Combination therapy

Abstract

The present invention relates to a combination therapy for treating cancer, optionally chemotherapy-resistant cancer, in a subject. The combination therapy comprises (a) an antibody or antigen-binding portion thereof that specifically binds to CD40, and (b) chemotherapy. The present invention also relates to pharmaceutical compositions, kits, and methods of using such therapies.

Description

Technical Field

[0001] The present invention relates to anti-CD40 antibodies and chemotherapy, and their combined use in the treatment of cancers such as chemotherapy-resistant cancers.

Background Art

[0002] Introduction to Pancreatic Ductal Adenocarcinoma Pancreatic ductal adenocarcinoma (PDAC) is a type of exocrine pancreatic cancer. It is the most common type of pancreatic cancer, accounting for 95 out of 100 (95%) of all pancreatic cancers. Pancreatic cancer is the fourth most common cause of cancer death in both the United States and the European Union [1]. The incidence of pancreatic cancer is highest in Europe (7.7 per 100,000) and North America (7.6 per 100,000), followed by Oceania (6.4 per 100,000) [2]. In the United States, the estimated number of new cases in 2020 was 57,600, and the estimated number of deaths was 47,050 (Cancer facts and figures, American Cancer Society, 2020). The incidence is slightly higher in men than in women.

[0003] The average 5-year survival rate of pancreatic cancer is approximately 5%, and this figure has not changed over the past 20 years. Surgical resection is the only curative method, but at best, only 1 in 5 patients is considered operable, and even in these cases, the 5-year survival rate is about 20%. More than three-quarters of patients with progressive disease, approximately half have distant metastases at the time of diagnosis, and the remaining quarter have locally inoperable disease. Over time, most of these locally advanced cancers also metastasize [3].

[0004] Therefore, there is a need for effective treatment of metastatic pancreatic cancer. Until the past decade, the standard first-line treatment for metastatic pancreatic cancer was gemcitabine, which resulted in an overall median survival (mOS) of about six months. The introduction of the FOLFIRINOX regimen led to a major advance in 2011, with an objective response rate (ORR) of 31% and an mOS of 11.1 months compared with gemcitabine monotherapy, which had an ORR of 9.4% and an mOS of 6.8 months, in the randomized trial PRODIGE 4 in which 342 patients were enrolled [4]. Two years later, the MPACT trial comparing gemcitabine + nab-paclitaxel with gemcitabine also showed superior activity of the combination, with an ORR of 23% and an mOS of 8.5 months compared with gemcitabine monotherapy, which had an ORR of 7% and an mOS of 6.7 months [5].

[0005] Mitazalimab, a CD40 agonist Mitazalimab (also known as JNJ-64457107 and ADC-1013) is a human monoclonal (IgG1) antibody that is an agonist targeting CD40. The agent is being investigated for the treatment of advanced solid tumors in two phase 1 trials: A-14-1013-C-01 (EudraCT number 2014-004556-56) and JNJ-64457107 CAN1001 (EudraCT number 2016-000969-23).

[0006] Overview of CD40 CD40 is a co-stimulatory receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily [6]. CD40 is expressed in a number of cell types and can be detected on the surface of antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages. In addition, CD40 is expressed in granulocytes, endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells [6-8]. CD40 is also present on the membranes of a wide range of malignant cells, including those of non-Hodgkin and Hodgkin lymphomas, myelomas, and several carcinomas, including those of the nasopharynx, bladder, cervix, kidney, and ovary [6, 9]. CD40 is a transmembrane protein expressed by activated T cells, B cells, platelets, mast cells, macrophages, basophils, natural killer (NK) cells, and non-hematopoietic cells (smooth muscle cells, endothelial cells, and epithelial cells) that interacts with a single ligand, CD40L (or CD154) [6, 7].

[0007] The molecular consequences of CD40 signaling depend on the cell type expressing CD40 and the microenvironment in which the CD40 signal is provided

[10] . Ligation and cross-linking of CD40 are required for an adaptive immune response through "licensing" of APCs, and particularly DCs, by inducing upregulation of co-stimulatory receptors and major histocompatibility complex molecules and production of pro-inflammatory cytokines. Thus, CD40 is involved in the functional maturation of APCs and, as a result, activation of antigen-specific T lymphocytes [11-13]. CD40 also plays a role in humoral immunity by activating resting B lymphocytes and increasing their antigen-presenting function [10, 14]. Furthermore, CD40 is involved in the induction of innate immunity through stimulation of cytotoxic myeloid cells such as NK cells, macrophages, and granulocytes [10, 14, 15].

[0008] Summary of non-clinical data The CD40 agonist properties of mitazalimab have been verified in vitro in human monocyte-derived DC cultures. Ligation of CD40 by mitazalimab leads to upregulation of activation markers on the surface of DCs such as CD80 and CD86, as well as the release of cytokines such as IL-12. Mitazalimab has also been shown to polarize tumor-associated macrophages (TAMs) isolated from human prostate and ovarian tumor samples from a more immunosuppressive phenotype to a more immunoinflammatory phenotype, for example, by upregulation of CD83.

[0009] The agonist effect of mitazalimab depends critically on the binding of the Fc portion of the antibody to the Fcγ receptor (FcγR). The ability of mitazalimab to activate DCs (i.e., upregulate CD86) was significantly reduced when using a deglycosylated variant of mitazalimab that contains an N297Q mutation in the γ chain and thus does not bind to FcγR. The CD40 agonist effect was restored again when this deglycosylated mitazalimab variant was cross-linked with anti-human IgG, conclusively demonstrating that mitazalimab depends on FcγR cross-linking for optimal activity. Mitazalimab has shown immune-mediated and antibody-dependent cell-mediated cytotoxicity (ADCC)-dependent antitumor efficacy in vivo in human tumors transplanted into NSG mice and human CD40 transgenic (hCD40tg) mice. Furthermore, mitazalimab induces T cell-dependent and tumor-specific immune memory in the bladder cancer cells of hCD40tg mice and has immunity against tumor rechallenge for at least 5 months. Mitazalimab has also shown a synergistically enhanced effect on tumor growth and survival when combined with other immunotherapies such as immune checkpoint inhibitors (e.g., PD-1), vaccination, or chemotherapy (e.g., mFOLFIRINOX) in experimental tumor models of hCD40tg mice. For additional and detailed information on non-clinical data, please refer to the mitazalimab investigational drug brochure.

[0010] Summary of Clinical Data Mitazalimab Monotherapy Trials First-in-Human Trial by Intratumoral Administration of Mitazalimab Study A-14-1013-C-01 (NCT02379741) was a first-in-human, multi-center, non-randomized, non-blinded, escalating-dose, repeat-dose Phase 1 study of mitazalimab (ADC-1013) in patients with advanced solid malignancies.

[0011] Mitazalimab was administered as an intratumoral bolus injection (intratumoral administration) to the same tumor lesion every 14 days in 18 patients and as an intravenous (IV) infusion over 2 hours every 14 days in 5 patients.

[0012] The maximum tolerated dose (MTD) was not identified. Increases in liver enzymes or bilirubin were observed in 9 of 18 patients who received intratumoral administration and in 3 of 5 patients who received IV administration. Only 2 patients had normal liver enzyme and bilirubin values at baseline. Two dose-limiting toxicities (DLTs) were observed, and both a patient with grade 3 abdominal pain and a patient with grade 3 cholecystitis received intratumoral administration. The best response was stable disease (SD) in 1 patient with renal cell carcinoma (intratumoral at 400 μg / kg). No patients with pancreatic cancer were enrolled in this study.

[0013] Dose Escalation Study of Mitazalimab by Intravenous Administration Study 64457107CAN1001 (NCT02829099) was a multi-center, non-randomized, non-blinded, dose escalation Phase 1 study of mitazalimab (JNJ-64457107) in patients with advanced solid tumors. Mitazalimab was administered as an IV infusion every 14 days at doses ranging from 75 μg / kg to 2000 μg / kg using corticosteroids included in premedication and at a maximum dose of 1200 μg / kg without using corticosteroids.

[0014] A total of 95 patients were exposed to mitazalimab. One patient remains on treatment. The MTD was not identified. Two patients experienced DLTs, one patient had a headache that persisted for 5 days, and one patient had grade 3 liver enzyme elevations along with grade 2 bilirubin elevation. Seven pancreatic cancer patients were enrolled in the trial, and one of them experienced SD for over 6 months. A partial response (PR) was observed in a patient with renal cell cancer who was administered 14 cycles (i.e., 28 doses) at a dose level of 1200 μg / kg and was on trial for 9.2 months

[16] .

[0015] Pharmacodynamic activity The pharmacodynamic biomarker data of trial 644577107CAN1001 were tested after the first mitazalimab administration and were consistent with CD40-mediated immune cell activation. After IV administration of mitazalimab, a marginal trend of B cells with dose-dependent B cell recovery was observed at all doses tested. Also, after injection of mitazalimab at all doses tested, NK cells and T cells in peripheral blood decreased, except for the lowest dose (75 μg / kg). The levels of both cell types had completely recovered by day 8 of the trial.

[0016] CD40 receptor occupancy was evaluated in B cells and was shown to be dose-dependent. CD40 remained engaged until day 8 after mitazalimab administration. The peripheral levels of MCP-1, IP-10, and MIP-1B chemokines reached a peak 1 - 4 hours after injection, consistent with myeloid cell activation. Other chemokines (such as MIP-1α and IL-8) and cytokines (such as IFN-γ, TNF-α, and IL12p70) were also observed, but to a lesser extent. IL-6 levels, which can be highly induced in subjects with cytokine release syndrome, did not increase after injection of mitazalimab. The pattern of biomarker changes was consistent with the proposed mechanism of mitazalimab as a CD40 agonist.

[0017] CD40 agonist administered to pancreatic cancer patients Serclidemab Serplulimab (CP-870.893, RO7009789) is a CD40 agonist monoclonal antibody with an IgG2 format that activates CD40 independent of cross-linking to Fcγ receptors

[17] . In agonist CD40 antibodies, systemic delivery by antibodies independent of cross-linking is generally thought to increase the risk of toxicity that can reduce the therapeutic window

[18] . Serplulimab has been evaluated in combination with gemcitabine in patients with previously untreated pancreatic cancer

[19] . The combination was well tolerated up to a maximum of 0.2 mg / kg. One DLT, a cerebrovascular accident, occurred at the 0.2 mg / kg dose level. Four of 22 patients had a PR, and no complete responses (CRs) were observed (response rate 19%). A cytokine release syndrome associated with serplulimab infusion was observed in 20 of 22 patients, with one event being grade 3 and all other events being grade 1 or 2. Immunological activation with increased inflammatory cytokines, increased expression of costimulatory molecules on B cells, and transient depletion of B cells was observed in all patients. Liver enzyme elevations occurred in approximately two-thirds of patients, all of which were grade 1-2, but hyperbilirubinemia occurred in some patients.

[0018] APX005M APX005M is a CD40 agonist monoclonal antibody with a mutated IgG1 format for improved binding to FcγR2b, which depends on cross-linking to FcγRs for its function. APX005M has a profile similar to mitazalimab, with the aim of improving efficacy and safety compared to previous CD40 antibodies. APX-005M is in clinical development for several malignancies.

