD40 antibody is preferably administered locally to the tumor.

[0057] The present invention also provides a kit for treating a solid tumor in a subject, the kit comprising (a) a therapeutically effective amount of an antibody that specifically binds to CD40 and remains at the tumor site after administration, and optionally (b) a therapeutically effective amount of an additional therapeutic agent suitable for systemic administration to the subject. The antibody that specifically binds to CD40 is preferably provided in a form suitable for local administration to the tumor.

[0058] Brief Description of Sequence Listing

[0059] SEQ ID NOs: 1, 2 and 3 are CDRs 1, 2 and 3, respectively, of the light chain of antibody G12 (defined according to IMGT numbering).

[0060] SEQ ID NOs: 4, 5 and 6 are CDRs 1, 2 and 3, respectively, of the heavy chain of antibody G12 (defined according to IMGT numbering).

[0061] SEQ ID NOs: 7 and 8 are the amino acid sequences of the light chain variable region and the heavy chain variable region of antibody G12, respectively.

[0062] SEQ ID NOs: 9 and 10 are the nucleic acid sequences of the light chain variable region and the heavy chain variable region of antibody G12, respectively.

[0063] SEQ ID NO: 11 is the amino acid sequence of an exemplary light chain constant region.

[0064] SEQ ID NO: 12 is the amino acid sequence of an exemplary heavy chain constant region.

[0065] SEQ ID NO: 13 is the amino acid sequence of human CD40. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Shown are the levels of antibodies detectable in the serum of hCD40tg mice bearing hCD40-negative bladder cancer cell MB49 tumors following administration of the antibody at a dose of 100 μg intratumorally (IT), peritumorally (PT), or intravenously (IV).

[0067] Figure 2 Shown are the levels of antibodies detectable in blood samples of cynomolgus monkeys after administration of the antibodies at the indicated doses via subcutaneous or intravenous routes.

[0068] Figure 3 Shown are the results of flow cytometric analysis of cell surface human CD40 expression of (A) B16.F10 (hCD40+) clone 5.G12.46 cells and (B) mock-transfected cells.

[0069] Figure 4A Shown are the survival rates of hCD40tg mice bearing subcutaneous B16.F10 (hCD40+) melanomas after treatment with control, ADC-1013 alone, or ADC-1013 and anti-PD-1 antibody ("PD-1").

[0070] Figure 4BThe tumor volume of each treatment group on the day the first mouse was sacrificed is shown. The figure shows the pooled data from two separate experiments (n=16). Treatment with ADC-1013 demonstrated significant anti-tumor efficacy and increased survival rate. Improved anti-tumor efficacy and improved survival rate were observed with anti-PD-1 combined treatment (*=p<0.05, **=<0.01, log-rank test for survival rate and Mann-Whitney test for tumor volume).

[0071] Figure 5A Results are shown for determination of CD11c positive cell activation in draining lymph nodes following different routes of administration of anti-CD40 antibodies in a mouse tumor model. Intratumoral administration (IT), intraperitoneal administration (IP). Activation is indicated by CD86 expression levels measured by mean fluorescence intensity (MFI).

[0072] Figure 5B Results are shown for determination of CD11b positive cell activation in draining lymph nodes following different routes of administration of anti-CD40 antibodies in a mouse tumor model. Intratumoral administration (IT), intraperitoneal administration (IP). Activation is indicated by CD86 expression levels measured by mean fluorescence intensity (MFI).

[0073] Figure 6 B16.F10.hCD40+ tumors were inoculated on one flank. Treatments were administered on days 3, 6, and 9. ADC-1013 (100 μg per dose) was administered intratumorally. 250 μg per dose of anti-PD-1 (clone RPM1-14, BioXcell) and 100 μg per dose of anti-CTLA-4 antibody (9D9, BioXcel) were injected intraperitoneally. Tumor volume on day 14 is shown.

[0074] Figure 7 B16.F10 tumors were inoculated on one flank. Treatments were administered on days 3, 6, and 9. ADC-1013, anti-CD137 (Lob 12.3), and anti-OX40 (CD86) antibodies and controls were administered intratumorally (100 μg per dose). Tumor volume was tracked over time.

[0075] Fig. 8A and Figure 8B Anti-tumor effect of ADC-1013 in B16.F10.hCD40+ tumors compared to that in B16.F10(wt) tumors after treatment with ADC-1013 or control (intratumoral, 100 μg, days 3, 6, and 9).

[0076] Fig.9A and Fig. 9BADC-1013 (30 μg) was administered intratumorally in hCD40tg-BalbC (F1) mice with established lymphoma tumors (A20) on days 10, 13, and 16. Changes in tumor volume and survival over time are presented in the graph.

[0077] Fig.10 ADC-1013 (100 μg) was administered intratumorally in hCD40tg mice with established tumors (LLC-1) on days 4, 7, and 10. The changes in tumor volume over time are presented in the graph.

[0078] Fig.11 . Anti-tumor effect in the B16.F10 melanoma metastasis model. hCD40tg mice with one tumor on each flank received 100 μg ADC-1013 peritumorally in the tumor on the right flank on days 3, 6, and 9 after tumor inoculation. The changes in tumor volume over time for injected (right tumor) and non-injected (left) tumors are shown in the figure.

[0079] Fig.12 Tumor volume at day 16. ADC-1013 100 μg was administered intravenously on days 3, 6 and 9 in hCD40tg mice with established melanoma tumors (B16.F10.hCD40+) (n=12).

[0080] Fig.13 Anti-tumor effects (measured by survival over time) in subcutaneous A20 lymphoma tumors in BalbC mice are shown. Mice were treated peritumorally with 9D9 (anti-CTLA-4 antibody, BioXcel) on days 5 and 8 and intratumorally with the TLR agonist CpG (1668) on days 5, 8, and 11. DETAILED DESCRIPTION

[0081] It will be appreciated that different applications of the disclosed combination therapies and methods can be tailored to specific needs in the art.It will also be appreciated that the terminology used herein is for the purpose of describing particular embodiments of the present invention only and is not intended to be limiting.

[0082] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes "antibodies", reference to "an antigen" includes two or more such antigens, reference to "a subject" includes two or more such subjects, and so forth.

[0083] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or other peptide mimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0084] "Retention at the solid tumor site" includes that the anti-CD40 antibody is only slowly released from the tumor area. The retention of the antibody at the tumor site can be assessed by monitoring the serum level of the antibody after administration (see Mangsbo et al., 2014, Clin. Cancer Res., 21(5): 1115-1126, the disclosure of which is incorporated herein by reference). For example, in one embodiment, the anti-CD40 serum level is less than 1 μL / ml 4 hours after intratumoral injection of 30 μg of antibody (60 μL).

[0085] A "therapeutically effective amount" of a substance refers to a given substance administered to a subject suffering from a condition in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. The effective amount for a given purpose and a given agent will depend on the severity of the disease or injury and the weight and general state of the subject. As used herein, the term "subject" includes any mammal, preferably a human.

[0086] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0087] method

[0088] The present invention provides a method of treating a solid tumor in a subject. The tumor is typically malignant and may be metastatic.

[0089] Solid tumors are traditionally defined by the tissue from which they originate, e.g., breast, colon, etc. However, since immunotherapy acts on the immune system, rather than on the tumor itself, the immune status of the tumor may be more predictive of response than the origin of the tumor. In the supporting studies presented herein, two different models were described in more detail, the melanoma model B16.F10 (with and without human CD40) and the MB49 bladder tumor model. The data presented in the Examples below show that MB49 is more immunogenic than B16.F10. However, in both models, significant anti-tumor effects were observed after treatment with anti-CD40 antibodies as described herein. Therefore, the present invention provides anti-tumor effects in immunogenic tumors (such as MB49) and low immunogenic models (such as B16.F10).

[0090] Therefore, in one embodiment of the invention, the tumor is immunogenic. These tumors are characterized by infiltration of immune cells such as T cells and bone marrow-derived cells. It has been shown that infiltration of CD8 T cells, i.e. a more immunogenic tumor spectrum, is associated with a good prognosis after treatment, for example in colon cancer (Galon et al., 2014, Journal of Pathology (J. Pathol.), 232 (2): 199-209).

[0091] In an alternative embodiment of the present invention, the tumor is non-immunogenic or low immunogenic. Low immunogenic tumors generally have low MHCI expression or no MHCI expression, and are characterized by low numbers of infiltrating immune cells such as T cells and bone marrow derived cells (Lechner et al., 2013, Journal of Immunotherapy, 36(9): 477-89). The tumor can be an adenoma, adenocarcinoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms tumor, lung tumor, colon tumor, lymphoma, breast tumor or melanoma.

[0092] The type of blastoma includes hepatoblastoma, glioblastoma, neuroblastoma or retinoblastoma. The type of carcinoma includes colorectal cancer or hepatocellular carcinoma, pancreatic cancer, prostate cancer, gastric cancer, esophageal cancer, cervical cancer and head and neck cancer and adenocarcinoma. The type of sarcoma includes Ewing sarcoma (Ewing sarcoma), osteosarcoma, rhabdomyosarcoma or any other soft tissue sarcoma. The type of melanoma includes lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentigo melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma, melanoma with small nevus-like cells, melanoma with Spitz nevus features and uveal melanoma. Types of lymphoma include precursor T-cell leukemia / lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma, Burkitt's lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosis form of Hodgkin lymphoma, mixed cellular subtype of Hodgkin lymphoma. Types of lung tumors include tumors of non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma and large cell carcinoma) and small cell lung cancer.

[0093] The incidence of melanoma is increasing, and approximately 200,000 new cases are diagnosed around the world each year. In addition, melanoma is very suitable for local administration in the clinic because primary tumors as well as metastatic tumors are generally easily accessible. Therefore, for the method of the present invention, the tumor is preferably a melanoma, and most preferably a metastatic melanoma. Due to a high lactate dehydrogenase test result, the tumor can be classified as metastatic. The tumor can be classified as any of the following stages, but preferably stage III or IV.

[0094] 0th issue : Melanoma in situ (Clark grade I), 99.9% survival rate

[0095] Phase I / II : Aggressive melanoma, 89-95% survival rate

[0096] T1a: Less than 1.0 mm primary tumor thickness, no ulceration, and mitoses < 1 / mm 2

[0097] T1b: Less than 1.0 mm primary tumor thickness, ulceration or mitosis ≥ 1 / mm 2

[0098] T2a: 1.01-2.0 mm primary tumor thickness, no ulceration

[0099] Phase II: High-risk melanoma, 45-79% survival

[0100] T2b: 1.01-2.0 mm primary tumor thickness, with ulceration

[0101] T3a: 2.01-4.0 mm primary tumor thickness, no ulceration

[0102] T3b: 2.01-4.0 mm primary tumor thickness, with ulceration

[0103] T4a: Primary tumor thickness greater than 4.0 mm, no ulceration

[0104] T4b: Primary tumor thickness greater than 4.0 mm, with ulceration

[0105] Stage III : Local metastasis, 24-70% survival rate

[0106] N1: Single positive lymph node

[0107] N2: Two to three positive lymph nodes or local skin / intravenous metastasis

[0108] N3: Four positive lymph nodes or one lymph node and local skin / intravenous metastasis

[0109] Stage IV : Distant metastasis, 7-19% survival rate

[0110] M1a: Distant skin metastasis, normal LDH

[0111] M1b: lung metastasis, normal LDH

[0112] M1c: Other distant metastases or any distant metastases with elevated LDH

[0113] The method of the present invention comprises: (a) administering to a subject a therapeutically effective amount of an antibody that specifically binds to CD40 and is retained at a tumor site after administration, and optionally (b) systemically administering to the subject a therapeutically effective amount of an additional therapeutic agent. Retention of the antibody at the tumor site is described in more detail below. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, as long as step (a) precedes step (b). In step (a), the anti-CD40 antibody is preferably administered locally to the tumor.

[0114] The method of the present invention has several advantages. First, because the anti-CD40 antibody is retained at the tumor site, it is very effective as a treatment. In addition, systemic exposure to the anti-CD40 antibody is reduced, allowing the use of lower doses of the antibody and resulting in fewer side effects. When step (b) is performed, the therapeutic effect is further improved.

[0115] The present invention also provides:

[0116] -An antibody that specifically binds to CD40 and remains at the tumor site after administration for use in a method of treating a solid tumor in a subject, the method comprising: (a) administering to the subject a therapeutically effective amount of the antibody that specifically binds to CD40, and optionally (b) systemically administering to the subject a therapeutically effective amount of an additional therapeutic agent. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, as long as step (a) precedes step (b). In step (a), the anti-CD40 antibody is preferably administered locally to the tumor.