[0019] Initial clinical data with APX005M in combination with gemcitabine + nab-paclitaxel with or without nivolumab in patients with metastatic pancreatic cancer that remained untreated were presented in 2019. See Table 1. In each 28-day cycle, gemcitabine + nab-paclitaxel was administered 3 times, nivolumab was administered 2 times, and APX005M was administered 1 time. Immunoprofiling of PBMCs showed remodeling of the myeloid compartment in response to treatment with rapid activation of DCs in most patients. Analysis of circulating mutant KRAS DNA showed significant and rapid reduction with therapy. Preliminary efficacy data from 24 patients showed promising data for the combination, and the randomized trial is ongoing

[20] .

Table 1

[0020] The listing or discussion of a document that is clearly publicly available prior to this specification should not necessarily be construed as an admission that the document is part of the state of the art or common general knowledge.

Summary of the Invention

[0021] Combination chemotherapy for metastatic pancreatic cancer has been pushed to the limits of tolerance, but survival outcomes remain poor. Pancreatic cancer is classified as immunologically "cold" compared to other tumors characterized by immune infiltration. The desmoplastic stroma formed around pancreatic cancer functions as a physical barrier to chemotherapy and hosts tumor fibroblasts and suppressive myeloid cells that suppress the immune response in the tumor microenvironment. Furthermore, pancreatic cancer has relatively few non-synonymous mutations compared to other cancers, so tumors are characterized by low expression of tumor neoantigens. These two factors contribute to the lack of activity seen with checkpoint inhibitors in pancreatic cancer.

[0022] Accordingly, an object of the present invention is to provide an improved combination therapy for treating cancer (e.g., pancreatic cancer), optionally chemotherapy-resistant cancer.

[0023] A first aspect of the present invention provides a combination therapy for use in treating cancer, optionally chemotherapy-resistant cancer, in a subject, the combination therapy comprising an antibody or antigen-binding portion thereof that specifically binds to CD40 and chemotherapy.

[0024] A second aspect of the present invention provides a combination therapy comprising an antibody or antigen-binding portion thereof and chemotherapy for use in a dosage regimen for treating cancer, optionally chemotherapy-resistant cancer, the dosage regimen comprising (a) a step of administering an antibody or antigen-binding portion thereof that specifically binds to CD40, and (b) a step of chemotherapy.

[0025] A third aspect of the present invention provides an antibody or antigen-binding portion thereof that specifically binds to CD40 for use in treating cancer, optionally chemotherapy-resistant cancer, in a subject, the antibody or antigen-binding portion thereof being for use in combination with chemotherapy.

[0026] In an alternative version of the third aspect, the present invention provides chemotherapy for use in treating cancer, optionally chemotherapy-resistant cancer, in a subject, the chemotherapy being for use in combination with an antibody or antigen-binding portion thereof that specifically binds to CD40.

[0027] In some embodiments, the subject may be treated with the antibody or antigen-binding portion thereof and then treated with chemotherapy to achieve the combination therapy. In one embodiment, the subject may be treated with chemotherapy and then treated with the antibody or antigen-binding portion thereof to achieve the combination therapy.

[0028] A fourth aspect of the invention provides for the use of an antibody or antigen-binding portion thereof that specifically binds to CD40 in the preparation of a medicament for treating cancer, optionally chemotherapy-resistant cancer, wherein the antibody or antigen-binding portion thereof is for use in combination with chemotherapy.

[0029] In an alternative version of the fourth aspect, the invention provides for the use of an antibody or antigen-binding portion thereof that specifically binds to CD40 in the preparation of a medicament in a combination therapy according to the first or second aspect of the invention.

[0030] A fifth aspect of the invention provides a method of treating cancer, optionally chemotherapy-resistant cancer, in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof that specifically binds to CD40 and chemotherapy.

[0031] In an alternative version of the fifth aspect, the invention provides a method of treating cancer, optionally chemotherapy-resistant cancer, in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination therapy according to the first aspect of the invention.

[0032] In a further alternative version of the fifth aspect, the invention provides a method of treating cancer, optionally chemotherapy-resistant cancer, in a subject, the method comprising administering to the subject a combination therapy according to the second aspect of the invention.

[0033] A sixth aspect of the invention provides a pharmaceutical composition comprising an antibody or antigen-binding portion thereof that specifically binds to CD40 and chemotherapy. The pharmaceutical composition may comprise an antibody or antigen-binding portion thereof and / or chemotherapy according to any preceding aspect of the invention. The pharmaceutical composition may comprise one or more components of chemotherapy (e.g., all 1, 2, 3, or 4 components of the FOLFIRINOX regimen) and may optionally further comprise an antibody or antigen-binding portion thereof.

[0034] The seventh aspect of the present invention provides a kit comprising an antibody or antigen-binding portion that specifically binds to CD40 and chemotherapy. The kit may comprise an antibody or antigen-binding portion thereof and / or chemotherapy according to any preceding aspect of the present invention.

[0035] The following paragraphs may relate to any of the foregoing aspects. Preferences and options regarding a given aspect, feature or parameter of the present invention should be considered to be disclosed in combination with any preferences and options regarding all other aspects, features and parameters of the present invention, unless the context otherwise indicates.

[0036] In some embodiments, the cancer, optionally a chemotherapy-resistant cancer, is pancreatic cancer. In some embodiments, the pancreatic cancer is an exocrine tumor such as an exocrine gland cancer or pancreatic ductal adenocarcinoma (PDAC), or an endocrine tumor. In some embodiments, the cancer is a solid tumor. The solid tumor can be selected from the group consisting of pancreatic tumors, adenomas, blastomas, carcinomas, desmoid tumors, fibromatosis round cell tumors, endocrine tumors, germ cell tumors, lymphomas, sarcomas, Wilms tumors, lung tumors, colon tumors, lymphoma tumors, breast tumors, and melanomas.

[0037] In some embodiments, the chemotherapy is selected from the group consisting of FOLFIRINOX or a variant thereof (such as mFOLFIRINOX), gemcitabine, nab-paclitaxel, and combinations thereof. In some embodiments, the chemotherapy is FOLFIRINOX or a variant thereof, and the antibody or antigen-binding portion thereof is mitazalimab.

[0038] In some embodiments, FOLFIRINOX or a variant thereof comprises an oxaliplatin (such as Eloxatin® or a generic drug) infusion, and optionally, the oxaliplatin is 85 mg / m 2administered intravenously at a dose of and / or over a period of 2 hours. In some embodiments, FOLFIRINOX or a variant thereof includes an infusion of folinate (such as leucovorin, calcium folate, calcium levofolinate, disodium folate, and disodium levofolinate), and optionally, leucovorin is administered at a dose of 400 mg / m 2 administered intravenously at a dose of and / or over a period of 2 hours. In some embodiments, FOLFIRINOX or a variant thereof includes an infusion of irinotecan (such as Campto®), and optionally, irinotecan is administered at a dose of 150 mg / m 2 administered intravenously at a dose of and / or 30 minutes after the end of the leucovorin infusion. In some embodiments, FOLFIRINOX or a variant thereof includes an infusion of 5-fluorouracil, and optionally, 5-fluorouracil is administered at a dose of 2400 mg / m 2 administered intravenously at a dose of and / or over a period of 46 - 48 hours (e.g., 2.4 g / m 2 / day).

[0039] In some embodiments, the subject receives at least one treatment cycle of FOLFIRINOX (e.g., mFOLFIRINOX). The treatment cycle may correspond to the OPTIMIZE-1 trial (see Example 2). In some embodiments, the subject receives 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more treatment cycles. In some embodiments, the subject does not receive more than 12 treatment cycles.

[0040] In some embodiments, oxaliplatin is administered over a period of 2 hours, immediately followed by folinate (e.g., leucovorin) administered over a period of 2 hours, irinotecan is started 30 minutes after the start of leucovorin and administered over a period of 90 minutes, and then 5-fluorouracil is administered over a period of 46 - 48 hours.

[0041] In some embodiments, the subject receives a pre-medication. In some embodiments, the subject receives a post-medication. In some embodiments, the subject receives both a pre-medication and a post-medication. The pre-medication may include (i) an NK1 receptor antagonist such as aprepitant, 125 mg PO, 60 minutes before infusion and / or continuously during the day of chemotherapy, (ii) a 5-HT3 receptor antagonist such as ondansetron, 8 mg PO, 30 minutes before infusion and / or continuously during the day of chemotherapy, and / or (iii) a corticosteroid such as dexamethasone, 8 mg IV or PO, 30 minutes before infusion. The post-medication may include G-CSF such as Neulasta, 6 mg SC, on the 4th day after the start of the FOLFORINOX regimen or its variant and / or at least 24 hours after the end of continuous 5-fluorouracil infusion.

[0042] In some embodiments, the antibody or antigen-binding portion thereof is administered at a dose of 50 μg / kg to 1200 μg / kg, such as 450 μg / kg to 900 μg / kg. In some embodiments, the antibody or antigen-binding portion thereof is administered at a dose of 50 μg / kg, 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1000 μg / kg, 1050 μg / kg, 1100 μg / kg, 1150 μg / kg, 1200 μg / kg, or more. In some embodiments, the antibody or antigen-binding portion thereof is administered at a dose of 450 μg / kg. In some embodiments, the antibody or antigen-binding portion thereof is administered at a dose of 900 μg / kg.

[0043] In some embodiments, the antibody or antigen-binding portion thereof is administered more than once. For example, the antibody or antigen-binding portion thereof may be administered 2, 3, 4, 5, 6, 7, or more times in any one or more treatment cycles.

[0044] In some embodiments, the antibody or antigen-binding portion thereof comprises the following CDRs. [Table 2]

[0045] In some embodiments, the antibody or antigen-binding portion thereof comprises (a) the light chain variable region of SEQ ID NO: 7 and / or the heavy chain variable region of SEQ ID NO: 8, (b) the light chain constant region of SEQ ID NO: 11 and / or the heavy chain constant region of SEQ ID NO: 12, or (c) the light chain of SEQ ID NO: 7 + SEQ ID NO: 11 and / or the heavy chain of SEQ ID NO: 8 + SEQ ID NO: 12.

[0046] In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to CD40 is mitazalimab.

[0047] In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to CD40 comprises or consists of an intact antibody, such as an IgG1 antibody.

[0048] In some embodiments, the antibody or antigen-binding portion thereof comprises or consists of an antigen-binding fragment selected from the group consisting of an Fv fragment (such as a single-chain Fv fragment or a disulfide-bonded Fv fragment), and a Fab-like fragment (such as a Fab fragment, a Fab' fragment or an F(ab)2 fragment).

[0049] In some embodiments, the antibody or antigen-binding portion thereof is human or humanized.

[0050] In some embodiments, the antibody or antigen-binding portion thereof and the chemotherapy are administered simultaneously, sequentially, or subsequently to each other.

[0051] In some embodiments, the antibody or antigen-binding portion thereof and / or chemotherapy is administered locally to the tumor site. In some embodiments, the antibody or antigen-binding portion thereof and / or chemotherapy is administered systemically. In some embodiments, the antibody or antigen-binding portion thereof is administered locally and the chemotherapy is administered systemically. In some embodiments, the chemotherapy is administered locally and the antibody or antigen-binding portion thereof is administered systemically. In some embodiments, a portion of the chemotherapy may be administered systemically while a different portion is administered locally. Systemic administration can be, for example, intravenous and / or subcutaneous.

[0052] In some embodiments, the antibody or antigen-binding portion thereof is administered on multiple separate occasions and the chemotherapy is administered continuously for the duration of the method.

[0053] Those skilled in the art will understand that the presence of the antibody or antigen-binding portion thereof and chemotherapy can provide a synergistic benefit in the treatment of cancer, optionally chemotherapy-resistant cancer, in a subject. "Synergistic" includes that the combined therapeutic effect (e.g., determined with reference to the tumor growth rate or size) is greater than the additive therapeutic effect of the two therapies administered separately. Such synergy can be identified by testing the antibody or antigen-binding portion thereof and chemotherapy, alone and in combination, in a cancer-related cell line model.

[0054] Optionally, the combination therapy further includes an additional agent effective in the treatment of cancer in addition to the antibody or antigen-binding portion thereof and chemotherapy.

[0055] In some embodiments, the subject is human.

Brief Description of the Drawings

[0056]

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Mode for Carrying Out the Invention

[0057] All publications, patents, and patent applications cited herein, regardless of whether above or below, are hereby incorporated by reference in their entirety.

[0058] It should be understood that the disclosed combination therapies, uses, methods, pharmaceutical compositions, and different uses of the kits can be adjusted according to specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments of the invention and are not intended to be limiting.

[0059] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context 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 on.

[0060] As used herein, the terms "combination therapy" or "combined treatment" or "combined" refer to any form of simultaneous or concurrent treatment with at least two different treatment groups. For example, the first group of treatments may be an antibody or an antigen-binding portion thereof as described herein, and the second group of treatments may be chemotherapy.

[0061] "An "antibody or antigen-binding portion thereof" may be referred to as an immunotherapy or immunotherapeutic agent. Thus, combination therapy may be considered a combination of immunotherapy and chemotherapy. As used herein, the term "antibody" includes whole antibodies and any antigen-binding portion (i.e., "antigen-binding fragment") or single chain thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed 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 an antigen. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). The constant region of an antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0062] The term "immunotherapeutic agent" is intended to include any antibody or antigen-binding portion thereof that can stimulate the host immune system to produce an immune response against a tumor or cancer in a subject. 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 brought about by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages.

[0063] The heavy chain can be of any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE.

[0064] Light chains include kappa chains and lambda chains.

[0065] Also related are antibodies and antigen-binding fragments thereof that are "isolated" such that they exist in a physical environment different from that in which they may naturally occur, or are modified such that they differ from antibodies that occur naturally in an amino acid sequence.

[0066] The antibody or its antigen-binding portion can be a polyclonal antibody or a monoclonal antibody. The antibody or its antigen-binding portion can be produced by any suitable method. For example, suitable methods 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 can also be used.

[0067] 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 such as CD40. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of binding fragments included 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 complementarity-determining regions (CDRs). Single-chain antibodies such as scFv, as well as heavy-chain antibodies such as VHH and camel antibodies, are also intended to be included within the scope of the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

[0068] The antibodies for use in the method of the present invention can be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. Further, when the antibody includes a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. Such antibodies are typically referred to as chimeric or humanized.

[0069] The human antibodies for use in the method of the present invention are typically human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic non-human animals, such as transgenic mice, having genomes that include human heavy chain transgenes and light chain transgenes fused to immortalized cells. Human antibodies can also be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus. The term "human antibody derivative" refers to any modified form of a human antibody, e.g., a conjugate of the antibody with another agent or antibody.

[0070] Alternatively, the antibody or antigen-binding portion thereof according to the present invention can be a humanized antibody.

[0071] The term "humanized" refers to antibody molecules generally prepared using recombinant techniques that have antigen-binding sites derived from immunoglobulin of non-human species and the remaining immunoglobulin structure based on the structure and / or sequence of human immunoglobulins. The antigen-binding site may comprise a complete non-human antibody variable domain fused to a human constant domain, or only the complementarity-determining regions (CDRs) of such variable domains grafted onto appropriate human framework regions of human variable domains. The framework residues of such humanized molecules may be wild-type (e.g., fully human), or they may be modified to contain one or more amino acid substitutions not found in human antibodies whose sequences serve as the basis for humanization. Humanization reduces or eliminates the potential for the constant region of the molecule to function as an immunogen in a human individual, but the potential for an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) “Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224).

[0072] Another approach focuses not only on providing human-derived constant regions but also on modifying the variable regions to bring them as close as possible to their human form. The variable regions of both the heavy and light chains vary in response to the antigen in question and contain three complementarity-determining regions (CDRs) that determine binding ability and are known to be flanked by four framework regions (FRs) that are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When non-human antibodies are prepared against a particular antigen, the variable regions can be "reshaped" or "humanized" by grafting the CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies is described in 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, C.A. et 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, S.D. et al. (1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185, Tempest, P.R. et al.(1991) "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo," Bio / Technology 9:266-271, Co, M.S. et al. (1991) "Humanized Antibodies For Antiviral Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873, Carter, P. et al. (1992) "Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289, and Co, M.S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen," J. Immunol. 148:1149-1154 have been reported.

[0073] In some embodiments, the humanized antibody preserves 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 (1, 2, 3, 4, 5, 6) modified with respect to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanize an antigen is well known (see, e.g., U.S. Pat. Nos. 5,225,539, 5,530,101, 5,585,089, 5,859,205, 6,407,213, 6,881,557).

[0074] Any antibody referred to herein may be provided in isolated form or, optionally, may be provided linked (directly or indirectly) to another moiety. The other moiety may be a cytotoxic moiety or a therapeutic molecule such as a drug.

[0075] Therapeutic molecules can be directly connected to the antibodies of the present invention, for example, by chemical conjugation. Methods for conjugating molecules to antibodies are known in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) can be used to conjugate various agents including doxorubicin to antibodies or peptides. The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), is particularly useful for conjugating functional moieties to linking moieties.

[0076] Other methods for conjugating a moiety to an antibody can also be used. For example, sodium periodate oxidation followed by reductive alkylation with a suitable reagent can be used, and glutaraldehyde cross-linking is also possible. However, it is recognized that regardless of how the method for producing the conjugate of the present invention is selected, a determination must be made that the antibody maintains its targeting ability and the functional moiety maintains its associated function.

[0077] The cytotoxic moiety can be cytotoxic directly and / or indirectly. "Cytotoxic directly" means that the moiety is a moiety that is cytotoxic per se. "Cytotoxic indirectly" means that the moiety is a moiety that is not cytotoxic per se but can induce cytotoxicity, for example, by its action on or action by a further molecule on a further molecule. The cytotoxic moiety may be cytotoxic only within the cell and is preferably not cytotoxic outside the cell.

[0078] The antibody or its antigen-binding portion may be directly linked to a cytotoxic moiety that is a cytotoxic chemotherapeutic agent. Optionally, the cytotoxic moiety is directly a cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art. In the context of the present invention, the antibody or its antigen-binding portion with a cytotoxic chemotherapeutic agent is for use in combination with different chemotherapies. In some embodiments, the antibody or its antigen-binding portion is not conjugated to a cytotoxic chemotherapeutic agent.

[0079] Whether conjugated to an antibody or its antigen-binding portion or for separate use as chemotherapy, cytotoxic chemotherapeutic agents such as anticancer agents include alkylating agents containing nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; ethyleneimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozocin (streptozotocin); and triazenes such as dacarbazine (DTIC, dimethyltriazenoimidazole-carboxamide); antimetabolites including folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracil (5-fluorouracil, 5-FU), floxuridine (fluorodeoxyuridine, FUdR), and cytarabine (cytosine arabinoside); and 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 (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenom. Other agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenediones such as mitoxantrone and anthracycline; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); and adrenal cortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogs / derivatives; and hormone agonists / antagonists such as flutamide and tamoxifen.

[0080] The cytotoxic moiety can be a cytotoxic peptide or a polypeptide moiety leading to cell death. Cytotoxic peptides and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, etc. Methods for conjugating them to targeting moieties such as antibodies are also known in the art. Other ribosome-inactivating proteins are described as cytotoxic agents in WO96 / 06641. Pseudomonas exotoxin can also be used as a cytotoxic polypeptide. Certain cytokines such as TNFα and IL-2 can also be useful as cytotoxic agents.

[0081] Certain radioactive atoms can also be cytotoxic when delivered in sufficient doses. Thus, the cytotoxic moiety can include radioactive atoms that deliver a sufficient amount of radioactivity to the target site to be cytotoxic during use. 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 sufficient energy to destroy adjacent cells, organs or nucleic acids. Preferably, the isotope and density of the radioactive atoms in the agents 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, preferably the cells of the target site and their organelles, particularly the nucleus.

[0082] The radioactive atoms can be connected to antibodies, antigen-binding fragments, variants, fusions or derivatives thereof by known methods. For example, EDTA or another chelating agent may be connected to the binding moiety and used to connect 111In or 90Y. Tyrosine residues may be directly labeled with 125I or 131I.

[0083] The cytotoxic moiety can be a suitable indirectly cytotoxic polypeptide. An indirectly cytotoxic polypeptide can be a polypeptide that has enzymatic activity and can convert a non-toxic and / or relatively non-toxic prodrug into a cytotoxic drug. In the case of antibodies, this type of system is often referred to as ADEPT (antibody-directed enzyme prodrug therapy). The system requires the antibody to place the enzyme moiety at the desired site in the patient's body, and after allowing time for the enzyme to localize at the site, administer the prodrug that is the substrate of the enzyme, and the end product of the catalysis is a cytotoxic compound. The aim of this approach 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 be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.

[0084] The enzyme and prodrug of the system using the targeted enzyme described herein can be any of those previously proposed. The cytotoxic substance can be any existing anti-cancer drug such as an alkylating agent; an agent that intercalates into DNA; an agent that inhibits any important enzyme such as dihydrofolate reductase, thymidine synthase, ribonucleotide reductase, nucleoside kinase or topoisomerase; or an agent that causes cell death by interacting with any other cellular component. Etoposide is an example of a topoisomerase inhibitor.

[0085] The reported prodrug systems include those listed in Table 2.

Table 3

[0086] Suitable enzymes for forming part of the enzyme moiety include exopeptidases such as carboxypeptidases G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs), and aminopeptidase (for 2-α-aminoacyl MTC prodrugs); endopeptidases such as thrombolysin (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; lactamases such as β-lactamase; glycosidases such as β-glucuronidase (for β-glucuronide anthracyclines), α-galactosidase (for amygdalin) and β-galactosidase (for β-galactose anthracyclines); 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 cyclodextrin.

[0087] Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, when measured in a suitable in vitro cytotoxicity test, it has less than 10%, preferably less than 1% of the toxicity.

[0088] The moiety capable of converting the prodrug to the cytotoxic drug is likely to be active when isolated from the remainder of the agent of the present invention, but (a) when combined with the remainder of the agent of the present invention, and (b) only when the agent of the present invention is attached to, adjacent to, or internalized within the target cell, it is necessary to be active.

[0089] When the moieties are polypeptides, the two moieties can be linked together by any of the conventional methods of cross-linking polypeptides. For example, an antibody or an antigen-binding portion thereof may have thiol groups concentrated, and a further moiety can react with those thiol groups with a bifunctional agent, such as N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). For example, amide and thioether bonds achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester are generally more stable in vivo than disulfide bonds.