[0117] - Use of an antibody that specifically binds to CD40 and remains at the tumor site after administration in the manufacture of a medicament for treating a solid tumor in a subject, wherein the treatment comprises: (a) administering to the tumor a therapeutically effective amount of the antibody that specifically binds to CD40, and optionally (b) systemically administering to the subject a therapeutically effective amount of an additional therapeutic agent. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, as long as step (a) precedes step (b). In step (a), the anti-CD40 antibody is preferably administered locally to the tumor.

[0118] A product containing: (1) an antibody that specifically binds to CD40 and is retained at a tumor site after administration, and optionally (2) an additional therapeutic agent for simultaneous, separate or sequential use in a method for treating a solid tumor in a subject, the method comprising (a) locally administering to the tumor a therapeutically effective amount of the antibody that specifically binds to CD40, and optionally (b) systemically administering to the subject a therapeutically effective amount of the additional therapeutic agent. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, as long as step (a) precedes step (b). In step (a), the anti-CD40 antibody is preferably administered locally to the tumor.

[0119] Timing and order of steps (a) and (b)

[0120] Steps (a) and (b) are preferably performed sequentially (ie at different times), wherein step (a) is performed before step (b).

[0121] Steps (a) and (b) are preferably spaced apart for a period of time so that the combined anti-tumor effect is optimized. This generally means that step (b) is performed at a sufficiently long interval after step (a) so that at least one physiological effect of step (a) is at or near its peak level. For example, the anti-CD40 antibody usually stimulates dendritic cells, which in turn leads to the activation of tumor-specific T cells. Activated T cells begin to express higher levels of immune system checkpoint molecules (such as PD-1) within about 24 hours of anti-CD40 treatment. These immune system checkpoint molecules can negatively regulate anti-tumor responses. The agent administered in step (b) may therefore preferably be an agent (such as anti-PD1 or anti-PDL1 antibody) that blocks or inhibits the activity of the immune system checkpoint. When the agent administered in step (b) is such an agent, step (b) is preferably performed at a sufficiently long time interval after step (a) so that the expression level of the immune system checkpoint molecule in the cells in the subject or the number of cells in the subject expressing the immune system checkpoint molecule is relative to the level or quantity in the subject before step (a) or relative to the level or quantity in a healthy subject. In this context, step (b) is preferably performed 24 hours to two weeks after step (a), 24 hours to one week after step (a), 24 hours to 72 hours after step (a), or 24 hours to 48 hours after step (a).

[0122] Optionally, step (b) may be performed at a time point after step (a), wherein the expression level of the immune system checkpoint molecule in the subject's cells or the number of cells in the subject expressing the immune system checkpoint molecule is determined to be increased relative to the level or number in the subject before step (a) or relative to the level or number in a healthy subject.

[0123] The expression level of an immune system checkpoint molecule in cells of a subject, or the number of cells in a subject that express such a molecule, can be determined by any suitable means, such as by flow cytometric analysis of a sample obtained from the subject.

[0124] Alternatively, steps (a) and (b) may be performed preferably simultaneously (i.e., at the same time) or within 24 hours of each other, so that both steps may be performed on the same day or during the same visit to a treatment center. This may be particularly advantageous where access to a treatment center is limited. In this case, steps (a) and (b) may be performed simultaneously, or may be performed less than 24 hours apart, less than 12 hours apart, less than 10 hours apart, less than 6 hours apart, less than 4 hours apart, less than 3 hours apart, or less than 2 hours apart.

[0125] In any of the above embodiments, step (a) may be performed in multiple other situations after the first situation. That is, the subject may receive a series of doses of anti-CD40 antibodies. These doses are administered so that the subject is only intermittently exposed to the anti-CD40 antibody, preferably so that the subject's immune cells do not become exhausted and / or the subject does not suffer from rapid tolerance to the anti-CD40 antibody. When any of these symptoms is detected, the next administration of the anti-CD40 antibody may be delayed or cancelled. If multiple doses of anti-CD40 are administered, step (b) is preferably performed in the following manner: after step (b) is initiated, the subject is allowed to be continuously exposed to the additional therapeutic agent during the duration of the method (including during any second and additional situations of step (a)). This may be particularly suitable when the additional agent is an agent that blocks or inhibits the activity of an immune system checkpoint (e.g., an anti-PD1 or anti-PDL1 antibody). Continuous receptor blocking may be particularly important for the therapeutic effects of these agents.

[0126] Step (a)

[0127] Step (a) of the method involves local or systemic administration of an anti-CD40 antibody to a subject having a solid tumor. Local administration to the tumor site is preferred and includes peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracranial and intracapsular administration by any suitable means (e.g., injection). Local administration may also include intracavitary infusion and inhalation, depending on the site of the tumor.

[0128] A high proportion of the anti-CD40 antibody will remain in the tumor site in vivo, i.e., within the tumor microenvironment, for an extended period of time following administration. That is, the antibody exhibits reduced leakage from the tumor site to the vascular or lymphatic circulation, particularly when administered topically to the tumor site. Preferably at least 30% of the antibody dose administered to the tumor according to the method is retained in the tumor site four hours after administration, more preferably at least 40% of the dose is retained four hours after administration and most preferably at least 50% of the dose is retained four hours after administration.

[0129] Antibody retention in the tumor microenvironment can be studied by injecting the antibody into the tumor in the murine model and measuring the change in serum levels of the antibody over time after administration. Alternatively, the distribution of the antibody can be measured using a radiolabeled antibody injected into the tumor in the murine model. Suitable techniques are described in the embodiments.

[0130] The pH in the tumor microenvironment in vivo is significantly more acidic than in healthy tissue. Tumors are reported to range from approximately pH 6.5 to 7.2 or 6.6 to 7.0, compared to 7.2 to 7.4 for healthy tissue. This acidity is primarily due to anaerobic glycolysis in tumor areas that are subject to short-term or long-term hypoxia due to poorly organized vasculature with reduced chaotic blood flow, and aerobic glycolysis (Warburg effect), which is a common cancer phenotypic property in which the glycolytic metabolic pathway is used even in the presence of oxygen. In view of this acidity, the anti-CD40 antibodies used in the methods of the present invention may preferably have a high isoelectric point, as this will result in improved retention in the tumor microenvironment relative to similar antibodies with lower isoelectric points.

[0131] The isoelectric point of an antibody can be determined by any suitable method. It can be determined, for example, in vitro by electrophoresis. Alternatively, the isoelectric point can be calculated according to basic principles. In this case, the isoelectric point obtained is generally referred to as the theoretical isoelectric point. There are numerous software programs for computer calculation of theoretical isoelectric points, such as GP-MAW (version 9.2, from Lighthouse Data). The anti-CD40 antibody used in the method of the present invention preferably has a theoretical isoelectric point (pI) of 9.0 or more, preferably 9.1 or more, more preferably 9.2 or more, or 9.25 or more, and most preferably 9.3 or more.

[0132] Step (b)

[0133] Step (b) of the method involves systemic administration of an additional therapeutic agent to a subject. Systemic administration of any agent described herein (including the anti-CD40 antibody of step (a)) refers to administration to the circulatory system (including the vascular and / or lymphatic system) of a subject. Such administration may be by any suitable route, but is typically parenteral.

[0134] As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, and is usually achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, intracerebral, intrathecal, intraosseous or other parenteral administration routes.

[0135] Antibody

[0136] summary

[0137] The term "antibody" as referred to herein includes complete antibodies and any antigen binding fragments thereof (i.e., "antigen binding portions") or single chains. Antibodies refer to glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen binding portions thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The VH and VL regions can be further subdivided into hypervariable regions known as complementary determining regions (CDRs), interspersed with more conservative regions known as framework regions (FRs). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0138] The heavy chain can be of any isotype, including IgG (IgGl, IgG2, IgG3, and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM, and IgE.

[0139] Light chains include kappa chains and lambda chains.

[0140] Of particular relevance are antibodies and antigen-binding fragments thereof that have been "isolated" so as to exist in a physical environment that is different from that in which it may be found in nature, or that have been modified so as to differ from a naturally occurring antibody in amino acid sequence.

[0141] The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be produced by any suitable method. For example, methods suitable for producing monoclonal antibodies are disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Hurrell (CRC Press, 1982). Recombinant techniques may also be used.

[0142] The term "antigen binding portion" or "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD40). It has been demonstrated that the antigen binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementary determining regions (CDRs). Single-chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utilization in the same manner as intact antibodies.

[0143] The antibody used in the method for the present invention can be a human antibody. As used herein, the term "human antibody" is intended to include antibodies with variable regions, wherein the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies wherein the CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences, which are generally referred to as chimeric or humanized.

[0144] The human antibodies used in the methods of the present invention are generally human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice) having a genome comprising a human heavy chain transgene and a light chain transgene fused to immortalized cells. Human antibodies can also be prepared by in vitro immunization of human lymphocytes followed by transformation of lymphocytes with Epstein-Barr virus. The term "human antibody derivative" refers to any modified form of a human antibody, e.g., a conjugate of an antibody with another agent or antibody.

[0145] The antibodies used in the methods of the invention may optionally be humanized antibodies.

[0146] The term "humanization" refers to an antibody molecule that is usually prepared using recombinant technology, which has an antigen binding site of an immunoglobulin derived from a non-human species and the remaining immunoglobulin structure based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may include a completely non-human antibody variable domain fused to a human constant domain, or only include the complementary determining regions (CDRs) of these variable domains of a suitable human framework region transplanted to a human variable domain. The framework residues of these humanized molecules may be wild type (e.g., completely human) or they may be modified to contain one or more amino acid substitutions that are not present in the human antibodies whose sequences are the basis of humanization. Humanization reduces or eliminates the possibility that the constant region of the molecule will be used as an immunogen in a human individual, but the possibility of an immune response to an external variable region still exists (LoBuglio, AF, et al., (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86: 4220-4224). Another approach focuses not only on providing human constant regions, but also on modifying variable regions so as to reconstruct them as closely as possible to human forms. It is known that the variable regions of heavy and light chains contain three complementary determining regions (CDRs), which vary in response to the antigen in question and determine the binding capacity, flanked by four framework regions (FRs) that are relatively conserved in a given species and presumably provide a skeleton for the CDRs. When non-human antibodies are prepared for a specific antigen, the variable regions can be "reconstructed" or "humanized" by transplanting CDRs derived from non-human antibodies into FRs present in the human antibodies to be modified. The application of this method to various antibodies has been reported by Sato, K. et al., (1993) Cancer Res, 53:851-856; Riechmann, L. et al., (1988) "Reshaping Human Antibodies for Therapy," Nature, 332:323-327; Verhoeyen, M. et al., (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science, 239:1534-1536; Kettleborough, CAet al., (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation," Protein Engineering, 4: 773-3783; Maeda, H. et al., (1991) "Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity," Human Antibodies Hybridoma, 2: 124-134; Gorman, SD et al., (1991) "Reshaping A Therapeutic CD4 Antibody," Proc. Natl. Acad. Sci. (USA) 88: 4181-4185; Tempest, PR et al., (1991) "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo," Bio / Technology, 9:266-271; Co, MS et al., (1991) "Humanized Antibodies For Antiviral Therapy," Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al., (1992) "Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al., (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen," J. Immunol.)》, 148:1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are changed relative to the original antibody, which is also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanize antigens is well known (see, e.g., U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557). .

[0147] Any antibody mentioned herein may be provided in isolated form or may optionally be provided linked to another moiety (directly or indirectly). The other moiety may be a therapeutic molecule, such as a cytotoxic moiety or a drug.

[0148] The therapeutic molecule can be directly connected to the antibody of the present invention, for example, by chemical bonding. Methods for combining molecules with antibodies are known in the art. For example, carbodiimide bonding (Bauminger and Wilchek (1980) Methods Enzymol., 70, 151-159) can be used to bind various agents (including doxorubicin) to antibodies or peptides. Water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly useful for combining functional moieties with binding moieties.

[0149] Other methods of conjugating moieties to antibodies can also be used. For example, sodium periodate oxidation can be used, followed by reductive alkylation with appropriate reactants, as can glutaraldehyde cross-linking. However, it should be recognized that no matter which method is chosen to produce the conjugate of the present invention, it must be ensured that the antibody retains its targeting ability and the functional moiety retains its associated function.

[0150] Cytotoxic moieties may be directly and / or indirectly cytotoxic. "Directly cytotoxic" refers to moieties that are themselves cytotoxic. "Indirectly cytotoxic" refers to moieties that, while not themselves cytotoxic, can induce cytotoxicity, e.g., through their action on another molecule or by acting on another molecule. A cytotoxic moiety may be cytotoxic only when intracellular and is preferably not cytotoxic when extracellular.

[0151] Preferably, the antibody or antigen binding fragment is linked to a cytotoxic moiety that is a direct cytotoxic chemotherapeutic agent. Optionally, the cytotoxic moiety is a direct cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art.