[0090] The cytotoxic moiety can be a radiosensitizer. Examples of radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanidazole, pimonidazole and misonidazole. Also, they can be radiosensitized by delivery of genes to cells, for example, delivery of the p53 gene or cyclin D. A further moiety can be a moiety that becomes cytotoxic upon irradiation or releases a cytotoxic moiety. For example, the boron-10 isotope releases cytotoxic alpha particles when appropriately irradiated. Similarly, the cytotoxic moiety can be useful in photodynamic therapy such as Photofrin.

[0091] "Therapeutically effective amount", "effective amount", or "therapeutically effective" means that a given substance is administered in an amount sufficient to cure, alleviate, or partially suppress one or more of the conditions or symptoms thereof in a subject suffering from such a condition. Such therapeutic treatment can result in a decrease in the severity of the disease symptoms, or an increase in the frequency or duration of the asymptomatic period. The effective amount of a given agent for a given purpose depends on the severity of the disease or injury, as well as the weight and general condition of the subject. This can be a predetermined amount of the active antibody calculated to produce the desired therapeutic effect in relation to the necessary additives and diluents, i.e., carriers or administration vehicles. Further, it is intended to mean an amount sufficient to reduce or prevent clinically significant deficiencies in the activity, function, and response of the host. Alternatively, a therapeutically effective amount is an amount sufficient to produce a clinically significant improvement in the host. As will be understood by those skilled in the art, the amount of the compound can vary depending on its specific activity. A suitable dosage can contain a predetermined amount of the active composition calculated to produce the desired therapeutic effect in relation to the necessary diluents. A therapeutically effective amount can be determined by a normally skilled medical or veterinary practitioner based on patient characteristics such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

[0092] "Cancer" includes solid tumors and hematological cancers (e.g., leukemia). Cancer can be malignant and / or metastatic. Solid tumors are classically defined by the tissue from which they originate, e.g., pancreas, breast, colon, etc. However, since immunotherapy acts on the immune system rather than the tumor itself, the immune status of the tumor can be more predictive of the response than the origin of the tumor.

[0093] Cancer can be immunogenic. Such cancers are characterized by infiltration of immune cells such as T cells and cells of bone marrow origin. Infiltration of CD8 T cells, i.e., a more immunogenic cancer profile, has been demonstrated to correlate with a favorable prognosis after therapy in, for example, colon cancer (Galon et al., 2014, J. Pathol. 232(2):199 - 209).

[0094] Cancer can be non-immunogenic or poorly immunogenic. Poorly immunogenic tumors are often characterized by low or absent MHC class I expression and low numbers of infiltrating immune cells such as T cells and cells of myeloid origin (Lechner et al., 2013, J Immunotherapy 36(9):477-89). The tumor can be a pancreatic tumor, adenoma, adenocarcinoma, blastoma, carcinoma, desmoid tumor, fibromatosis, small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms tumor, lung tumor, colon tumor, lymphoid tumor, breast tumor, or melanoma.

[0095] In some embodiments, the cancer is a CD40-positive cancer. "CD40-positive cancer" includes any cancer that expresses CD40, albeit at different levels.

[0096] Types of pancreatic tumors include exocrine tumors such as exocrine adenocarcinoma or pancreatic ductal adenocarcinoma (PDAC), or endocrine tumors. Types of blastomas include hepatoblastoma, glioblastoma, neuroblastoma, or retinoblastoma. Types of carcinomas include colorectal cancer or hepatocellular carcinoma, pancreatic cancer, prostate cancer, gastric cancer, esophageal cancer, cervical cancer, and head and neck cancer, and adenocarcinoma. Types of sarcomas include Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, or any other soft tissue sarcoma. Types of melanomas include lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, neurotropic melanoma, amelanotic melanoma, soft tissue melanoma, melanoma with small nevus-like cells, melanoma with features of Spitz nevus, and uveal melanoma. Types of lymphomas 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 lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosis type of Hodgkin lymphoma, and mixed cellularity 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.

[0097] Each of the cancers described above is well known, and the symptoms and cancer diagnostic markers are well described as they are therapeutic agents used to treat those cancers. Thus, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat cancers of the type described above would be known to those of ordinary skill in the medical field.

[0098] The clinical definitions of diagnosis, prognosis, and progression of many cancers rely on specific classifications known as staging. Those staging systems function to collate several different cancer diagnostic markers and cancer symptoms to provide an overview of cancer diagnosis, and / or prognosis, and / or progression. How to evaluate cancer diagnosis, and / or prognosis, and / or progression using a staging system, and which cancer diagnostic markers and cancer symptoms should be used to do so would be known to those of ordinary skill in the oncology field.

[0099] "Cancer staging" includes the Rai staging, which includes stage 0, stage I, stage II, stage III, and stage IV, and / or the Binet staging, which includes stage A, stage B, and stage C, and / or the Ann Arbor staging, which includes stage I, stage II, stage III, and stage IV.

[0100] Cancer is known to be able to cause abnormalities in cell morphology. These abnormalities often occur reproducibly in specific cancers, meaning that examination of these changes in morphology (or histological examination, as it is also known) can be used for cancer diagnosis or prognosis. Techniques for visualizing samples to examine cell morphology and for preparing samples for visualization, such as light microscopy or confocal microscopy, are well known in the art.

[0101] "Chemotherapy-resistant cancer" includes cancer that does not respond to chemotherapy. For example, cancer can continue to grow and / or metastasize despite treatment with chemotherapy. The selection of a particular chemotherapy can be made by a physician after the cancer diagnosis of a subject or patient. Cancer can initially respond to chemotherapy and then develop resistance to chemotherapy. Thus, a subject may be undergoing treatment with chemotherapy and then develop resistance to chemotherapy, and then the subject may be treated with an antibody or an antigen-binding portion thereof as described herein, thereby providing a combination therapy for treating chemotherapy-resistant cancer.

[0102] In some embodiments, the cancer is a cancer that is resistant to treatment with a therapeutic anti-cancer antibody. Such resistant cancers can be recurrent cancers and / or refractory cancers. A recurrent cancer is a cancer that has been previously treated and as a result of that treatment, the subject has recovered completely or partially (i.e., the subject is said to be in remission), but after the treatment is discontinued, the cancer has recurred or worsened. In other words, a recurrent cancer is a cancer that has become resistant to treatment after a period during which it was effective and the subject recovered completely or partially. A refractory cancer is a cancer that is being treated but does not respond to that treatment and / or is being treated but progresses during treatment. In other words, a refractory cancer is a cancer that is resistant to treatment. It will be understood that cancer can be a refractory cancer due to intrinsic resistance. "Intrinsic resistance" includes the meaning that the cancer, and / or the subject, and / or the target cells are resistant to a particular treatment from the time it is first administered or even before it is administered. Recurrent cancers and / or refractory cancers will be readily diagnosed by those of ordinary skill in the medical art.

[0103] The term "subject" (used interchangeably with "patient" herein) includes any animal, including a human, in need of treatment with an antibody that specifically binds to CD40 or an antigen-binding portion thereof, and / or chemotherapy. The subject or patient can be a mammal or a non-mammal. Preferably, the subject is a mammal such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog, or a cat. Most preferably, the mammalian patient is a human.

[0104] Preferably, the subject is a subject diagnosed with cancer, optionally a chemotherapy-resistant cancer, and / or a subject identified as likely to have, optionally, a chemotherapy-resistant cancer, and / or a subject presenting, optionally, symptoms of a chemotherapy-resistant cancer. "Presenting" includes the subject being able to exhibit cancer symptoms and / or cancer diagnostic markers, and / or being able to measure, and / or evaluate, and / or quantify cancer symptoms and / or cancer diagnostic markers. Those of ordinary skill in the medical art will readily appreciate what cancer symptoms and cancer diagnostic markers are, and how to measure, and / or evaluate, and / or quantify whether there is a decrease or increase in the severity of cancer symptoms or a decrease or increase in cancer diagnostic markers, and how to form a prognostic diagnosis regarding cancer using cancer symptoms and / or cancer diagnostic markers.

[0105] The term "regimen" as used herein is synonymous with regime or regiment. "Dosing regimen" includes the meaning that the antibody or an antigen-binding portion thereof and chemotherapy are administered in steps where the plurality of steps form a regimen.

[0106] In some embodiments, the dosing regimens described herein can be repeated as many times as necessary in a particular subject. For example, this dosing regimen can be used each time an antibody or antigen-binding portion thereof that specifically binds to CD40 is administered to the subject. In some embodiments, the exact form of the dosing regimen (with respect to time and dose) can vary during repeated administrations to the subject. The advantage of repeatedly using the dosing regimens described herein is that it enhances the anti-cancer effect.

[0107] However, as will be understood by those skilled in the art, repeated dosing can also utilize higher or lower total doses, as guided by the patient's tolerance. Dosing regimens based on similar flat dosing or receptor occupancy-guided can be used.

[0108] It will be understood that the dosage and dosing regimen of each of the therapeutic antibodies discussed and contemplated herein depends on the approved dosage / regimen of these therapeutic antibodies and also varies according to the indication (e.g., cancer type / stage) and / or the subject (e.g., BMI or age).

[0109] The term "chemotherapy" includes the meaning of chemotherapy regimens (which may include treatment duration, treatment cycles, and overall treatment time), chemotherapeutic agents (and combinations thereof), chemotherapeutic drugs (and combinations thereof), chemotherapeutic agents (and combinations thereof), and chemotherapeutic drugs (and combinations thereof).

[0110] As used herein, a "treatment period" with a particular preparation or treatment means the period during which the particular preparation or treatment is administered to a patient. For example, if chemotherapy (e.g., a chemotherapeutic drug) is administered continuously for 8 days, followed by 2 days without chemotherapy, the treatment period by chemotherapy is 8 days.

[0111] As used herein, the term "treatment cycle" means the passage of one or more treatments or treatment periods that are repeated on a regular schedule and may include rest periods. For example, 8 days of treatment followed by 2 days of rest is one treatment cycle. The treatment cycles may be repeated either identically or in a modified form, e.g., at different dosages and / or schedules, or with different additional treatments. The "treatment interval" is the interval between the start and completion of a treatment cycle.

[0112] "Total treatment time" means the period that includes the entire treatment cycle. As described above, the treatment cycle may include periods without treatment (intervals during which treatment is not administered to the patient, i.e., no chemotherapy and no antibody, and optionally no other drugs). Thus, as used herein, the total treatment time may also include such intervals without treatment within the treatment cycle. For example, if a patient undergoes 8 treatment cycles of 10 days each, the total treatment time is 80 days. The total treatment time may include at least 1, or 2 or more cycles, or up to 12 cycles. In one embodiment, the total treatment time includes 3, 4, 5, 6, 7, 8, 9, 10, or 11 cycles.

[0113] Within a given treatment cycle, the antibody or antigen-binding portion thereof and the chemotherapy may be administered simultaneously, sequentially, or separately. As used herein, "simultaneously" means that the agents should be taken together on at least one treatment day and may or may not be formulated as a single composition. "Simultaneously" also encompasses partial overlap of the treatment days on which the agents are administered. For example, the chemotherapy may be administered for one or more consecutive days, and then both the chemotherapy and the antibody or antigen-binding portion thereof may be administered on subsequent consecutive days. "Sequentially" means that the drugs are administered on consecutive treatment days but not on the same treatment day. For example, the chemotherapy may be administered for one or more consecutive days, and the antibody or antigen-binding portion thereof may be administered for one or more consecutive days immediately thereafter. As used herein, "separate" administration means that the antibody or antigen-binding portion thereof and the chemotherapy are administered as part of the same overall dosing regimen but are not administered on the same day. For example, the chemotherapy may be administered for one or more consecutive days, and then there may be one or more days during which neither the chemotherapy nor the antibody or antigen-binding portion thereof is administered, and then the antibody or antigen-binding portion thereof may be administered on subsequent days for one or more days. Typically, the antibody or antigen-binding portion thereof is administered simultaneously with the chemotherapy (or a portion thereof), and more typically, the treatment period of the antibody or antigen-binding portion thereof and the chemotherapy partially overlap.

[0114] In some embodiments, chemotherapy is in the form of FOLFIRINOX. FOLFIRINOX is a chemotherapy regimen that includes oxaliplatin, folinate (also known as folic acid; e.g., leucovorin), irinotecan, and 5-fluorouracil, each of which may be administered by infusion, e.g., IV infusion. The FOLFIRINOX regimen is known in the art and may be modified for any one or more of its components, thereby resulting in a modified FOLFIRINOX (mFOLFIRINOX) regimen (also referred to herein as a FOLFIRINOX variant). This modification may be made before initiating the FOLFIRINOX regimen, during the FOLFIRINOX regimen (e.g., to reduce the side effects of a particular component of the FOLFIRINOX regimen), and / or after a treatment cycle of the FOLFIRINOX regimen (e.g., to reduce the side effects of a particular component of the FOLFIRINOX regimen if the treatment cycle is repeated).

[0115] The term "FOLFIRINOX" as used herein can be replaced with the term "mFOLFIRINOX".

[0116] Exemplary and preferred mFOLFIRINOX corresponds to the following (as shown in Example 2). [Table 4]

[0117] Modified forms of FOLFIRINOX are known in the art. For example, the following (the complete details of the authors can be found in the references section of this specification, see References 41-51; derived from Tong et al., 2018). [Table 5]

[0118] Thus, in some embodiments, FOLFIRINOX (or mFOLFIRINOX) is selected from Regimen 1, Regimen 2, Regimen 3, Regimen 4, Regimen 5, Regimen 6, Regimen 7, Regimen 8, Regimen 9, Regimen 10, and / or Regimen 11 as described in the table above. In some embodiments, the treatment cycle may start with a specific regimen but may be switched to an alternative regimen.

[0119] Further variations of FOLFIRINOX include the following (LV = leucovorin, exemplary folic acid); OX = oxaliplatin; Irino = irinotecan; Freq = frequency of cycle; q2W = every 2 weeks (i.e., FOLFIRINOX is repeated every 2 weeks); mCyc = number of cycles of FOLFIRINOX; Met = cohort of patients with metastatic pancreatic cancer; LA = cohort of patients with locally advanced pancreatic cancer): [Table 6]

[0120] In some embodiments, FOLFIRINOX comprises oxaliplatin at a dose of 85 mg per square meter given as a 2-hour intravenous infusion, followed by leucovorin at a dose of 400 mg per square meter given as a 2-hour intravenous infusion, and, 30 minutes later, in addition, irinotecan at a dose of 180 mg per square meter given as a 90-minute intravenous infusion via a Y-connector. Immediately following this treatment, fluorouracil was administered by intravenous bolus at a dose of 400 mg per square meter, followed by a continuous intravenous infusion of 2400 mg per square meter over 46 hours every 2 weeks.

[0121] In some embodiments, the patient may receive palonosetron, aprepitant, and dexamethasone for nausea prevention. Pegfilgrastim was scheduled on day 3 or 4 of each cycle in the absence of severe leukocytosis, for 12 cycles in Met and 8 cycles in LAPC, and / or.

[0122] In some embodiments, FOLFIRINOX or a variant thereof includes an oxaliplatin (such as Eloxatin® or a generic drug) infusion, and optionally, the oxaliplatin is at least 85 mg / m 2 and is administered intravenously and / or over 2 hours at a dose of. In some embodiments, the oxaliplatin is at least 40 mg / m 2 , for example, at least 45 mg / m 2 , at least 50 mg / m 2 , at least 55 mg / m 2 , at least 60 mg / m 2 , at least 65 mg / m 2 , at least 70 mg / m 2 , at least 75 mg / m 2 , and / or at least 80 mg / m 2 and is administered at a dose of. In some embodiments, the oxaliplatin is administered at a dose of 40 mg / m 2 to 85 mg / m 2 . The dose of oxaliplatin may vary between treatment cycles and / or may be adapted during a treatment cycle.

[0123] In some embodiments, FOLFIRINOX or a variant thereof includes a folinate (such as leucovorin, calcium folate, calcium levofolinate, disodium folate, and disodium levofolinate) infusion, and optionally, the folinate (e.g., leucovorin) is at least 400 mg / m 2 and is administered intravenously and / or over 2 hours at a dose of. The dose of folinate may vary between treatment cycles and / or may be adapted during a treatment cycle.

[0124] In some embodiments, FOLFIRINOX or a variant thereof includes an irinotecan (such as Campto®) infusion, and optionally, the irinotecan is at least 150 mg / m 2It is administered intravenously at the dose of 2 and / or within 30 minutes after the end of leucovorin infusion. In some embodiments, irinotecan is at least 80 mg / m 2 , for example, 90 mg / m 2 , 100 mg / m 2 , 110 mg / m 2 , 120 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 140 mg / m 2 , 150 mg / m 2 , 160 mg / m 2 , or 180 mg / m 2 . In some embodiments, irinotecan is administered at a dose of 80 mg / m 2 ~180 mg / m 2 . The dose of irinotecan may vary between treatment cycles and / or may be adapted during a treatment cycle.

[0125] In some embodiments, FOLFIRINOX or a variant thereof includes a 5-fluorouracil (''5-FU'') infusion, and optionally, 5-fluorouracil is at least 2400 mg / m 2 administered intravenously at a dose of and / or over a period of 46 - 48 hours (e.g., 2.4 g / m 2 / day). In some embodiments, 5-FU is at least 300 mg / m 2 , for example, 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , 1100 mg / m 2 , 1200 mg / m 2 , 1300 mg / m 2 , 1360 mg / m 2 , 1400 mg / m 2 , 1500 mg / m 2 , 1600 mg / m 2, 1700 mg / m 2 , 1800 mg / m 2 , 1900 mg / m 2 , or 1920 mg / m 2 is administered at a bolus dose. In some embodiments, 5-FU is administered at a dose of 1360 mg / m 2 to 2400 mg / m 2 . The dose of 5-FU may vary between treatment cycles and / or may be adapted during a treatment cycle.

[0126] Side effects associated with chemotherapy (including each component related to FOLFIRINOX) are well known. It is a routine matter for a physician to identify the onset of side effects associated with chemotherapy (or its components) and to adjust the concentration of chemotherapy (or its components) to a lower dose that reduces the risk of side effects while maintaining an appropriate level of efficacy. Furthermore, additional agents that compensate for the side effects of a particular chemotherapy (or its components) are known and may be included as premedication and / or postmedication.

[0127] "Premedication" includes the meaning that a pharmaceutical form is administered before a treatment or procedure. For example, the premedication may be before treatment with an antibody or its antigen-binding portion, before chemotherapy or a part thereof, or before both. The selected premedication may be to counteract or alleviate the symptoms of side effects that occur in the subject before the antibody or its antigen-binding portion, or the known side effects of chemotherapy.

[0128] "Postmedication" includes the meaning that a pharmaceutical form is administered after a treatment or procedure. For example, the postmedication may be after treatment with an antibody or its antigen-binding portion, after chemotherapy or a part thereof, or after both. The selected postmedication may be to counteract or alleviate the symptoms of side effects observed in the subject after the antibody or its antigen-binding portion, or the known side effects of chemotherapy.

[0129] Additional pharmaceuticals can be before one agent (or its dosing regimen) of combination therapy and after a different agent, in which case the pharmaceuticals can be considered premedication and postmedication. The presence of the pharmaceuticals can be in a subject such that they can be considered to be present during the combination therapy (or its dosing regimen). Thus, premedication and / or postmedication can be considered to be administered during a particular step of the combination therapy (or its dosing regimen).

[0130] Treatment with chemotherapy is often associated with cytopenia. Depending on the cell lineage, affected patients can suffer from different symptoms. Neutropenia is associated with an increased risk of infections that can be severe and even life-threatening. Thrombocytopenia can lead to an increased risk of bleeding. Anemia can cause fatigue, dyspnea, and tachycardia. Subjects can be monitored with regular blood tests to evaluate these laboratory parameters and the dose of chemotherapy can be adjusted accordingly. Patients treated with mFOLFIRINOX can receive primary prophylaxis with G-CSF as postmedication. When using the gemcitabine + nab-paclitaxel chemotherapy regimen, the use of G-CSF is permitted, but its use is at the discretion of the investigator-in-charge of the trial. High-grade neutropenia and febrile neutropenia should be managed according to the facility's criteria, for example with regard to the application of antibiotics. Transfusion of blood components and administration of erythropoietin are alternative postmedications that can be approved at the discretion of the investigator-in-charge of the trial. Red blood cell transfusion can be considered for hemoglobin < 9.5 g / dL or for prominent symptoms of anemia.

[0131] Subjects treated with gemcitabine + nab-paclitaxel are at high risk of non-neutropenic sepsis. In the Phase 3 trial at the first occurrence of fever ≥ 38.5 °C (irrespective of neutrophil count), the practice of ciprofloxacin (500 mg orally, twice daily) or amoxicillin / clavulanic acid (Augmentin®, 500 mg orally, two to three times daily) in patients with an allergy to fluoroquinolones can be initiated as postmedication.

[0132] Generally, chemotherapy-induced diarrhea results from the death of rapidly dividing intestinal cells, combined with inflammation and loss of absorptive function, and changes in gastrointestinal osmotic gradients resulting in loss of fluid and electrolyte secretion. Treatment is generally supportive and may require hospitalization with administration of post-dosing parenteral fluids in severe or persistent cases. When coinciding with neutropenia, the patient is at high risk of infectious complications and consideration can be given to the application of broad-spectrum antibiotics following the facility's standard practice. In particular, the irinotecan component of mFOLFIRINOX causes both acute (within 24 hours) and delayed (2 to 14 days after dosing) diarrhea.

[0133] Cytotoxic chemotherapy targets rapidly dividing cells such as the epithelial lining of the gastrointestinal tract, including the oral mucosa, resulting in tissue damage and inflammation, mucositis. This can be severe enough to limit intake and compromise nutrition, so symptomatic support care and nutritional monitoring can be continued as post-dosing. Of the components of mFOLFIRINOX, 5-fluorouracil (5-FU) is the strongest contributing factor to mucositis.