[0152] Cytotoxic chemotherapeutic agents (e.g., anticancer agents) include: alkylating agents, including nitrogen mustards such as dichloromethane (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; ethyleneimines and methylmelamines, such as hexamethylmelamine, thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin (streptozotocin); and triazenes, such as dacarbazine. The invention relates to inhibitors of the present invention, including dimethyltriazene imidazole (DTIC; dimethyltriazene imidazole-carboxamide); antimetabolites, including folate analogs such as methotrexate (aminopterin); pyrimidine analogs, such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside); and purine analogs and related inhibitors, such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin). Natural products, including vinca alkaloids, such as vinblastine (VLB) and vincristine; epipodophyllotoxins, such as etoposide and teniposide; antibiotics, such as dactinomycin (actinomycin D), daunorubicin (daunorubicin; erythromycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes, such as L-asparaginase; and biological response modifiers, such as interferon alphenome. Various agents, including platinum coordination complexes, such as cisplatin (cis-DDP) and carboplatin; anthracenediones, such as mitoxantrone and anthracyclines; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, such as procarbazine (N-methylhydrazine; MIH); and adrenocortical suppressants, such as mitotane (o, p'-DDD) and aminoglutethimide; paclitaxel and analogs / derivatives; and hormone agonists / antagonists, such as flutamide and tamoxifen.

[0153] The cytotoxic moiety can be a cytotoxic peptide or polypeptide moiety that causes cell death. Cytotoxic peptides and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, etc. It is also known in the art to connect them to a targeting moiety such as an antibody. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96 / 06641. Pseudomonas exotoxins can also be used as cytotoxic polypeptides. Some cytokines such as TNF α and IL-2 can also be used as cytotoxic agents.

[0154] If delivered in sufficient doses, some radioactive atoms may also be cytotoxic. Therefore, the cytotoxic portion may include radioactive atoms that deliver a sufficient amount of radioactivity to the target site in use so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188 or yttrium-90, or any other isotope that emits enough energy to destroy adjacent cells, organelles or nucleic acids. Preferably, the isotope and density of the radioactive atoms in the medicament of the present invention are such that a dose exceeding 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site, and preferably delivered to the cells and organelles thereof, particularly the nucleus, of the target site.

[0155] Radioactive atoms can be attached to antibodies, antigen-binding fragments, variants, fusions or derivatives thereof in a known manner. For example, EDTA or another chelating agent can be attached to the binding moiety and used to attach 111In or 90Y. Tyrosine residues can be directly labeled with 125I or 131I.

[0156] The cytotoxic moiety can be a suitable indirect cytotoxic polypeptide. The indirect cytotoxic polypeptide can be a polypeptide with enzymatic activity and can convert non-toxic and / or relatively non-toxic prodrugs into cytotoxic drugs. With regard to antibodies, this type of system is generally referred to as ADEPT (antibody-directed enzyme prodrug therapy). The system requires antibodies to locate the enzyme moiety to the desired site in the patient's body, and after allowing the enzyme to be located at the site, the prodrug is administered as an enzyme substrate, and the final product of the catalysis is a cytotoxic compound. The purpose of the method is to maximize the concentration of the drug at the desired site and minimize the concentration of the drug in normal tissues. The cytotoxic moiety can convert non-cytotoxic drugs into cytotoxic drugs.

[0157] The enzymes and prodrugs using the system of targeted enzymes as described herein can be any enzyme previously proposed. The cytotoxic substance can be any existing anticancer drug, such as an alkylating agent; an agent that inserts into DNA; an agent that inhibits any key enzyme such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinase or topoisomerase; or an agent that achieves cell death by interacting with any other cellular component. Etoposide is an example of a topoisomerase inhibitor.

[0158] Reported prodrug systems include those listed in Table 1.

[0159] Table 1

[0160]

[0161]

[0162] Enzymes suitable for forming part of the enzyme portion include: exopeptidases, such as carboxypeptidases G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs), and aminopeptidases (for 2-α-aminoacyl MTC prodrugs); endopeptidases, such as thrombolytics (for thrombin prodrugs); hydrolases, such as phosphatases (e.g., alkaline phosphatase) or sulfatases (e.g., arylsulfatase) (for phosphorylated or sulfated prodrugs); amidases, such as penicillin amidase and aryl acyl amidase; lactamase, such as β-lactamase; glycosidase, such as β-glucuronidase (for β-glucuronide anthracycline), α-galactosidase (for amygdalin) and β-galactosidase (for β-galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for ganciclovir); reductases, such as nitroreductase (for CB1954 and analogs), azoreductase (for azobenzene mustard) and DT-diaphorase (for CB1954); oxidases, such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemase, catalytic antibodies and cyclodextrins.

[0163] Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% toxicity, preferably less than 1% toxicity as measured in a suitable in vitro cytotoxicity assay.

[0164] It is possible that the portion capable of converting a prodrug into a cytotoxic drug will be active apart from the remainder of the agent of the invention, but necessarily only when (a) it is combined with the remainder of the agent of the invention and (b) the agent of the invention is attached, adjacent to or internalized in a target cell.

[0165] When each moiety is a polypeptide, the two moieties may be linked together by any conventional means for cross-linking polypeptides. For example, the antibody or antigen-binding fragment may be enriched in thiol groups and another moiety that reacts with a bifunctional reagent capable of reacting with those thiol groups, such as N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Amide and thioether bonds, such as those achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulfide bonds.

[0166] The cytotoxic moiety may be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzamide, etanidazole, pimonidazole and misonidazole. In addition, gene delivery to cells can sensitize them radioactively, such as the delivery of the p53 gene or cyclin D. The additional moiety may be one that becomes cytotoxic or releases a cytotoxic moiety when irradiated. For example, when properly irradiated, the boron-10 isotope releases alpha particles that are cytotoxic. Similarly, the cytotoxic moiety may be one that can be used in photodynamic therapy, such as photofrin.

[0167] Antibodies specific for CD40

[0168] The combination therapies and methods of the present invention utilize antibodies that immunospecifically bind to CD40, i.e., "anti-CD40 antibodies." In one embodiment, the antibody is retained at the tumor site after administration to a subject (see discussion under step (a) above). The antibody preferably binds specifically to CD40, that is, it binds to CD40 but does not bind or binds to other molecules with lower affinity (e.g., 10-fold lower affinity). Unless otherwise indicated, the term CD40 as used herein refers to human CD40. The sequence of human CD40 is shown in SEQ ID NO: 13. The anti-CD40 antibodies of the present invention may have some binding affinity for CD40 from other mammals (e.g., primate or murine CD40). The antibody preferably binds to human CD40 when localized on the surface of a cell.

[0169] In particular, the anti-CD40 antibodies used in the combination therapy of the present invention compete for binding to human CD40 with a 'reference antibody' comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8 (optionally and light chain and heavy chain constant regions of SEQ ID NO: 11 and SEQ ID NO: 12, respectively). Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using a BIAcore chip with immobilized human CD40 and incubating in the presence of the reference antibody with and without the antibody polypeptide to be tested. Alternatively, a paired localization method can be used, in which the reference antibody is immobilized on the surface of the BIAcore chip, human CD40 is bound to the immobilized antibody, and then the secondary antibody is tested for simultaneous binding to human CD40 (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00AA 05 / 2012; the disclosure of which is incorporated herein by reference).

[0170] Exemplary anti-CD40 antibodies are disclosed in WO 2013 / 034904 to Alligator Bioscience AB (the disclosure of which is incorporated herein by reference).

[0171] The antibody is preferably capable of binding to CD40 in its native state and in particular to CD40 located on the surface of a cell. Preferably, the antibody will bind specifically to CD40. That is, the antibody used in the methods of the invention will preferably bind to CD40 with a greater binding affinity than it binds to another molecule.

[0172] "Locate on the surface of a cell" means that CD40 is associated with a cell such that one or more regions of CD40 are present on the outer surface of the cell surface. For example, CD40 may be inserted into the plasma membrane of a cell with one or more regions present on the outer surface of the cell (i.e., oriented as a transmembrane protein). This may occur during the process of CD40 expression by the cell. Thus, in one embodiment, "located on the surface of a cell" may mean "expressed on the surface of a cell". Alternatively, CD40 may be external to the cell with covalent and / or ionic interactions that localize it to one or more specific regions on the cell surface.

[0173] The anti-CD40 antibodies used in the combination therapy and methods of the present invention can induce and / or enhance ADCC-mediated lysis of cells expressing CD40 and / or enhance apoptosis of cells expressing CD40. The cells are typically tumor cells. "Enhancement" means that the number of cells that are lysed or apoptotic increases in the presence of the antibodies of the present invention relative to the number of cells that are lysed or apoptotic in the presence of an appropriate control substance. Methods for determining the level of ADCC-mediated lysis or apoptosis in a cell sample are well known in the art. For example, chromium-51 release assays, europium release assays, or sulfur-35 release assays can be used. In these assays, a previously labeled target cell line expressing an antigen (in this case CD40) is incubated with the antibody to be tested. After washing, effector cells (usually expressing the Fc receptor CD16) are co-incubated with antibody-labeled target cells. Target cell lysis is then measured by the release of intracellular markers using a scintillation counter or spectrophotometer.

[0174] Preferably, the antibody or antigen-binding fragment comprises an antibody Fc region. It will be appreciated by the skilled person that the Fc portion may be from an IgG antibody, or from antibodies of different classes (e.g., IgM, IgA, IgD, or IgE). For example, the Fc region may be from an IgG1, IgG2, IgG3, or IgG4 antibody. However, advantageously, the Fc region is from an IgG1 antibody.

[0175] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or may be artificial (e.g., comprising one or more point mutations relative to a naturally occurring Fc region). Fc regions with point mutations that increase their ability to bind to FcRs may be advantageous, for example, by altering serum half-life or improving binding to Fcγ receptors (FcγRs) involved in ADCC and CDC. In particular, mutations that enhance binding to FcγRIIB, such as S267E (Strohl et al., 2009, Curr Opin Biotechnol, 20:685-691) may be advantageous for the present invention, providing a correlation between FcγRIIB binding and the functional activity of CD40 antibodies (Li et al., 2011, Science, 333:1030-1034).

[0176] As an alternative to the radioisotope labeling required in these assays, a method can be used in which the cleavage is detected by measuring the release of an enzyme naturally present in the target cell. This can be achieved by detecting (e.g., bioluminescent detection) the product of the enzyme catalyzed reaction. No prior labeling of cells is required in such an assay. A typical cellular enzyme detected by this assay is GAPDH.

[0177] The anti-CD40 antibodies used in the combination therapies and methods of the present invention can modulate the activity of cells expressing CD40, wherein the modulation is an increase or decrease in the activity of the cells. The cells are typically dendritic cells or B cells.

[0178] When signaling occurs through CD40, dedicated APCs (e.g., dendritic cells) are activated, which triggers several biological events, including immune cell activation, proliferation, and production of cytokines and chemokines. Methods for determining dendritic cell activation associated with CD40 are known in the art (e.g., discussed in Schonbeck et al., 2001, Cell Mol Life Sci., 58:40-43; van Kooten et al., 2000, J. Leuk., Biol., 67:2-17) and are further described below.

[0179] Stimulation of human B cells with recombinant CD40L or anti-CD40 antibodies induces upregulation of surface markers such as CD23, CD30, CD80, CD86, Fas, and MHC II, secretion of soluble cytokines such as IL-6, TNF-γ, and TNF-α, and homotypic aggregation. Methods for determining CD40-related B cell activation are known in the art (e.g., discussed in Schonbeck et al., 2001, supra) and are further described below.

[0180] Methods and assays for determining the ability of antibodies to regulate the activity of dendritic cells and B cells are well known in the art. For example, the activation of dendritic cells can be assessed by measuring the levels of cell surface markers such as CD86 and CD80 and / or by measuring the IFNγ secretion from T cells induced by anti-CD40 antibodies, wherein an increase in any of these parameters indicates an increase in activation and a decrease in activation. Similarly, the ability of antibodies to regulate B cell activity can be assessed by measuring the levels of cell surface markers (e.g., CD86) and / or by measuring the B cell proliferation induced by anti-CD40 antibodies (see Example 3 below), wherein an increase in any of these parameters indicates an increase in activation and a decrease in activation.

[0181] Preferably, the anti-CD40 antibodies used in the combination therapies and methods of the invention that increase activation of dendritic cells or B cells have potency for activation of dendritic cells or B cells. Cell activation can generally be measured at the EC50 level in an assay that involves incubating isolated dendritic or B cells with a test stimulus and then detecting cell proliferation as a measure of activation.

[0182] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of an antibody molecule to bind or not bind to a target. Binding affinity can be quantified by measuring the dissociation constant (Kd) of the antibody and its target. Similarly, the binding specificity of an antibody to its target can be defined based on the comparative dissociation constant (Kd) of the antibody for its target compared to the dissociation constant of the antibody and another non-target molecule.