[0134] Both taxanes and platinum-containing chemotherapy are associated with nerve damage resulting from changes in microtubule transport that primarily interfere with axonal function. Typically, this affects peripheral neurons that are more distal than proximal and more sensory than motor. Sensory neuropathy can begin with paresthesia but progress to impair activities of daily living. Toxicity accumulates over time and may persist or worsen after dose modification. Predisposing factors include a history of peripheral neuropathy, diabetes, advanced age, or previous exposure to neurotoxic drugs. The main cause of neuropathy in the gemcitabine / nab-paclitaxel regimen is the paclitaxel component, while oxaliplatin is involved in most of the neurotoxicity of the mFOLFIRINOX regimen. In addition to this systemic peripheral neuropathy, a specific acute neuropathy, laryngopharyngeal sensory disturbances, rarely occur in patients within hours of treatment with oxaliplatin. This may present as an unpleasant sensation in the pharyngolaryngeal region, and patients may experience dyspnea and anxiety. Patients with a history of oxaliplatin-related laryngeal convulsions are recommended to receive a sedative before oxaliplatin infusion and the infusion is administered over 6 hours.

[0135] mFOLFIRINOX is considered a moderately emetogenic regimen and prophylaxis with antiemetics is recommended according to the discretion of the treating physician and the facility's criteria. Gemcitabine + nab-paclitaxel is considered to have a low potential for emesis. It should be noted that the use of corticosteroids should be restricted when administered as a prophylactic agent. Nausea as a symptom of infusion-related reactions has been observed in mitazalimab therapy, and antiemetics may be part of premedication and / or postmedication associated with mitazalimab administration.

[0136] If chemotherapy leaks into the tissue surrounding the intended blood vessel, severe local tissue necrosis can occur, which has been reported for irinotecan and oxaliplatin. If this occurs, it is necessary to immediately stop the infusion. With the needle or catheter left in place, as much of the drug as possible can be aspirated as a post-dose. Ice can be applied to the infiltration area as an alternative or additional post-dose, for example, for 15 - 20 minutes every 4 - 6 hours over a 72-hour period. Alternatively, or in addition, local corticosteroid therapy can be applied.

[0137] In some embodiments, the subject receives at least one treatment cycle of FOLFIRINOX. The FOLFIRINOX treatment cycle may be as defined in the examples. In some embodiments, the subject receives a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 FOLFIRINOX treatment cycles. In some embodiments, the subject does not receive more than 12 FOLFIRINOX treatment cycles. Since the number of FOLFIRINOX cycles can vary from individual to individual, the need for additional repetitions can be evaluated on a case-by-case basis. Thus, a physician competent to administer FOLFIRINOX cycles can determine based on responsiveness (e.g., evaluated by reduction / prevention of cancer growth and / or metastasis) and / or the risk of side effects (e.g., if a particular side effect becomes a problem for the recipient of FOLFIRINOX or its components, a decision can then be made to discontinue further treatment cycles).

[0138] In some embodiments, the FOLFIRINOX treatment cycle may be as follows: · Oxaliplatin IV over 2 hours, immediately followed by; · Leucovorin or a similar approved folinate over 2 hours; · Irinotecan over 90 minutes (starting 30 minutes after the start of the leucovorin infusion), followed by; · A 5-FU infusion over 46 - 48 hours, for example, over 46 hours, over 47 hours, or over 48 hours.

[0139] Local administration to the tumor site is preferred and includes administration around, near, within the tumor, within the lesion, around the lesion, intracranially, and intravesically, by any suitable means such as injection. Local administration may also include intracavitary infusion and inhalation, depending on the location of the tumor.

[0140] Systemic administration of any of the agents described herein (e.g., an antibody or antigen-binding portion thereof and / or chemotherapy) means administration to the circulatory system of a subject, including the vascular and / or lymphatic systems. Such administration can be by any suitable route, but is typically parenteral.

[0141] As used herein, the phrase "parenteral administration" means an administration mode other than enteral administration and local administration, typically achieved by injection, infusion, or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, intracerebral, intrathecal, intraosseous, or other parenteral administration routes.

[0142] In some embodiments, the antibody or antigen-binding portion thereof is formulated and / or adapted for delivery by a route selected from the group including intravenous, intramuscular, and subcutaneous. Preferably, the antibody or antigen-binding portion thereof is formulated and / or adapted for intravenous (i.e., "i.v." or "IV") delivery.

[0143] In some embodiments, the antibody or antigen-binding portion thereof is delivered to a subject by a route selected from the group including intravenous, intramuscular, and subcutaneous. Preferably, the antibody or antigen-binding portion thereof is delivered intravenously.

[0144] Thus, in preferred embodiments, the first and / or second and / or further doses of the antibody or antigen-binding portion thereof are formulated for intravenous delivery to a subject and / or are delivered by intravenous delivery to the subject.

[0145] Methods and formulations for intravenous administration of an antibody or antigen-binding portion thereof are well known in the art. In the present invention, any type of intravenous administration such as injection or infusion can be used.

[0146] In some embodiments, the chemotherapy is formulated and / or adapted for delivery by a route selected from the group including intravenous, intramuscular, and subcutaneous.

[0147] In embodiments of the present invention, the chemotherapy is delivered to a subject by a route selected from the group including intravenous, intramuscular, and subcutaneous.

[0148] Thus, in a preferred embodiment, the first and / or second and / or further doses of chemotherapy are formulated for intravenous delivery to a subject and / or are delivered by intravenous delivery to the subject.

[0149] Depending on the route of administration, the antibody or antigen-binding portion thereof and / or the chemotherapy may be coated with a material for protecting the agent(s) from the action of acids and other natural conditions that may inactivate or denature the antibody or antigen-binding portion thereof and / or the chemotherapy. 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 physiological saline. Examples of other carriers include ethanol, polyols (such as 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 such as lecithin to maintain the particle size required in the case of a dispersion and by using a surfactant. In many cases, it is preferred to include in the composition an isotonicity agent, such as a sugar, a polyhydric alcohol such as mannitol, sorbitol, or sodium chloride.

[0150] Methods and formulations for various routes of administration are well known in the art.

[0151] The combination therapies and methods of the present invention utilize an antibody that immunospecifically binds to CD40, i.e., an "anti-CD40 antibody". In one embodiment, the antibody is retained at the tumor site after administration to a subject. The antibody preferably specifically binds to CD40, i.e., it binds to CD40 but not to other molecules or binds to them with a lower affinity (e.g., one-tenth the affinity). Unless otherwise defined, 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, such as primates or mouse CD40. The antibody preferably binds to human CD40 when localized on the surface of a cell.

[0152] In particular, the anti-CD40 antibody used in the combination therapies of the present invention competes for binding to human CD40 with a "reference antibody" that includes the light chain variable region of SEQ ID NO: 7 and the heavy chain variable region of SEQ ID NO: 8 (optionally, together with the light chain and heavy chain constant regions of SEQ ID NO: 11 and SEQ ID NO: 12, respectively). Such competitive binding inhibition can be tested using assays and methods well known in the art, for example, using a BIAcore chip having immobilized human CD40, with the antibody polypeptide being tested, and also by incubating in the presence or absence of the reference antibody. Alternatively, a pairwise mapping approach can be used where the reference antibody is immobilized on the surface of a BIAcore chip, human CD40 is bound to the immobilized antibody, and then a secondary antibody is tested for its ability to bind simultaneously to human CD40 (see, for example, 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05 / 2012, the disclosure of which is incorporated herein by reference).

[0153] Exemplary anti-CD40 antibodies are disclosed in WO2013 / 034904 and WO2016 / 023960 to Alligator Bioscience AB (the disclosures of which are incorporated herein by reference).

[0154] The antibody preferably has the ability to bind to CD40 in its native state, particularly CD40 localized on the surface of the cell. Preferably, the antibody will specifically bind to CD40. That is, the antibody used in the method of the present invention will preferably bind to CD40 with a binding affinity higher than its binding affinity for binding to another molecule.

[0155] "Localized on the surface of the cell" means that CD40 is associated with the cell such that one or more regions of CD40 are present on the outer surface of the cell surface. For example, CD40 can be inserted into the cell plasma membrane along with one or more regions presented on the extracellular surface (i.e., oriented as a transmembrane protein). This can occur during the process of CD40 expression by the cell. Thus, in one embodiment, "localized on the surface of the cell" can mean "expressed on the surface of the cell". Alternatively, CD40 can be present outside the cell by covalent and / or ionic interactions and localized to a specific region or regions of the cell surface.

[0156] The anti-CD40 antibodies described herein 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. "Enhance" means that the number of cells induced to undergo lysis or apoptosis in the presence of the antibody of the present invention is increased compared to the number of cells induced to undergo lysis or apoptosis 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, a chromium-51 release assay, a europium release assay, or a sulfur-35 release assay may be used. In such an assay, a target cell line labeled prior to expressing the antigen (in this case, CD40) is incubated with the antibody being tested. After washing, effector cells (typically expressing the Fc receptor CD16) are incubated with the antibody-labeled target cells. Subsequently, target cell lysis is measured by a scintillation counter or by release of intracellular label by spectrophotometry.

[0157] Preferably, the antibody, its antigen-binding portion, comprises the antibody Fc region. One of ordinary skill in the art will understand that the Fc portion can be derived from an IgG antibody, or an antibody of a different class (such as IgM, IgA, IgD, or IgE). For example, the Fc region can be derived from an IgG1, IgG2, IgG3, or IgG4 antibody. However, advantageously, the Fc region is derived from an IgG1 antibody.

[0158] The Fc region may be a naturally occurring one (e.g., a part of an endogenously produced antibody) or an artificial one (e.g., containing one or more point mutations relative to a naturally occurring Fc region). An Fc region having point mutations that improve their ability to bind to FcRs can be advantageous, for example, by modifying the serum half-life or improving the binding to Fcγ receptors (FcγRs) involved in ADCC and CDC. In particular, mutations that enhance the binding to FcγRIIB, such as S267E (Strohl et al., 2009, Curr Opin Biotechnol, 20:685-691), may be advantageous for the present invention considering the linkage between the binding of FcγRIIB and the functional activity of the CD40 antibody (Li et al., 2011, Science, 333:1030-1034).

[0159] As an alternative to radiolabeling required in such assays, methods can be used in which lysis is detected by measuring the release of an enzyme that naturally exists in the target cells. This can be achieved by detecting the product of an enzyme-catalyzed reaction (e.g., bioluminescence detection). In such assays, prior labeling of the cells is not required. A typical cellular enzyme detected in such assays is GAPDH.

[0160] The anti-CD40 antibodies described herein can regulate the activity of cells expressing CD40, and such regulation is an increase or decrease in the activity of the cells. The cells are typically dendritic cells or B cells.

[0161] Professional APCs such as dendritic cells are activated when signaling via CD40 occurs, which induces several biological events including activation, proliferation of immune cells, and production of cytokines and chemokines. Methods for determining activation of dendritic cells related to CD40 are known in the art (e.g., as 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.

[0162] Stimulation of human B cells with recombinant CD40L or anti - CD40 antibodies induces up - regulation 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., as discussed in Schonbeck et al., 2001, supra) and are further described below.

[0163] Methods and assays for determining the ability of antibodies to modulate the activity of dendritic cells and B cells are well - known in the art. For example, activation of dendritic cells can be evaluated by measuring the levels of cell surface markers such as CD86 and CD80 and / or by measuring the anti - CD40 antibody - induced secretion of IFNγ from T cells, and an increase in any of these parameters indicates increased activation and a decrease indicates decreased activation. Similarly, the ability of an antibody to modulate the activity of B cells can be evaluated by measuring the levels of cell surface markers (such as CD86) and / or by measuring anti - CD40 antibody - induced B cell proliferation (see Example 3 of WO2016 / 023960), and an increase in any of these parameters indicates increased activation and a decrease indicates decreased activation.

[0164] Preferably, the anti-CD40 antibodies described herein that increase activation of dendritic cells or B cells have efficacy for activation of dendritic cells or B cells. Cell activation can typically be measured as an EC50 level in an assay involving incubating isolated dendritic cells or B cells with a test stimulant and then detecting cell proliferation as a measure of activation.

[0165] 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 determining the dissociation constant (Kd) of the antibody and its target. Similarly, the specificity of binding of an antibody to its target can be defined in terms of the comparative dissociation constant (Kd) of the antibody for its target as compared to the dissociation constants for the antibody and another non-target molecule.

[0166] Typically, the Kd of an antibody for its target will be less than 2-fold, preferably less than 5-fold, more preferably less than 10-fold that of the Kd for other non-target molecules such as irrelevant substances or accompanying substances in the environment. More preferably, the Kd will be less than 50-fold, even more preferably less than 100-fold, and even more preferably less than 200-fold.

[0167] This dissociation constant value can be determined directly by well-known methods and can also be calculated for complex mixtures, for example, by methods such as those described in Caceci et al. (Byte 9:340-362, 1984). For example, it can be established using a dual filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays for evaluating the binding ability of a ligand such as an antibody for its target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding rate of an antibody (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as by BIAcore™ system analysis.

[0168] A competitive binding assay may be performed in which the binding of an antibody to a target is compared to the binding of the target by another known ligand of the target, such as another antibody. The concentration at which 50% inhibition occurs is known as Ki. Under ideal conditions, Ki is equivalent to Kd. Since the Ki value does not fall below Kd, the measured value of Ki can be conveniently substituted to provide an upper limit for Kd.

[0169] The anti-CD40 antibodies described herein can preferably bind to their target with an affinity that is at least 2-fold, 10-fold, 50-fold, 100-fold or more greater than their affinity for binding to another non-target molecule.

[0170] The antibodies used in the combination therapies and methods of the present invention typically (i) the ability to specifically bind to human CD40 when localized on the surface of a cell, (ii) the ability to enhance antibody-dependent cell-mediated cytotoxicity (ADCC)-mediated lysis of cells expressing CD40, (iii) the ability to enhance apoptosis of cells expressing CD40, and / or (iv) the ability to regulate the activity of cells expressing CD40, wherein the regulation is an increase or decrease in the activity of the cell.

[0171] The antibody may be a variant or fragment of one of the specific anti-CD40 antibodies disclosed herein, or may contain them, provided that the variant or fragment retains specificity for CD40 and at least one of the functional characteristics (i)-(iv).

[0172] The fragment is preferably the antigen-binding portion of the antibody. The fragment can be produced by terminal truncation, for example, by removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. In this way, up to 10, up to 20, up to 30, up to 40, or more amino acids may be removed from the N-terminus and / or C-terminus. The fragment can also be generated by one or more internal deletions.

[0173] The variant can include one or more substitutions, deletions, or additions with respect to the sequence of the specific anti-CD40 antibody disclosed herein. The variant can include 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 sequence disclosed herein. A "deletion" variant may include deletion of individual amino acids, deletion of small groups of amino acids, such as 2, 3, 4, or 5 amino acids, or deletion of larger amino acid regions, such as deletion of a specific amino acid domain or other feature. A "substitution" variant preferably involves replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid may be replaced with an alternative amino acid having similar properties, such as 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.

[0174] Some properties of 20 major amino acids that can be used to select suitable substituents are as follows.

Table 7

[0175] Preferred "variants" include those in which the amino acids appearing in the sequence are structural analogs of the naturally occurring amino acids. The amino acids used in the sequence may also be derivatized or modified, such as labeled, provided that the function of the antibody is not significantly adversely affected.

[0176] Variants may be prepared during the synthesis of the antibody or by post-production modification, or, if the antibody is in recombinant form, by site-directed mutagenesis, random mutagenesis, or using known techniques of enzymatic cleavage and / or ligation of nucleic acids.

[0177] Preferably, the variant antibody has an amino acid sequence having greater than 60%, or greater than 70% (e.g., 75% or 80%), preferably greater than 85%, e.g., greater than 90% or 95% amino acid identity to the VL or VH domain of the antibodies disclosed herein. This level of amino acid identity can be seen over the full length of the relevant SEQ ID NO sequence or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200, or more amino acids depending on the size of the full-length polypeptide.

[0178] In the context of amino acid sequences, "sequence identity" refers to sequences having the recited values when evaluated using ClustalW (Thompson et al., 1994, supra) with the following parameters. Pairwise alignment parameters - method: exact, matrix: PAM, gap open penalty: 10.00, gap extension penalty; 0.10; Multiple alignment parameters - matrix: PAM, gap open penalty: 10.00, % identity for delay: 30, end gap penalty: on, gap separation distance: 0, negative matrix: none, gap extension penalty: 0.20, residue-specific gap penalty: on, hydrophilic gap penalty: on, hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include only the identical residue that has been derivatized.

[0179] The anti-CD40 antibody of the present invention can bind to the same epitope as the specific antibodies disclosed herein. This is because such antibodies are likely to mimic the action of the disclosed antibodies. Whether one antibody binds to the same epitope as another can be determined by routine methods. For example, the binding of each antibody to the target may use a competitive binding assay. Methods for performing a competitive binding assay are well known in the art. For example, they may involve contacting the antibody and the target molecule together under conditions where the antibody can bind to the target molecule. Subsequently, the antibody / target complex may be contacted with a second (test) antibody, and the degree to which the test antibody can replace the first antibody from the antibody / target complex may be evaluated. Such evaluation 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 indicates that the test antibody can compete with the first antibody for binding to the target, and thus the test antibody binds to the same epitope or region on the target as the first antibody, and thus may mimic the action of the first antibody.

[0180] The anti-CD40 antibody of the present invention can be an antibody comprising 1, 2, or all 3 of the CDR sequences of SEQ ID NOs: 1-3 and / or 1, 2, or all 3 of the CDR sequences of SEQ ID NOs: 4-6. The antibody can comprise all 6 CDR sequences of SEQ ID NOs: 1-6.

[0181] The antibody can comprise the light chain variable region sequence of SEQ ID NO: 7 and / or the heavy chain variable region sequence of SEQ ID NO: 8.

[0182] The antibody may be an antibody comprising the light chain variable region sequence of SEQ ID NO: 7 and / or the heavy chain variable region sequence of SEQ ID NO: 8, or may bind to the same epitope as it. In addition, the antibody can comprise the light chain constant region sequence of SEQ ID NO: 11 and / or the heavy chain constant region sequence of SEQ ID NO: 12.

[0183] The anti-CD40 antibody or any variant or fragment thereof used in the combination therapy and method of the present invention preferably has a theoretical isoelectric point (pI) of 9.0 or higher, preferably 9.1 or higher, more preferably 9.2 or higher, or 9.25 or higher, and most preferably 9.3 or higher.

[0184] According to certain embodiments, the antibody or antigen-binding portion thereof and the chemotherapy are administered simultaneously, either in the same composition or in separate compositions. According to other embodiments, the antibody or antigen-binding portion thereof and the chemotherapy are administered sequentially, i.e., the antibody or antigen-binding portion thereof is administered either before, during, and / or after the administration of the chemotherapy. In some embodiments, the administration of the antibody or antigen-binding portion thereof and the chemotherapy is simultaneous, i.e., the administration period of the antibody or antigen-binding portion thereof overlaps with the administration period of the chemotherapy. In some embodiments, the administration of the antibody or antigen-binding portion thereof and the chemotherapy is not simultaneous. For example, in some embodiments, the administration of the antibody or antigen-binding portion thereof ends before the chemotherapy is administered. In some embodiments, the administration of the chemotherapy ends before the antibody or antigen-binding portion thereof is administered.

[0185] In some embodiments, the antibody or antigen-binding portion thereof and the chemotherapy are administered within a single therapeutic composition (e.g., a pharmaceutical composition). In some embodiments, the therapeutic composition further comprises a therapeutically acceptable diluent or carrier. In some embodiments, the antibody or antigen-binding portion thereof is administered as a pharmaceutical composition and the chemotherapy is not administered as a pharmaceutical composition.

[0186] The present invention also provides a kit for treating cancer, optionally chemotherapy-resistant cancer, in a subject, the kit comprising the combination therapy as defined above. For example, the kit can comprise (a) a therapeutically effective amount of an antibody that specifically binds to CD40 as described herein or an antigen-binding portion thereof, and optionally (b) a therapeutically effective amount of chemotherapy suitable for administration to the subject (e.g., systemic administration). The antibody or antigen-binding portion thereof is preferably provided in a form suitable for local administration to the tumor site.

[0187] The kit of the present invention may further include one or more other reagents or instruments that enable the implementation of any of the above-described embodiments. Such reagents or instruments include one or more of a suitable buffer (aqueous solution), and means for administering an anti-CD40 antibody and / or chemotherapy (e.g., a container or instrument containing a needle). The kit may include instructions for performing the combination therapy or method described herein.

[0188] The anti-CD40 antibody and chemotherapy described herein or provided in the kit of the present invention may each be provided as a separate pharmaceutical composition formulated with a pharmaceutically acceptable carrier. When chemotherapy consists of multiple agents, each may be provided as a separate pharmaceutical composition formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are physiologically compatible and compatible with the required route of administration.

[0189] The pharmaceutical composition may include a pharmaceutically acceptable antioxidant. These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both sterilization procedures (see above) and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, phenol, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, long-term absorption of injectable dosage forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0190] Pharmaceutical compositions typically must be sterile and stable under manufacturing and storage conditions. The compositions can be formulated as solutions, microemulsions, liposomes, or other regular structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the active agent (e.g., an antibody) into a suitable solvent containing one or a combination of the ingredients listed above, as necessary, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are high vacuum drying and lyophilization (freeze drying), which yield powders of the active agent and any additional desired ingredients from its pre-sterilized and filtered solution. Pharmaceutical compositions can include additional active ingredients and those described above.

[0191] 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).

[0192] The term "buffer solution" is intended to contain an aqueous solution containing an acid-base mixture for the purpose of stabilizing pH. Examples of buffer solutions 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 lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

[0193] The term "diluent" is intended to contain an aqueous or non-aqueous solution for the purpose of diluting a drug in a pharmaceutical preparation. The diluent may be one or more of physiological saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0194] The term "adjuvant" is intended to include any compound added to a formulation to increase the biological effect of the drug of the present invention. The adjuvant can be one or more of zinc, copper, or silver salts having different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetate having different acyl compositions. The adjuvant can also be a cationic polymer such as a cationic cellulose ether, a cationic cellulose ester, a deacetylated hyaluronic acid, chitosan, a cationic dendrimer, a cationic synthetic polymer such as poly(vinylimidazole), and a cationic polypeptide such as polyhistidine, polylysine, polyarginine, and a peptide containing these amino acids.

[0195] The excipient can be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, cyclodextrin, etc., which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ether, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinyl pyrrolidone, all of which have different molecular weights and are added to the formulation, for example, for viscosity control, to achieve bioadhesion, or to protect lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono, di, and triglycerides, ceramides, sphingolipids and glycolipids, all with different acyl chain lengths and degrees of saturation, egg lecithin, soy lecithin, hydrogenated egg, and soy lecithin, which are added to the composition for the same reasons as for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain advantages such as reduction of liquid accumulation or favorable pigment properties.