[0183] Typically, the Kd of an antibody for a target will be 2-fold, preferably 5-fold, more preferably 10-fold smaller than the Kd of the antibody for other non-target molecules such as unrelated substances or substances accompanying the environment. The Kd will more preferably be 50-fold smaller, even more preferably 100-fold smaller, and still more preferably 200-fold smaller.

[0184] The dissociation constant value can be determined directly by well-known methods, and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (Byte 9:340-362, 1984). For example, a double-filtration nitrocellulose filter binding assay such as that disclosed by Wong and Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993) can be used to establish Kd. Other standard assays for evaluating the binding ability of ligands such as antibodies to targets are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of antibodies can also be determined by standard assays known in the art, such as by Biacore.TM System analysis to evaluate.

[0185] Competitive binding assays can be performed in which the binding of an antibody to a target is compared to the binding of the target to another known ligand for the target, such as another antibody. The concentration at which 50% inhibition occurs is called the Ki. Under ideal conditions, the Ki is equal to the Kd. The Ki value is never less than the Kd, so it is convenient to replace the measurement of the Ki to provide an upper limit for the Kd.

[0186] The anti-CD40 antibodies used in the combination therapies and methods of the invention are preferably capable of binding to their target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than their affinity for binding to another non-target molecule.

[0187] Antibodies used in the combination therapies and methods of the invention will generally exhibit the following abilities:

[0188] (i) specifically binds to human CD40 when localized on the surface of a cell; and / or

[0189] (ii) enhancing antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis of cells expressing CD40; and / or

[0190] (iii) enhancing apoptosis of cells expressing CD40; and / or

[0191] (iv) regulating the activity of cells expressing CD40, wherein the regulation is an increase or decrease in the activity of the cells.

[0192] The antibody may be or may comprise a variant or fragment of one of the specific anti-CD40 antibodies disclosed herein, as long as the variant or fragment retains specificity for CD40 and at least one of the functional properties (i) to (iv).

[0193] The fragment is preferably an antigen-binding portion of the antibody. Fragments can be prepared by truncation, for example by removing one or more amino acids from the N and / or C termini of the polypeptide. In this way, up to 10, up to 20, up to 30, up to 40 or more amino acids can be removed from the N and / or C termini. Fragments can also be produced by one or more internal deletions.

[0194] Variants may comprise one or more substitutions, deletions or additions to the sequences of the specific anti-CD40 antibodies disclosed herein. Variants may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the specific sequences disclosed herein. "Deletion" variants may include the deletion of a single amino acid, the deletion of a small group of amino acids such as 2, 3, 4 or 5 amino acids, or the deletion of a larger amino acid region, such as the deletion of a specific amino acid domain or other feature. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids, and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid.

[0195] Some properties of the 20 major amino acids that can be used to select suitable substituents are as follows:

[0196] Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral

[0197] Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral

[0198] Asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral

[0199] Glu polar, hydrophilic, charged (-) Gln polar, hydrophilic, neutral

[0200] Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+)

[0201] Gly aliphatic, neutral Ser polar, hydrophilic, neutral

[0202] His is aromatic, polar, hydrophilic, and charged (+)Thr is polar, hydrophilic, and neutral

[0203] Ile Aliphatic, hydrophobic, neutral Val Aliphatic, hydrophobic, neutral

[0204] Lys polar, hydrophilic, charged (+) Trp aromatic, hydrophobic, neutral

[0205] Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic.

[0206] Preferred "variants" include those in which the amino acid appearing in the sequence instead of the naturally occurring amino acid is a structural analog thereof. The amino acids used in the sequence may also be derivatized or modified (eg, labeled) as long as the function of the antibody is not significantly adversely affected.

[0207] Variants may be prepared during antibody synthesis or by post-production modification, or when the antibody is in recombinant form, using site-directed mutagenesis, random mutagenesis, or known techniques of enzymatic cleavage and / or nucleic acid ligation.

[0208] Preferably, the variant antibody has an amino acid sequence with greater than 60%, or greater than 70%, such as 75% or 80%, preferably greater than 85%, such as greater than 90% or 95% amino acid identity with the VL or VH domain of the antibodies disclosed herein. This level of amino acid identity can be found over the entire length of the relevant SEQ ID NO sequence or over a portion of the sequence, such as 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0209] With respect to amino acid sequences, "sequence identity" refers to sequences having the stated values ​​when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

[0210] Pairwise alignment parameters—method: exact, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10;

[0211] Multiple alignment parameters - Matrix: PAM, Gap Open Penalty: 10.00, Delayed Identity %: 30, Penalty End Gaps: On, Gap Interval Distance: 0, Negative Matrix: None, Gap Extension Penalty: 0.20, Residue Specific Gap Penalty: On, Hydrophilicity Gap Penalty: On, Hydrophilic Residues: GPSNDQEKR. Sequence identities at specific residues are intended to include identical residues that have been simply derivatized.

[0212] The anti-CD40 antibodies used in the combination therapies and methods of the present invention may bind to the same epitope as the specific antibodies disclosed herein, because such antibodies may mimic the effects of the disclosed antibodies. Whether an antibody binds to the same epitope as another antibody can be determined by conventional methods. For example, the binding of each antibody to a target can be determined using a competitive binding assay. Methods for conducting competitive binding assays are well known in the art. For example, they may include contacting the antibody and the target molecule together under conditions where the antibody can bind to the target molecule. The antibody / target complex can then be contacted with a second (test) antibody and the extent to which the test antibody can displace the first antibody from the antibody / target complex can be assessed. Such assessments may use any suitable technique, including, for example, surface plasmon resonance, ELISA, or flow cytometry. The ability of the test antibody to inhibit the binding of the first antibody to the target demonstrates that the test antibody can compete with the first antibody for binding to the target, and therefore the test antibody binds to the same epitope or region on the target as the first antibody and therefore can mimic the effects of the first antibody.

[0213] The anti-CD40 antibody used in the combination therapy and method of the present invention may be an antibody comprising one, two or all three CDR sequences of SEQ ID NOs: 1 to 3 and / or one, two or all three CDR sequences of SEQ ID NOs: 4 to 6. The antibody may comprise all six CDR sequences of SEQ ID NOs: 1 to 6.

[0214] The antibody may comprise a light chain variable region sequence of SEQ ID NO:7 and / or a heavy chain variable region sequence of SEQ ID NO:8.

[0215] The antibody may be an antibody comprising a light chain variable region sequence of SEQ ID NO: 7 and a heavy chain variable region sequence of SEQ ID NO: 8, or may bind to the same epitope as an antibody comprising a light chain variable region sequence of SEQ ID NO: 7 and a heavy chain variable region sequence of SEQ ID NO: 8. In addition, the antibody may comprise a light chain constant region sequence of SEQ ID NO: 11 and / or a heavy chain constant region sequence of SEQ ID NO: 12.

[0216] The anti-CD40 antibody or any variant or fragment thereof used in the combination therapies and methods of the present invention preferably has a theoretical isoelectric point (pI) of 9.0 or above, preferably 9.1 or above, more preferably 9.2 or above or 9.25 or above, most preferably 9.3 or above.

[0217] Combination therapy

[0218] The present invention provides a combination therapy for treating a solid tumor in a subject, comprising: (a) an antibody or an antigen-binding portion thereof that specifically binds to CD40, and (b) another immunotherapeutic agent with cancer treatment efficacy, which is not an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 competes with an antibody comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8 for binding to human CD40, and wherein the additional immunotherapeutic agent specifically binds to an immune checkpoint molecule.

[0219] The term "combination therapy" or "combination treatment" or "combination" as used herein means any form of simultaneous or concurrent treatment with at least two different therapeutic agents.

[0220] According to certain embodiments, the anti-CD40 antibody or its antigen-binding fragment and the immunotherapeutic agent that specifically binds to immune checkpoint molecules other than CD40 are administered simultaneously in the same composition or in separate compositions. According to other embodiments, the anti-CD40 antibody or its antigen-binding fragment and the immunotherapeutic agent that specifically binds to immune checkpoint molecules other than CD40 are administered sequentially, that is, the anti-CD40 antibody or its antigen-binding fragment is administered before or after the administration of the immunotherapeutic agent that specifically binds to immune checkpoint molecules other than CD40. In some embodiments, the administration of the anti-CD40 antibody or its antigen-binding fragment and the immunotherapeutic agent that specifically binds to immune checkpoint molecules other than CD40 is simultaneous, that is, the administration period of the anti-CD40 antibody or its antigen-binding fragment and the administration period of the immunotherapeutic agent that specifically binds to immune checkpoint molecules other than CD40 overlap each other. In some embodiments, the administration of the anti-CD40 antibody or its antigen-binding fragment and the immunotherapeutic agent that specifically binds to immune checkpoint molecules other than CD40 is not simultaneous. For example, in some embodiments, administration of an anti-CD40 antibody or antigen-binding fragment thereof is terminated prior to administration of an immunotherapeutic agent that specifically binds to an immune checkpoint molecule other than CD40. In some embodiments, administration of an immunotherapeutic agent that specifically binds to an immune checkpoint molecule other than CD40 is terminated prior to administration of an anti-CD40 antibody or antigen-binding fragment thereof.

[0221] According to certain typical embodiments, the anti-CD40 antibody or antigen-binding fragment thereof and the immunotherapeutic agent that specifically binds to an immune checkpoint molecule other than CD40 are administered in a single therapeutic composition. According to some embodiments, the therapeutic composition further comprises a therapeutically acceptable diluent or carrier.

[0222] The other component of the combination therapy of the present invention is an immunotherapeutic agent having cancer therapeutic efficacy, which is not an anti-CD40 antibody or antigen-binding fragment thereof.

[0223] The term "immunotherapeutic agent" is intended to include any molecule, peptide, antibody or other agent that can stimulate the host immune system to produce an immune response to a tumor or cancer in a subject. Various immunotherapeutic agents can be used in the compositions and methods described herein. In one embodiment, the immunotherapeutic agent is an antibody or an antigen-binding fragment thereof.

[0224] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses indirectly achieved by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0225] The term "immune checkpoint molecule" is intended to include a group of proteins on the cell surface of immune cells (e.g., CD4+ and / or CD8+ T cells, dendritic cells, NK cells, and macrophages) and on certain tumor cells that regulate the immune response. It will be understood by those skilled in the art that immune checkpoint proteins can be inhibitory, such as CTLA-4 and PD-1, or stimulatory, such as OX40 and CD137. Exemplary immune checkpoint molecules include, but are not limited to, PD-1, CTLA-4, OX40 (CD134), CD137, VISTA, B7-H2, B7-H3, PD-L 1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR. In a preferred embodiment, the immune checkpoint molecule is PD-1, CTLA-4, OX40 (CD134), or CD137.

[0226] Blocking or neutralizing one or more inhibitory immune checkpoint molecules can block or otherwise neutralize inhibitory signaling, thereby upregulating the immune response to more effectively treat cancer. Exemplary agents that can be used to block inhibitory immune checkpoints include antibodies, small molecules, peptides, peptide mimetics, natural ligands, and derivatives of natural ligands that can bind and / or inactivate or inhibit inhibitory immune checkpoint proteins, or fragments thereof; and RNA interference, antisense, nucleic acid aptamers, etc., or fragments thereof that can downregulate the expression and / or activity of inhibitory immune checkpoint nucleic acids. Exemplary agents for upregulating immune responses include antibodies to one or more inhibitory immune checkpoint proteins that block the interaction between proteins and their natural receptors; inactive forms of one or more immune checkpoint inhibitor proteins (e.g., dominant negative polypeptides); small molecules or peptides that block the interaction between one or more inhibitory immune checkpoint proteins and their natural receptors; fusion proteins that bind to their natural receptors (e.g., the extracellular portion of immune checkpoint inhibitor proteins fused to the Fc region of antibodies or immunoglobulins); nucleic acid molecules that block transcription or translation of inhibitory immune checkpoint nucleic acids; and the like. These agents can directly block the interaction between one or more inhibitory immune checkpoints and their natural receptors (such as antibodies) to prevent inhibitory signaling and upregulating immune responses. Alternatively, the agent can indirectly block the interaction between one or more inhibitory immune checkpoint proteins and their natural receptors to prevent inhibitory signaling and upregulating immune responses. For example, immune checkpoint protein ligands such as soluble forms of stabilized extracellular domains can bind to their receptors to indirectly reduce the effective concentration of receptors bound to appropriate ligands. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies are used alone or in combination to inhibit immune checkpoint inhibitors.

[0227] Thus, in one embodiment, the immunotherapeutic agent that specifically binds to an immune checkpoint molecule other than CD40 is an antibody or antigen-binding fragment thereof that binds to an inhibitory immune checkpoint molecule and inhibits the function of the inhibitory immune checkpoint molecule.

[0228] Activation of one or more stimulatory immune checkpoint molecules can upregulate immune responses to more effectively treat cancer. Exemplary agents that can be used to activate stimulatory immune checkpoints include antibodies, small molecules, peptides, peptide mimetics, natural ligands, and derivatives of natural ligands, or fragments thereof that can activate stimulatory immune checkpoint proteins; and RNA interference, antisense, nucleic acid aptamers, etc., or fragments thereof that can enhance immune checkpoint nucleic acids. Exemplary agents for upregulating immune responses include soluble forms of stimulatory immune checkpoint protein ligands, or ligands fused to Fc or domains that promote ligand multimerization. In one embodiment, anti-CD137 and anti-OX40 antibodies are used alone or in combination to activate stimulatory immune checkpoint inhibitors.

[0229] Therefore, in an alternative embodiment, the immunotherapeutic agent that specifically binds to an immune checkpoint molecule other than CD40 is an antibody or antigen-binding fragment thereof that binds to a stimulatory immune checkpoint molecule and activates the function of the stimulatory immune checkpoint molecule.

[0230] Preferred examples of checkpoint molecules include PD1, which acts as a negative regulator of T cell activation when engaged with its ligand PD-L1 or PD-L2. PD-L1 is particularly expressed by many solid tumors (including melanoma). These tumors can therefore downregulate immune-mediated anti-tumor effects by activating inhibitory PD-1 receptors on T cells. By blocking the interaction between PD1 and PD-L1, the checkpoint of the immune response can be removed, resulting in an enhanced anti-tumor T cell response. This interaction can be blocked by antibodies or any other suitable agents that are specific to PD1 or PD-L1. These antibodies and agents may generally be referred to as PD1 inhibitors. PD1 inhibitors are particularly preferred as additional therapeutic agents in step (b) of the method of the present invention.

[0231] Anti-PD1 antibodies include nivolumab, pembrolizumab, lambliazumab, pidilizumab, and AMP-224. Anti-PD-L1 antibodies include MEDI-4736 and MPDL3280A.

[0232] Since tumors express PD-L1, the combination of systemic PD1 inhibitors and local anti-CD40 antibodies is not only attractive. This combination can also be beneficial for regulating the general immunosuppressive environment by affecting regulatory T cells and bone marrow-derived suppressor cells (MSDC). For example, CD40 agonists such as anti-CD40 antibodies used in the methods of the present invention will regulate CD40-positive tumor-associated M2 macrophages and MSDC, while PD1 inhibitors will regulate regulatory T cells and MSDC. In addition, as explained above (for the timing of steps (a) and (b), see discussion), it is reported that antibody connection to CD40 can activate and mature dendritic cells, which will upregulate PD-L 1 and 2 when mature, thereby providing a further rationale for combining two treatments. In addition, CD40 agonists such as anti-CD40 antibodies used in the methods of the present invention will indirectly upregulate PD-1 on T cells and PD-L1 on tumors and tumor-infiltrating cells. Therefore, treatment of solid tumors such as melanoma with a combination of a systemic PD1 inhibitor and an anti-CD40 antibody that remains at the tumor site would have pleiotropic effects both by activating the immune system and by inhibiting counter-regulatory signals.

[0233] Another example of a checkpoint molecule is the T cell receptor CTLA-4, which acts as a negative regulator of T cell activation. It is usually upregulated on the T cell surface after initial activation. The ligand of the CTLA-4 receptor is the B7 protein (B7-1 and B7-2), which is expressed by antigen presenting cells. The corresponding receptor responsible for the upregulation of T cell activation is CD28, which competes with CTLA-4 for binding to B7 protein. Therefore, by blocking the interaction of CTLA-4 with B7 protein, rather than the interaction of CD28 with B7 protein, one of the normal checkpoints of the immune response can be removed, thereby leading to enhanced anti-tumor T cell responses. Blocking the interaction of CTLA-4 with B7 protein can be achieved with anti-CTLA-4 antibodies or other suitable agents. Anti-CTLA-4 antibodies include ipilumumab, tremelimumab, or any antibody disclosed in PCT / EP2014 / 063442. Other molecules include polypeptides, or soluble mutant CD86 polypeptides.

[0234] Therefore, the additional therapeutic agent may preferably be an antibody or other agent that specifically binds to at least one of PD1, PD-L1 or CTLA-4.

[0235] The additional therapeutic agent is most preferably a PD1 inhibitor, such as an anti-PD1 or anti-PD-L1 antibody selected from the group consisting of Nivolumab, Pembrolizumab, Lamborlizumab, Pdilizumab, MEDI-4736 and MPDL3280A.

[0236] When the additional therapeutic agent is an antibody or a bispecific molecule comprising an antibody, it will be understood that all general considerations set forth above with respect to the definition of an antibody, an antigen-binding fragment of an antibody, optional association with other therapeutic moieties, etc., also apply to antibodies as additional therapeutic agents. Similarly, it will be understood that the definition of target specificity / affinity and methods for determining specificity / affinity set forth above for anti-CD40 antibodies will likewise apply to antibodies as additional therapeutic agents, except that the specific target of the agent will be understood instead of CD40. Variants and fragments of antibodies as additional therapeutic agents may also be defined in the same manner as variants and fragments of anti-CD40 antibodies.

[0237] Kits and pharmaceutical compositions

[0238] The present invention also provides a kit for treating a solid tumor in a subject, the kit comprising a combination therapy as defined above. For example, the kit may comprise (a) a therapeutically effective amount of an antibody that specifically binds to CD40 and is retained at the tumor site after administration, and optionally (b) a therapeutically effective amount of an additional therapeutic agent, which is suitable for systemic administration to the subject. The antibody that specifically binds to CD40 is preferably provided in a form suitable for local administration to the tumor site.

[0239] The kit of the present invention may further comprise one or more other reagents or instruments capable of implementing any of the above embodiments. These reagents or instruments include one or more of the following: a suitable buffer (aqueous solution) and a device for administering an anti-CD40 antibody and / or additional therapeutic agent (e.g., a blood vessel or an instrument comprising a needle).

[0240] The anti-CD40 antibody and additional therapeutic agent used in the methods of the invention or provided in the kits of the invention can each be provided as a separate pharmaceutical composition formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and also compatible with the desired route of administration.

[0241] Thus, carriers for anti-CD40 antibodies and additional therapeutic agents may be suitable for systemic administration, which as defined above refers to administration into the circulatory system (including the vascular and / or lymphatic system) of a subject. Such administration may be by any suitable route, but is typically parenteral. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, and is typically achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes.

[0242] However, carriers for anti-CD40 antibodies are preferably suitable for local administration, which as defined above includes peritumoral, juxta-tumoral, intratumoral, intralesional, perilesional, intracranial and intracapsular administration by any suitable means (e.g., injection). Local administration may also include intracavitary infusion and inhalation, depending on the site of the tumor.

[0243] Depending on the route of administration, the antibody and / or the agent may be coated in a material to protect the antibody from the effects of acid and other natural conditions that may inactivate or denature the antibody and / or the agent. Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water, and saline. Examples of other carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using a coating material (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In many cases, it will be preferred to include an isotonic agent in the composition, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride.

[0244] It will be appreciated by those skilled in the art that the antibody component of the combination therapy of the present invention is generally provided in the form of one or more pharmaceutical compositions, each of which contains a therapeutically effective amount of the antibody component and a pharmaceutically acceptable buffer, excipient, diluent or carrier.

[0245] As used herein, 'therapeutically effective amount' or 'effective amount' or 'therapeutically effective' refers to an amount that provides a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of active antibody calculated to produce the desired therapeutic effect together with the required additives and diluents (i.e., carriers or administration vehicles). In addition, it means an amount sufficient to reduce or prevent clinically significant defects in the activity, function, and response of the host. Alternatively, the therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of the compound may vary according to its specific activity. A suitable dose may contain a predetermined amount of active composition calculated to produce the desired therapeutic effect and the required diluent.

[0246] The therapeutically effective amount can be determined by a medical or veterinary practitioner of ordinary skill based on patient characteristics such as age, weight, sex, condition, complications, other diseases, etc. as is well known in the art.

[0247] The pharmaceutical composition may include a pharmaceutically acceptable antioxidant. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be prevented by the above sterilization procedures and by including various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be necessary to include isotonic agents in the composition, such as sugar, sodium chloride, etc. In addition, the extended absorption of injectable drug forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0248] Pharmaceutical compositions must generally be sterile and stable under manufacturing and storage conditions. The compositions may be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injection solutions may be prepared by incorporating the required amount of an active agent (e.g., an antibody) into an appropriate solvent having a composition or combination of components listed above as required, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active agent into a sterile vehicle containing an alkaline dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active agent plus any additional required components from its previously sterile filtered solution. Pharmaceutical compositions may include additional active ingredients as well as those described above.

[0249] Suitable pharmaceutically acceptable buffers, diluents, carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, ed., Mack Publishing Company (1990), and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

[0250] The term "buffer" is intended to include aqueous solutions containing acid-base mixtures for stabilizing pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0251] The term "diluent" is intended to include aqueous or non-aqueous solutions for diluting a medicament in a pharmaceutical preparation. The diluent can be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (e.g., safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0252] The term "adjuvant" is intended to include any compound added to a formulation to increase the biological effect of the agent of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetate of different acyl compositions. The adjuvant may also be a cationic polymer, such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine and peptides containing these amino acids.

[0253] Excipient can be one or more in carbohydrate, polymer, lipid and mineral.The example of carbohydrate comprises lactose, glucose, sucrose, mannitol and cyclodextrin, and it is added in the composition, for example, for promoting freeze drying.The example of polymer is starch, cellulose ether, cellulose carboxymethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivative, polyacrylic acid, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / propylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate with different hydrolysis degree, and polyvinyl pyrrolidone (all with different molecular weight), it is added in the composition, for example, for viscosity control, for realizing bioadhesion, or for protecting lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, monosaccharides, mono-, di- and triglycerides, ceramides, sphingolipids and glycolipids (all with varying acyl chain lengths and degrees of saturation), lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for similar reasons to polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced fluid accumulation or favorable pigment properties.

[0254] The active antibody-based agents of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for their delivery.

[0255] In one embodiment, the pharmaceutical composition of the present invention can be in the form of liposomes, wherein the medicament is combined with an amphiphilic agent such as a lipid in addition to other pharmaceutically acceptable carriers, and the amphiphilic agent is present in the aggregated form of micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposome formulations include (but are not limited to) monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, etc. Suitable lipids also include the above lipids modified by poly (ethylene glycol) in the polar head group to extend the blood circulation time. The preparation of such liposome formulations can be found in, for example, US 4,235,871, the disclosure of which is incorporated herein by reference.

[0256] The pharmaceutical composition of the present invention can also be in the form of biodegradable microspheres. Aliphatic polyesters such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly (caprolactone) (PCL) and polyanhydrides have been widely used as biodegradable polymers in microsphere production. The preparation of such microspheres can be found in US 5,851,451 and EP 0 213303, the disclosures of which are incorporated herein by reference.

[0257] In another embodiment, the pharmaceutical composition of the present invention is provided in the form of nanoparticles, for example, based on poly-gamma glutamic acid. The details of the preparation and use of this nanoparticle can be found in WO 2011 / 128642, the disclosure of which is incorporated herein by reference. It will be appreciated by those skilled in the art that one or more active components of the combination therapy of the present invention can be formulated in separate nanoparticles, or two active components can be formulated in the same nanoparticles.

[0258] In another embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, wherein polymers such as starch, cellulose ethers, cellulose carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carrageenan, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / propylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with varying degrees of hydrolysis, and polyvinyl pyrrolidone are used to thicken the solution containing the medicament. The polymer may also include gelatin or collagen.

[0259] Alternatively, the agent may be simply dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (eg, safflower, corn, peanut, cottonseed or sesame oils), tragacanth gum, and / or various buffers.

[0260] It should be understood that pharmaceutical compositions of the present invention may include ions and a limited pH for enhancing the effect of the active agent. In addition, the compositions may be subjected to conventional pharmaceutical operations such as sterilization, and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0261] The pharmaceutical composition according to the present invention can be administered by any suitable route known to those skilled in the art. Therefore, possible routes of administration include parenteral (intravenous, subcutaneous and intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, vaginal and rectal. It can also be administered from an implant.

[0262] Advantageously, the pharmaceutical composition is suitable for administration at or near a tumor site, such as within or around a tumor.

[0263] The pharmaceutical composition is preferably suitable for parenteral administration. Methods for formulating the antibodies into pharmaceutical compositions will be well known to those skilled in the medical and pharmaceutical arts, and preferred compositions are described in the accompanying examples.

[0264] The combination therapy of the present invention can be delivered using an injectable sustained release drug delivery system. These are specifically designed to reduce the frequency of injections. An example of such a system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, slowly release rhGH over a sustained period. Preferably, delivery is performed intramuscularly (im) and / or subcutaneously (sc) and / or intravenously (iv).

[0265] The combination therapy of the present invention can be administered by a surgical implant device that releases the drug directly to the desired site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. Applying the toxic agent directly to the disease site achieves effective treatment without the significant systemic side effects of the drug.

[0266] The combination therapy of the invention may also be administered using an electroporation therapy (EPT) system. Devices that deliver pulsed electric fields to cells increase the permeability of cell membranes to drugs, resulting in significant enhancement of intracellular drug delivery.

[0267] The combination therapy of the present invention can also be delivered by electroporation (EI). EI occurs when small particles up to 30 microns in diameter on the surface of the skin are subjected to an electrical pulse identical or similar to that used in electroporation. In EI, these particles are driven through the stratum corneum and into the deeper layers of the skin. The particles can be loaded or coated with drugs or genes, or can simply act as "bullets" that create holes in the skin that drugs can enter.

[0268] An alternative combination therapy of the present invention is the thermosensitive ReGel injection system. Below body temperature, ReGel is an injectable liquid, while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into a known safe biodegradable polymer. The active substance is delivered over time as the biopolymer dissolves.

[0269] The combination therapy of the present invention can also be delivered orally. 12 and / or vitamin D to co-deliver proteins and peptides. 12 and / or vitamin D absorption system, the agents, medicaments and pharmaceutical compositions of the present invention can move through the intestinal wall. Synthetic vitamin B 12 Analogs and / or complexes between vitamin D analogs and drugs, which in the complex contain vitamin B 12 A significant affinity for intrinsic factor (IF) is retained in the β-lactamase / vitamin D portion and a significant biological activity is retained in the active substance of the complex.

[0270] The combination therapy of the present invention can be introduced into cells through "Trojan peptides". These are a class of polypeptides known as penetratin, which have transport properties and can carry hydrophilic compounds across the plasma membrane. This system allows oligopeptides to be directly targeted to the cytoplasm and nucleus and can be non-cell type specific and efficient. See Derossi et al., (1998), Trends Cell Biol., 8, 84-87.

[0271] Preferably, the combination therapies of the invention are presented as unit doses containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof of the active ingredients.

[0272] The combination therapy of the present invention will generally be administered orally or by any parenteral route in a pharmaceutically acceptable dosage form, in the form of a pharmaceutical composition comprising an active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base addition salt. Depending on the disorder and patient to be treated, and the route of administration, the composition can be administered in different doses.

[0273] In human therapy, the combination therapies of the invention may be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0274] For example, the combination therapy of the present invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions that may contain flavorings or coloring agents for immediate release, delayed release or controlled release applications. The medicaments, drugs and pharmaceutical compositions of the present invention can also be administered by intracavernous injection.

[0275] These tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinyl pyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and acacia. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0276] Similar solid compositions can also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the medicaments, drugs and pharmaceutical compositions of the present invention can be combined with various sweeteners or flavorings, coloring substances or dyes, emulsifying and / or suspending agents and diluents such as water, ethanol, propylene glycol and glycerol, and combinations thereof.

[0277] Combination therapy of the present invention can be parenteral, for example intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular or subcutaneous administration, or they can be used by infusion technique.They are preferably used in the form of sterile aqueous solution, and the sterile aqueous solution can contain other substances, for example enough salts or glucose to make the solution isotonic with blood.If necessary, the aqueous solution should be appropriately buffered (preferably buffered to 3 to 9 pH).Easily realize the preparation of suitable parenteral preparations under aseptic conditions by standard pharmaceutical techniques well known to those skilled in the art.

[0278] Drugs and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Drugs and pharmaceutical compositions may be presented in unit dose or multi-dose containers (e.g., sealed ampoules and vials), and may be stored in freeze-dried (lyophilized) conditions that require addition of sterile liquid carriers (e.g., water for injection) only immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0279] The combination therapy of the present invention can also be administered intranasally or by inhalation and is conveniently delivered in the form of an aerosol spray from a dry powder inhaler or from a pressurized container, pump, sprayer or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. The pressurized container, pump, sprayer or nebulizer may contain a solution or suspension of the active agent, for example using a mixture of ethanol and a propellant as a solvent, which may additionally contain a lubricant, for example sorbitan trioleate. Capsules and cartridges (e.g. made of gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent of the present invention and a suitable powder base such as lactose or starch.

[0280] Alternatively, the combination therapy of the present invention can be applied in the form of suppositories or vaginal suppositories, or they can be topically applied in the form of lotions, solutions, creams, gels, ointments or dusting powders. Medicaments, drugs and pharmaceutical compositions of the present invention can also be applied transdermally, for example, by using a skin patch. They can also be applied by ocular routes, particularly for the treatment of eye diseases.

[0281] For ophthalmic use, the combination therapy of the invention can be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or preferably as a solution in isotonic, pH-adjusted sterile saline, optionally in combination with a preservative such as benzalkonium chloride. Alternatively, they can be formulated in an ointment such as petrolatum.

[0282] For topical application to the skin, the combination therapy of the present invention can be formulated as a suitable ointment containing an active agent suspended or dissolved in a mixture such as one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream suspended or dissolved in a mixture such as one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0283] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0284] Generally, in humans, local administration of the combination therapy of the invention at or near the tumor site is the preferred route, particularly intratumoral or peritumoral administration.

[0285] For veterinary use, the combination therapies of the invention are administered in a suitably acceptable formulation in accordance with normal veterinary practice, and the veterinarian will determine the dosing regimen and route of administration that will be most appropriate for the particular animal.

[0286] Embodiments of the present invention include (but are not limited to) the following:

[0287] A. A method of treating a solid tumor in a subject, the method comprising: (a) administering to the subject a therapeutically effective amount of an antibody, or an antigen-binding portion thereof, that specifically binds CD40 and is retained at the tumor site after administration, and optionally (b) systemically administering to the subject a therapeutically effective amount of an additional therapeutic agent.

[0288] B. The method according to embodiment A, wherein the additional therapeutic agent of step (b) is an immunotherapeutic agent for treating cancer, which is not an anti-CD40 antibody.

[0289] C. The method according to embodiment b, wherein the immunotherapeutic agent is a PD1 inhibitor, optionally wherein the PD1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0290] D. A method according to any of the preceding embodiments, wherein the solid tumor is an adenoma, a blastoma, a carcinoma, a desmoid tumor, a desmoid small round cell tumor, an endocrine tumor, a germ cell tumor, a lymphoma, a sarcoma, a Wilms' tumor, a lung tumor, a colon tumor, a lymphoid tumor, a breast tumor, or a melanoma.

[0291] E. The method according to any one of the preceding embodiments, wherein the solid tumor is a melanoma, preferably a metastatic melanoma.

[0292] F. The method according to any one of the preceding embodiments, wherein the antibody of step (a) comprises at least one CDR selected from SEQ ID NOs 1, 2, 3, 4, 5 and 6.

[0293] G. The method according to any one of the preceding embodiments, wherein the antibody of step (a) comprises the CDR sequences of SEQ ID NOs: 1, 2 and 3 and / or SEQ ID NOs: 4, 5 and 6.

[0294] H. The method according to any one of the preceding embodiments, wherein the antibody of step (a) comprises a light chain variable region of SEQ ID NO:7 and / or a heavy chain variable region of SEQ ID NO:8.

[0295] I. The method according to any one of the preceding embodiments, wherein the antibody of step (a) comprises a light chain constant region of SEQ ID NO:11 and / or a heavy chain constant region of SEQ ID NO:12.

[0296] J. The method according to any one of the preceding embodiments, wherein the antibody of step (a) competes for binding to human CD40 with an antibody comprising a light chain variable region of SEQ ID NO:7 and a heavy chain variable region of SEQ ID NO:8.

[0297] K. A method according to any of the preceding embodiments, wherein steps (a) and (b) are performed simultaneously or wherein step (b) is performed 24 hours to two weeks after step (a), 24 hours to one week after step (a), 24 hours to 72 hours after step (a), or 24 hours to 48 hours after step (a).

[0298] L. A method according to any of the preceding embodiments, wherein step (a) comprises locally administering the antibody to the tumor site, optionally wherein the antibody is formulated into a composition suitable for local administration with at least one pharmaceutically acceptable diluent or carrier, and / or the antibody is conjugated to an additional therapeutic moiety.

[0299] M. A method according to any of the preceding embodiments, wherein at least 30% of the amount of antibody administered in step (a) remains at the tumor site four hours after administration, preferably wherein at least 40% of the amount remains at the tumor site four hours after administration.

[0300] N. The method according to any one of the preceding embodiments, wherein the additional therapeutic agent of step (b) is formulated with at least one pharmaceutically acceptable diluent or carrier into a composition suitable for systemic administration.

[0301] O. The method according to one of the preceding embodiments, wherein step (a) is performed on multiple separate occasions and step (b) is performed so that the subject's exposure to the additional therapeutic agent is continuous for the duration of the method.

[0302] P. The method according to any one of the preceding embodiments, wherein the subject is a human.

[0303] Q. A kit for treating a solid tumor in a subject, the kit comprising (a) a therapeutically effective amount of an antibody that specifically binds CD40 and is retained at the tumor site after administration, and optionally (b) a therapeutically effective amount of an additional therapeutic agent suitable for systemic administration to the subject.

[0304] R. An antibody, or antigen-binding portion thereof, that specifically binds CD40 and is capable of being retained at a tumor site after administration, for use in treating a solid tumor in a subject.

[0305] S. The antibody, or antigen-binding portion thereof, of embodiment R, for use in combination with one or more additional therapeutic agents, wherein the additional therapeutic agent is not an anti-CD40 antibody.

[0306] T. The antibody or antigen-binding portion thereof according to embodiment S, wherein the additional therapeutic agent is a cancer treatment selected from the group consisting of conventional radiotherapeutics, pathway inhibitors (e.g., tyrosine kinase inhibitors or serine / threonine kinase inhibitors), cytokines (e.g., IL-2, IL-12, IL-15 or IL-21), chemotherapeutics and immunotherapeutics.

[0307] U. The antibody or antigen-binding portion thereof according to embodiment S or T, used in combination with one or more of the following additional therapeutic agents:

[0308] (i) anti-PD1 immunotherapeutic agents;

[0309] (ii) anti-CTLA-4 immunotherapeutic agents;

[0310] (iii) anti-OX40 immunotherapeutic agents; and / or

[0311] (iv) Anti-CD137 immunotherapeutic agents.

[0312] V. The antibody, or antigen binding portion thereof, of embodiment T or U, wherein the immunotherapeutic agent is a PD1 inhibitor, optionally wherein the PD1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0313] W. The antibody, or antigen binding portion thereof, of any one of embodiments R to V, wherein the solid tumor is an adenoma, a blastoma, a carcinoma, a desmoid tumor, a desmoid small round cell tumor, an endocrine tumor, a germ cell tumor, a lymphoma, a sarcoma, a Wilms' tumor, a lung tumor, a colon tumor, a lymphoid tumor, a breast tumor, or a melanoma.

[0314] X. The antibody, or antigen binding portion thereof, according to any one of embodiments R to W, wherein the solid tumor is a melanoma, preferably a metastatic melanoma.

[0315] Y. The antibody, or antigen binding portion thereof, according to any one of embodiments R to X, comprising at least one CDR selected from SEQ ID NOs 1, 2, 3, 4, 5 and 6.

[0316] Z. The antibody, or antigen binding portion thereof, according to any one of embodiments R to Y, comprising the CDR sequences of SEQ ID NOs: 1, 2 and 3 and / or SEQ ID NOs: 4, 5 and 6.

[0317] AA. The antibody, or antigen binding portion thereof, according to any one of embodiments R to Z, which comprises the light chain variable region of SEQ ID NO:7 and / or the heavy chain variable region of SEQ ID NO:8.

[0318] BB. The antibody, or antigen binding portion thereof, according to any one of embodiments R to AA, comprising the light chain constant region of SEQ ID NO:11 and / or the heavy chain constant region of SEQ ID NO:12.

[0319] CC. The antibody, or antigen binding portion thereof, of any one of embodiments R to BB, wherein the antibody, or antigen binding portion thereof, competes for binding to human CD40 with an antibody comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8.

[0320] DD. The antibody, or antigen-binding portion thereof, of any one of embodiments R to CC, wherein the antibody is formulated into a composition suitable for topical administration with at least one pharmaceutically acceptable diluent or carrier, and / or the antibody is conjugated to an additional therapeutic moiety.

[0321] EE. A combination therapy composition comprising the antibody, or antigen binding portion thereof, according to any one of embodiments R to DD and one or more additional therapeutic agents, wherein the additional therapeutic agent is not an anti-CD40 antibody.

[0322] FF. A combination therapy composition according to embodiment EE, wherein the additional therapeutic agent is a cancer treatment selected from the following: conventional radiotherapeutics, pathway inhibitors (e.g., tyrosine kinase inhibitors or serine / threonine kinase inhibitors), cytokines (e.g., IL-2, IL-12, IL-15 or IL-21), chemotherapeutics and immunotherapeutics.

[0323] GG. A combination therapy composition according to embodiment EE or FF, comprising:

[0324] (i) anti-PD1 immunotherapeutic agents;

[0325] (ii) anti-CTLA-4 immunotherapeutic agents;

[0326] (iii) anti-OX40 immunotherapeutic agents; and / or

[0327] (iv) Anti-CD137 immunotherapeutic agents.

[0328] HH. The combination therapy composition according to any one of embodiments EE or GG, wherein the immunotherapeutic agent is a PD1 inhibitor, optionally wherein the PD1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0329] II. Use of the antibody, or antigen-binding portion thereof, according to any one of embodiments R to DD for the preparation of a medicament for treating a solid tumor in a subject.

[0330] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of all figures and all references, patents and published patent applications cited in this application are expressly incorporated herein by reference.

[0331] Example

[0332] Example 1 - Sequence Information

[0333] Anti-CD40 Antibody Clone G12 (Antibody ADC-1013)

[0334] (a) CDR sequences (defined according to IMGT numbering, with the core CDR sequences underlined)

[0335] V L CDR1:CTGS SSNIGAGYN VY[SEQ ID NO:1];

[0336] V L CDR2: GNI NRPS[SEQ ID NO:2];

[0337] V L CDR3:C AAWDKSISGLV [SEQ ID NO:3];

[0338] V H CDR1: GFTFSTYG MH[SEQ ID NO:4];

[0339] V H CDR2:GKGLEWLSY ISGGSSYI FYADSVRGR[SEQ ID NO:5];

[0340] V H CDR3:C ARILRGGSGMDL [SEQ ID NO:6];

[0341] (b)Variable region sequences

[0342] Variable light chain (V L ) amino acid sequence - SEQ ID NO: 7

[0343] QSVLTQPPSASGTPGQRVTIS CTGSSSNIGAGYNVY WYQQLPGTAPKLLIY GNINRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYY CAAWDKSISGLV FGGGTKLTVLG

[0344] Variable heavy chain (V H ) amino acid sequence - SEQ ID NO: 8

[0345] EVQLLESGGGLVQPGGSLRLSCAAS GFTFSTYGMH WVRQAP GKGLEWLSYISGGSSYIFYADSVRGR FTISRDNSENALYLQMNSLRAEDTAVYY CARILRGGSGMDL WGQGTLVTVSS

[0346] Variable light chain (V L ) Nucleotide sequence-SEQ ID NO:9

[0347] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGCACTGGGAGCAGCTCCAACATCGGGGCGGGTTACAATGTATACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACATCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATAAGAGCATTTCTGGTCTGGTTTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT

[0348] Variable heavy chain (V H ) Nucleotide sequence-SEQ ID NO:10

[0349] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTTTCATATATTAGTGGTGGTAGTAGTTACATTTTCTACGCAGACTCAGTGAGGGGCCGATTCACCATCTCCAGAGACAACTCCGAGAACGCGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAATATTAAGAGGCGGGAGCGGTATGGACCTCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA

[0350] (c) Exemplary constant region amino acid sequences

[0351] Human Ig lambda light chain C2 region (NCBI AAA59107.1) - SEQ ID NO: 11

[0352] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Human Igγ-1 heavy chain constant region (Uniprot P01857.1) - SEQ ID NO:12

[0353] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0354] Human CD40 sequence - SEQ ID NO: 13

[0355] >gi|117606560|gb|ABK41937.1|CD40 molecule, TNF receptor superfamily member 5 [Homo sapiens]MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ.

[0356] Example 2 - Bioavailability after local or systemic administration

[0357] Preclinical studies were conducted in cynomolgus monkeys and mice to investigate the bioavailability of ADC-1013 following peritumoral, intratumoral, or intravenous administration.

[0358] Materials and methods

[0359] The pharmacokinetics of ADC-1013 were evaluated in a human CD40-positive transgenic mouse model inoculated with human CD40-negative bladder cancer cells MB49. ADC-1013 was injected intratumorally (IT), peritumorally (PT), or intravenously (IV) at a dose of 100 μg, and serum was collected before treatment and 4 and 24 hours after treatment. Pharmacokinetic profile ( Figure 1 ) showed that systemic exposure of ADC-1013 at 4 hours post-administration was reduced 100- to 1000-fold following IT or PT administration compared with IV.

[0360] Cynomolgus monkeys were exposed to ADC-1013 via subcutaneous or intravenous routes. Subcutaneous administration here is similar to topical administration to melanoma.

[0361] Results and Conclusions

[0362] Blood samples were collected from all animals for toxicokinetic analysis at the following time points: pre-dose and 15 min and 2, 6, 24, 48, 72, 96 and 168 h post-dose, and ADC1013 levels were measured ( Figure 2 ). The bioavailability of ADC1013 after subcutaneous administration was calculated based on the mean area under the curve (AUC-96). The range of AUC-96 for subcutaneous relative to intravenous administration was 28-42%, indicating a systemic bioavailability of 28%-42%.

[0363] These observations were used to determine dose levels for administration of immunotherapeutic antibody combinations in the B16.F10 mouse model as disclosed in Example 3.

[0364] Example 3 - In vivo murine melanoma model

[0365] B16.F10 is the tumor cell line most commonly used as a melanoma model in preclinical mouse models (Grosso 2013 Cancer Immunity Review). The mouse B16.F10 melanoma cell line is well compared to human melanoma because it expresses MHC at low levels and is considered to be low immunogenic (Lechner J immunother 2013). Low MHC levels may, for example, be associated with insufficient T cell activation, which may make these tumors more difficult to treat with immunotherapy. In order to increase the translational relevance of B16.F10, the cell line was transfected with human CD40. The transfected cell line (referred to as B16.F10 (hCD40+) was used for these experiments.

[0366] Materials and methods

[0367] Melanoma cell line B16.F10 was obtained from American Type Cell Collection (ATCC). The hCD40 expressing line B16.F10 (hCD40+) was obtained by transfecting B16.F10 with a linearized vector containing human CD40 and by using Lipofectamine (Invitrogen). The vector contains an element that confers neomycin resistance. The transfected cells were cultured in DMEM (containing 4.5 g / L glucose, Ultraglutamine I and sodium pyruvate), 10% FCS, Hepes and 1 mg / ml G418 to select stable transfectants. CD40 positive clones were selected using CD40 biotin and magnetic beads (Miltenyi). A single clone (clone 5.G12.46) with verified hCD40 expression measured by flow cytometry was selected.

[0368] B16.F10 (hCD40+) cells growing in logarithmic phase were injected subcutaneously into the right flank of hCD40 transgenic mice (hCD40Tg) at day 0 (D0) in a volume of 100 μL. The average inoculation number of cells was 0.1×10 per mouse. 6 Tumor growth was measured in width, length and height using a digital caliper, and tumor volume was calculated (w / 2 x l / 2 x h / 2 x pi x (4 / 3)). Treatment was administered on days 3, 6 and 9. ADC-1013 (20 μL, 100 μg per dose) was administered intratumorally and anti-PD-1 (clone RPM1-14, BioXcell) (100 μL, 250 μg per dose) was administered intraperitoneally.

[0369] Results and Conclusions

[0370] like Figure 3 As shown in Figure A, the transfected B16.F10 (hCD40+) melanoma cell line showed abundant expression of human CD40. Figure 4A ) and the tumor volume on the day the first mouse was sacrificed ( Figure 4B ) The anti-tumor effect of ADC-1013 was determined in hCD40tg mice bearing subcutaneous B16.F10 (hCD40+) melanoma. ADC-1013 showed a significant reduction in tumor growth measured by increased survival time and reduced tumor volume. Anti-tumor efficacy was increased by treating animals with a combination of ADC-1013 and anti-PD-1 antibodies.

[0371] Example 4 - In vivo murine bladder cancer model

[0372] Materials and methods

[0373] MB49 bladder cancer cells were used to initiate tumors in 8-week-old female hCD40Tg mice. On day 0, 0.25 × 10 6 Tumor cells were inoculated subcutaneously into the right flank of mice. On day 14, mice were injected intratumorally or intraperitoneally with test anti-CD40 antibodies (a total of 1 μg and 30 μg antibody per mouse, or PBS; 4 mice per group). On day 16, 40 mice were killed by cervical dislocation. Tumor-draining lymph nodes were collected into complete medium, and two tumors or lymph nodes from each experimental group were pooled together. The collected tissues were enzymatically and mechanically homogenized using Liberase TL (Roche) and a nylon mesh filter (100 μm; Fischer Scientific). The membranes were washed thoroughly with RPMI medium containing 3-10 mM EDTA and 0.1% fetal bovine serum to prepare a single cell suspension. The isolated cells were washed in PBS containing 0.5% bovine serum albumin, and nonspecific Fc binding was blocked by treating the cells with mouse anti-CD16 / 32 (BD Bioscience).

[0374] CD86 expression levels (as a marker of activation) were analyzed by flow cytometry for CD11c positive cells and CD11b positive cells, respectively. CD11c is a marker for dendritic cells. CD11b is expressed on monocytes, macrophages, and dendritic cell subsets. The cells were stained with live / dead fixable stain FVS450 (BD Bioscience) and antibodies specific for CD11c-PE, CD11b-PECy7, and CD86-APC (BD Bioscience) diluted 1:100. After staining, all cells were paraformaldehyde fixed using Cellfix (BD Bioscience). The staining of each sample was measured and calculated as CD86 (sample) -FMO (sample) and expressed as % positive cells minus PBS control. The stained cells were analyzed using FACS Verse (Becton Dickinson) and FlowJo vX analysis software. The results are shown in Figure 5A (CD11c cells) and Figure 5B (CD11b cells).

[0375] Results and Conclusions

[0376] Figure 5A The data in show that treatment with anti-CD40 antibodies increases dendritic cell activation as measured by CD86 expression in tumors. Figure 5BThe data in show that treatment with anti-CD40 antibodies increases CD11b-positive cell activation measured by CD86 expression in tumors. In summary, stronger activation was obtained after intratumoral (IT) treatment compared with intraperitoneal (IP) treatment. Treatment with a low dose of 1 μg given intratumorally (IT) produced unexpectedly high dendritic cell activation.

[0377] Example 5 - In vivo effects of combination therapy I

[0378] Materials and methods

[0379] B16.F10 (hCD40+) tumor cells in logarithmic phase were injected subcutaneously into the right flank of hCD40 transgenic mice (hCD40Tg) at day 0 (D0) in a volume of 100 μL. The number of cells inoculated was 0.1×10 per mouse. 6 Tumor growth was measured in width, length and height using a digital caliper and tumor volume was calculated (w / 2 x l / 2 x h / 2 x pi x (4 / 3)). Treatment was administered on days 3, 6 and 9. ADC-1013 (100 μg per dose) was administered intratumorally. Anti-PD-1 (250 μg per dose) (clone RPM1-14, BioXcell) and anti-CTLA-4 antibodies (100 μg per dose) (9D9, BioXcel) were injected intraperitoneally. Tumor volume on day 14 is shown in Figure 6 middle.

[0380] Results and Conclusions

[0381] It has been shown that treatment with antibodies targeting PD-1 in combination with antibodies targeting CTLA-4 can confer an additive effect in clinical trials (Wolchok et al., New Eng J Med, 2013). In this example, we disclose preclinical data showing that adding ADC-1013 treatment to the CTLA-4 / PD-1 combination can further increase the therapeutic effect in melanoma patients. The anti-tumor effect, measured by the reduction in tumor volume at day 14, was significantly better for CTLA-4, PD-1, and ADC-1013 compared to CTLA-4 and PD-1 alone ( Figure 6 ).

[0382] In a clinical setting, the CTLA-4 antibody may be any of ipilimumab, tremelimumab or other CTLA-4 targeting antibodies. The PD-1 targeting antibody may be an antibody that binds to human PD-1, such as nivolumab or pembrolizumab or others. It may also be an antibody that binds to a ligand of PD-1, such as an antibody that targets PD-L1 and PD-L2, such as durvalumab and avelumab.

[0383] Example 6 - In vivo effects of combination therapy II

[0384] Materials and methods

[0385] B16.F10 tumor cells in logarithmic phase were injected subcutaneously into the right flank of hCD40 transgenic mice (hCD40Tg) at day 0 (D0) in a volume of 100 μl. The number of cells inoculated was 0.1×10 per mouse. 6 Tumor growth was measured in width, length and height using a digital caliper and tumor volume was calculated (w / 2xl / 2xh / 2xpi x(4 / 3)). Treatments were administered on days 3, 6 and 9. ADC-1013, anti-CD137 (clone Lob 12.3) and anti-OX40 (CD86) antibodies and controls were administered intratumorally (100 μg per dose). Figure 7 The change in tracking volume over time is shown in .

[0386] Results and Conclusions

[0387] Treatment with ADC-1013 produced a significant antitumor response compared to controls. The antitumor effect was greater than that obtained with antibodies targeting CD137 and OX40 ( Figure 7 ). The combination of ADC-1013 and OX40 and the combination of ADC-103 and CD137 resulted in a stronger anti-tumor effect than monotherapy. This suggests that combining CD40 and CD137 or combining CD40 and OX40 will have clinical benefits.

[0388] Example 7 - Effect of ADC-1013 on wild-type B16 melanoma in mice

[0389] Materials and methods

[0390] B16.F10.hCD40+ cells or B16.F10(wt) cells (1×10 5 ), and mice were treated intratumorally with ADC-1013 on days 3, 6, and 9 (100 μg per dose).

[0391] The mice used in this study were hCD40tg mice.

[0392] Results and Conclusions

[0393] ADC-1013 also produced significant antitumor effects in B16.F10 tumors. The antitumor effects obtained in B16.F10 (wt) and B16.F10.hCD40+ tumors were similar ( Fig. 8A and Figure 8B). This does not exclude that direct induction of ADCC plays a role in human CD40-positive melanoma therapy. There may be other qualitative differences in hCD40-positive tumors, such as lower immune cell infiltration. In addition, human CD40 levels were lower on B16 tumors, as measured ex vivo on tumor samples.

[0394] Example 8 - Effect of ADC-1013 in a lymphoma model (A20)

[0395] Materials and methods

[0396] A20 lymphoma cell line growing in logarithmic phase was injected subcutaneously into the right flank of hCD40tg-BalbC(F1) mice in a volume of 100 μL on day 0 (D0). hCD40tg-BalbC(F1) mice were generated by crossing hCD40tg mice with BalbC mice. The number of cells inoculated was 5×10 per mouse. 6 Tumor growth was measured in width, length and height using a digital caliper and tumor volume was calculated (w / 2 x l / 2 x h / 2 x pi x (4 / 3)). ADC-1013 (30 μg per dose) was administered peritumorally in mice with established lymphoma tumors (A20) on days 10, 13 and 16.

[0397] Results and Conclusions

[0398] Treatment with ADC-1013 produced significant antitumor effects in the A20 lymphoma model ( Fig.9A and Fig. 9B ). This model is hCD40 negative and therefore the anti-tumor effects shown here are solely caused by the activation of immune cells by ADC-1013.

[0399] Example 9 - Effect of ADC-1013 in a lung cancer model (LLC-1)

[0400] Materials and methods

[0401] A lung cancer cell line (LLC-1) growing in the logarithmic phase was injected subcutaneously into the right flank of hCD40tg mice at day 0 (D0) in a volume of 100 μL. The number of cells inoculated was 0.25×10 per mouse. 6 Tumor growth was measured in width, length and height using a digital caliper and tumor volume was calculated (w / 2 x l / 2 x h / 2 x pi x (4 / 3)). ADC-1013 (100 μg) was administered peritumorally in hCD40tg mice with established tumors (LLC-1) on days 4, 7 and 10.

[0402] Results and Conclusions

[0403] Treatment with ADC-1013 produced significant (p<0.05, one-sided Mann Whitney t-test) antitumor effects in the lung cancer model ( Fig.10 ). This model is hCD40 negative and the anti-tumor effects shown here are solely caused by the activation of immune cells by ADC-1013.

[0404] Example 10 - Distal effects of topically administered ADC-1013

[0405] Materials and methods

[0406] B16.F10 tumor cells in logarithmic phase were injected subcutaneously into the right and left flanks of hCD40 transgenic mice (hCD40Tg) at day 0 (D0) in a volume of 100 μL. The number of cells inoculated was 0.1×10 per mouse. 6 Tumor growth was measured in width, length, and height using a digital caliper and tumor volume was calculated (w / 2 x l / 2 x h / 2 x pi x (4 / 3)). Treatments were administered on days 3, 6, and 9. ADC-1013 and controls were administered intratumorally (100 μg per dose) into tumors in the right flank. Fig.11 Tumor volume changes over time were tracked for injected and non-injected tumors as shown in .

[0407] Results and Conclusions

[0408] Local intratumoral treatment also produced antitumor effects in distant non-injected tumors. The free ADC-1013 levels after 100 μg treatment were far below the EC50 of ADC-1013 in the in vitro assay, suggesting that the antitumor effects on non-injected tumors depend in part on the migration of immune cells activated in the injected tumor area to the non-injected tumors.

[0409] This suggests that ADC-1013 injected into one tumor can have a significant anti-tumor effect on other non-injected tumors (eg, metastases).

[0410] Example 11 - Effects of systemic (iv) administration of ADC-1013

[0411] Materials and methods

[0412] B16.F10 (hCD40+) tumor cells in logarithmic phase were injected subcutaneously into the right flank of hCD40 transgenic mice (hCD40Tg) at day 0 (D0) in a volume of 100 μL. The number of cells inoculated was 0.1×10 per mouse. 6Tumor growth was measured in width, length and height using a digital caliper and tumor volume was calculated (w / 2 x l / 2 x h / 2 x pi x (4 / 3)). Treatment was administered on days 3, 6 and 9. ADC-1013 (100 μg per dose) was administered intravenously.

[0413] Results and Conclusions

[0414] Systemic, intravenous administration of ADC-1013 resulted in significant inhibition of tumor growth ( Fig.12 ).

[0415] Example 12 - In vivo effects of combination therapy III

[0416] Materials and methods

[0417] Female BalbC mice were inoculated subcutaneously with 5 × 10 6 A20 lymphoma cells. Mice were treated peritumorally with 9D9 (anti-CTLA-4 antibody, BioXcel) on days 5 and 8 and intratumorally with the TLR agonist CpG (1668) on days 5, 8, and 11. Survival was followed over time.

[0418] Results and Conclusions

[0419] The 9D9 antibody targets the checkpoint receptor CTLA-4 and CpG binds to Toll-like receptor 9. TLRs are transmembrane proteins expressed on antigen presenting cells such as dendritic cells. Ligation of TLR9 with CpG induces dendritic cell activation. The results are shown in Fig.13 middle.

[0420] The anti-tumor effect of the combination of treatments targeting TLR9 and CTLA4 did not result in an increased anti-tumor effect compared to treatment with CpG alone. The data therefore suggest that treatment combinations that activate dendritic cells in combination with checkpoint inhibitors do not always result in an increased anti-tumor effect.

[0421] Therefore, the efficacy of the combination therapies of the present invention cannot be predicted or reasonably anticipated.

Claims

1. Use of a combination of an antibody or an antigen binding portion thereof that specifically binds to CD40 and an immunotherapeutic agent in the preparation of a medicament for treating solid tumors, wherein the immunotherapeutic agent is selected from: a) Immunotherapeutic agents that bind to PD-1; b) immunotherapeutic agents that bind to CTLA-4; c) an immunotherapeutic agent that binds OX40; and d) Immunotherapeutic agents that bind CD137.

2. The use according to claim 1, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain CDR sequences of SEQ ID NOs: 1, 2 and 3 and the heavy chain CDR sequences of SEQ ID NOs: 4, 5 and 6.

3. The use according to claim 1, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 is in the same composition or in separate compositions as the immunotherapeutic agent. 4 . The use according to claim 1 , wherein the immunotherapeutic agent that binds to PD-1 is an anti-PD-1 antibody that blocks the interaction between PD1 and PD-L1.

5. The use according to claim 1, wherein the solid tumor is melanoma.

6. The use according to claim 1, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 comprises or consists of an intact antibody.

7. The use according to claim 1, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 comprises or consists of an antigen-binding fragment selected from the group consisting of an Fv fragment and a Fab-like fragment.

8. The use of claim 1, wherein the antibody or antigen-binding portion thereof is human or humanized.

9. The use according to claim 1, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain variable region of SEQ ID NO: 7 and / or the heavy chain variable region of SEQ ID NO:

8.

10. The use according to claim 1, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain constant region of SEQ ID NO: 11 and / or the heavy chain constant region of SEQ ID NO:

12.

11. The use according to claim 1, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of: SEQ ID NO:7 plus the light chain of SEQ ID NO:11, and / or SEQ ID NO:8 plus the heavy chain of SEQ ID NO:

12.

12. The use according to claim 4, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, lambulatuzumab, pidilizumab and AMP-224.

13. The use of claim 1, wherein the immunotherapeutic agent that binds to CTLA-4 is an anti-CTLA-4 antibody or an antigen-binding portion thereof.

14. The use of claim 1, wherein the immunotherapeutic agent that binds OX40 is an anti-OX40 antibody or an antigen-binding portion thereof.

15. The use according to claim 1, wherein the immunotherapeutic agent that binds to CD137 is an anti-CD137 antibody or an antigen-binding portion thereof.

16. A pharmaceutical composition for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) Anti-PD-1 antibodies that block the interaction between PD1 and PD-L1.

17. A pharmaceutical composition for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) an anti-CTLA-4 antibody or an antigen-binding portion thereof.

18. A pharmaceutical composition for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) an anti-OX40 antibody or an antigen-binding portion thereof.

19. A pharmaceutical composition for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) an anti-CD137 antibody or an antigen-binding portion thereof.

20. The pharmaceutical composition of any one of claims 16 to 19, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain CDR sequences of SEQ ID NOs: 1, 2 and 3 and the heavy chain CDR sequences of SEQ ID NOs: 4, 5 and 6.

21. The pharmaceutical composition of any one of claims 16 to 19, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 comprises or consists of an intact antibody.

22. The pharmaceutical composition according to any one of claims 16 to 19, wherein the antibody or antigen-binding portion thereof that specifically binds to CD40 comprises or consists of an antigen-binding fragment selected from the group consisting of an Fv fragment and a Fab-like fragment.

23. The pharmaceutical composition of any one of claims 16 to 19, wherein the antibody, or antigen binding portion thereof, is human or humanized.

24. The pharmaceutical composition of any one of claims 16 to 19, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises a light chain variable region of SEQ ID NO: 7 and / or a heavy chain variable region of SEQ ID NO:

8.

25. The pharmaceutical composition of any one of claims 16 to 19, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain constant region of SEQ ID NO: 11 and / or the heavy chain constant region of SEQ ID NO:

12.

26. The pharmaceutical composition of any one of claims 16 to 19, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of: SEQ ID NO:7 plus the light chain of SEQ ID NO:11, and / or SEQ ID NO:8 plus the heavy chain of SEQ ID NO:

12.

27. The pharmaceutical composition according to claim 16, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, lambulatuzumab, pidilizumab and AMP-224.

28. A kit for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) Anti-PD-1 antibodies that block the interaction between PD1 and PD-L1.

29. A kit for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) an anti-CTLA-4 antibody or an antigen-binding portion thereof.

30. A kit for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) an anti-OX40 antibody or an antigen-binding portion thereof.

31. A kit for treating solid tumors, comprising: a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and b) an anti-CD137 antibody or an antigen-binding portion thereof.

32. The kit of any one of claims 28 to 31, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain CDR sequences of SEQ ID NOs: 1, 2 and 3 and the heavy chain CDR sequences of SEQ ID NOs: 4, 5 and 6.

33. The kit of any one of claims 28 to 31, wherein the solid tumor is a melanoma.

34. The kit of any one of claims 28 to 31, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of an intact antibody.

35. The kit of any one of claims 28 to 31, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of an antigen binding fragment selected from the group consisting of an Fv fragment and a Fab-like fragment.

36. The kit of any one of claims 28 to 31, wherein the antibody, or antigen binding portion thereof, is human or humanized.

37. The kit of any one of claims 28 to 31, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises a light chain variable region of SEQ ID NO: 7 and / or a heavy chain variable region of SEQ ID NO:

8.

38. The kit of any one of claims 28 to 31, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises the light chain constant region of SEQ ID NO: 11 and / or the heavy chain constant region of SEQ ID NO:

12.

39. The kit of any one of claims 28 to 31, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of: SEQ ID NO:7 plus the light chain of SEQ ID NO:11, and / or SEQ ID NO:8 plus the heavy chain of SEQ ID NO:

12.

40. The kit of claim 28, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, lambulatuzumab, pidilizumab, and AMP-224.

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