[0196] The active antibody-based agent and / or chemotherapy of the present invention can be formulated into any type of pharmaceutical composition known in the art to be suitable for its delivery.

[0197] In one embodiment, the pharmaceutical composition of the present invention may be in the form of liposomes, and the agent may be combined with an amphiphilic agent such as micelles, insoluble monolayers, and lipids present in an aggregated form as liquid crystals, in addition to other pharmaceutically acceptable carriers. Lipids suitable for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysophosphatidylcholine, phospholipids, saponins, bile acids, and the like. Also, suitable lipids include the above lipids modified with poly(ethylene glycol) in the polar head group to extend the blood circulation time. The preparation of such liposomal formulations can be found, for example, in US4,235,871, the disclosure of which is incorporated herein by reference.

[0198] The pharmaceutical composition of the present invention can also be in the form of biodegradable microparticles. Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides are widely used as biodegradable polymers in the production of microparticles. Preparations of such microparticles can be found in US5,851,451 and EP0213303, the disclosures of which are incorporated herein by reference.

[0199] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of nanoparticles based on, for example, poly-gamma-glutamic acid. Details of the preparation and use of such nanoparticles can be found in WO2011 / 128642, the disclosure of which is incorporated herein by reference. Those skilled in the art will understand that one or more of the active ingredients of the combination therapy of the present invention may be formulated into separate nanoparticles, or both active ingredients may be formulated into the same nanoparticles.

[0200] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymeric gel, and polymers such as starch, cellulose ether, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinyl imidazole, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinyl pyrrolidone are used for thickening the solution containing the drug. The polymer may also include gelatin or collagen.

[0201] Alternatively, the drug may be simply dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and / or various buffer solutions.

[0202] It will be understood that the pharmaceutical composition of the present invention may contain ions and a defined pH to enhance the action of the active drug. In addition, the composition may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents, buffer solutions, fillers, etc.

[0203] The pharmaceutical composition according to the present invention can be administered by any suitable route known to those skilled in the art. Thus, the routes of administration include parenteral (intravenous, subcutaneous, intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, vaginal, and rectal. Administration from an implant is also possible.

[0204] Advantageously, the pharmaceutical composition is suitable for administration to or in the vicinity of the tumor site, for example, intratumorally or peritumorally.

[0205] In some embodiments, the pharmaceutical composition is suitable for parenteral administration. Methods for formulating an antibody into a pharmaceutical composition will be well known to those skilled in the pharmaceutical and pharmaceutical arts.

[0206] The combination therapy of the present invention can be delivered using injectable sustained-release drug delivery systems. These are designed, in particular, to reduce the frequency of injections. An example of such a system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in biodegradable microparticles that gradually release rhGH over a period of time once injected. Preferably, the delivery is carried out intramuscularly (i.m.) and / or subcutaneously (s.c.) and / or intravenously (i.v.).

[0207] The combination therapy of the present invention can be administered by surgically implanted devices that directly release the drug to the required site. For example, Vitrasert directly releases ganciclovir to the eye to treat CMV retinitis. By directly applying this toxic agent to the disease site, an effective therapy is achieved without significant systemic side effects of the drug.

[0208] Electroporation therapy (EPT) systems can also be used for the administration of the combination therapy of the present invention. Devices that deliver pulsed electric fields to cells increase the permeability of the cell membrane to drugs, resulting in a significant enhancement of intracellular drug delivery.

[0209] The combination therapy of the present invention can also be delivered by electronic incorporation (EI). EI occurs when small particles up to 30 microns in diameter on the surface of the skin experience an electric pulse identical or similar to that used in electroporation. In EI, these particles are driven through the stratum corneum into deeper layers of the skin. The particles can carry or coat drugs or genes, or can act as "bullets" that create pores in the skin through which drugs can enter.

[0210] An alternative combination therapy of the present invention is a temperature-sensitive ReGel injectable system. Below body temperature, ReGel is an injectable liquid, but at body temperature it immediately forms a gel reservoir, gradually erodes, and dissolves in a known safe biodegradable polymer. The active substance is delivered over time as the biopolymer dissolves.

[0211] The combination therapy of the present invention can also be delivered orally. This process uses the natural process for oral uptake of vitamins B 12 and / or D in the body to co-deliver proteins and peptides. By hitching a ride on the vitamin B 12 and / or D uptake system, the agents, medicaments and pharmaceutical compositions of the present invention can move through the intestinal wall. The complex is synthesized between a vitamin B 12 analogue and / or a vitamin D analogue and a drug that retains both a significant affinity for the intrinsic factor (IF) in the vitamin B 12 portion / vitamin D portion of the complex and a significant biological activity of the active substance of the complex.

[0212] The combination therapy of the present invention can be introduced into cells by "Trojan peptides". These are a type of polypeptide called penetratin that have migratory properties and can carry hydrophilic compounds across the plasma membrane. This system allows for direct targeting of oligopeptides to the cytoplasm and nucleus and can be very efficient without being cell-type specific. See Derossi et al. (1998), Trends Cell Biol. 8, 84 - 87.

[0213] The combination therapy of the present invention may be a unit dose containing the daily dose or unit of the active ingredient, a partial daily dose, or an appropriate fraction thereof.

[0214] The combination therapy of the present invention is usually administered in the form of a pharmaceutical composition containing the active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base addition salt of a pharmaceutically acceptable dosage form, by oral administration or any parenteral route. Depending on the disorder to be treated, the patient, and the route of administration, the composition may be administered in various dosages.

[0215] In human therapy, the combination therapy of the present invention can be administered alone, but is generally administered in admixture with suitable pharmaceutical excipients, diluents or carriers selected with respect to the intended route of administration and standard pharmaceutical practice.

[0216] For example, the combination therapy of the present invention may be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions for immediate release, delayed release or controlled release applications, and may contain flavoring or coloring agents. The agents, medicaments and pharmaceutical compositions of the present invention may also be administered via intracorporal injection.

[0217] Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0218] Solid compositions of the same type may also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactulose or high molecular weight polyethylene glycol. In the case of aqueous suspensions and / or elixirs, the agents, medicaments and pharmaceutical compositions of the present invention can be combined with various sweeteners or flavoring agents, coloring agents or dyes, emulsifying agents and / or suspending agents, and diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

[0219] The combination therapies of the present invention may be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intramedullary, intracardially, intrasternal, intracranial, intramuscularly or subcutaneously, or by infusion techniques. They are most preferably used in the form of a sterile aqueous solution which may contain other substances, for example salts or glucose sufficient to render the solution isotonic with blood. Where necessary, the aqueous solution should be suitably buffered (preferably to a pH of 3 to 9). The preparation of suitable parenteral formulations under sterile conditions can be readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0220] Pharmaceuticals and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions (which can contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient) and aqueous and non-aqueous sterile suspensions (which can contain suspending agents and thickening agents). The pharmaceuticals and pharmaceutical compositions may be provided in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state which requires only the addition of a sterile liquid carrier, for example water for injection, immediately prior to use. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the previously described types.

[0221] The combination therapy of the present invention can also be administered intranasally or by inhalation, and can be conveniently in the form of a dry powder inhalant, or, for example, in an aerosol spray delivery form from a pressurized container, pump, spray or nebulizer using a suitable propellant such as hydrofluoroalkane such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA 134A) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for delivering a measured amount. The pressurized container, pump, spray, or nebulizer may contain a solution or suspension of the active agent, and for example, a mixture of ethanol and a propellant may be used as a solvent, and may further contain a lubricant such as sorbitan trioleate. Capsules and cartridges (for example, made of gelatin) used in inhalers or insufflators can be formulated to contain a powder mixture of the agent of the present invention and a suitable powder base such as lactose or starch.

[0222] Alternatively, the combination therapy of the present invention can be administered in the form of a suppository or pessary, or may be topically applied in the form of a lotion, solution, cream, gel, ointment or spray. Also, the agents, medicaments, and pharmaceutical compositions of the present invention may be administered transdermally, for example, by use of a skin patch. They may also be administered by the ocular route, particularly for treating eye diseases.

[0223] For use in the eye, the combination therapy of the present invention can be formulated, optionally, as a micronized suspension in isotonic, pH-adjusted, sterile physiological saline combined with a preservative such as benzalkonium chloride, or preferably, as a solution in isotonic, pH-adjusted, sterile physiological saline. Alternatively, it can be incorporated into an ointment such as petrolatum.

[0224] For topical application to the skin, the combination therapy of the present invention can be formulated as a suitable ointment containing the active agent suspended or dissolved, for example, in a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agents, emulsifying wax, and water. Alternatively, they can be suspended or dissolved, for example, in a suitable lotion or cream, such as a mixture of one or more of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0225] Suitable formulations for topical administration in the oral cavity include lozenges containing the active ingredient in a flavor base, usually sucrose and acacia or tragacanth, gelatin and glycerin, or pastes containing the active ingredient in an inert base such as sucrose and acacia, and oral rinses containing the active ingredient in a suitable liquid carrier.

[0226] Generally in humans, topical administration of the combination therapy of the present invention at or near the site of the tumor, particularly intratumoral or peritumoral administration, is the preferred route.

[0227] For veterinary use, the combination therapy of the present invention is administered as a suitable formulation that is preferably acceptable according to normal veterinary practice, and the veterinarian determines the dosing regimen and route of administration that would be most appropriate for a particular animal.

Example

[0228] Example 1 - Sequence Information Anti-CD40 antibody clone G12 (antibody ADC-1013) This clone is described in WO2016 / 023960. An exemplary antibody containing the CDR sequences of ADC-1013 is known as mitazalimab. (a) CDR sequences (defined according to IMGT numbering, with the core CDR sequences underlined)

Table 8

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0229] Example 2 - OPTIMIZE - 1 Clinical Trial General Information · Trial Title: A Phase 1b / 2 Open-Label, Multicenter Trial to Evaluate the Clinical Efficacy of Mitazalimab in Combination with Chemotherapy in Metastatic Pancreatic Ductal Adenocarcinoma. · Region: Europe. · Estimated Number of Sites: 6 - 8 sites in Part 1 and up to 15 sites in Part 2. · Estimated Number of Patients: Up to 80 patients. · Patient Population: Patients with measurable and histologically confirmed, previously untreated metastatic pancreatic ductal adenocarcinoma. Patients must be 18 years of age or older, have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and have adequate organ function as determined by laboratory evaluation.

[0230] Investigational Agent(s), Dose, and Administration Mitazalimab was evaluated in combination with modified FOLFIRINOX (mFOLFIRINOX) chemotherapy. Mitazalimab and mFOLFIRINOX were administered by intravenous infusion according to a 14-day cycle schedule, except for the first 21-day treatment cycle in which mitazalimab was administered on Days 1 and 10 and the infusion of mFOLFIRINOX was started on Day 8.

[0231] Mitazalimab: A human monoclonal (IgG1) antibody, an agonist targeting CD40, developed for cancer immunotherapy. In Part 1 (Phase 1b) of the trial, the dose of mitazalimab was escalated from a starting dose of 450 μg / kg to a maximum of 900 μg / kg (evaluating 2 dose levels) to obtain the recommended Phase 2 dose (RP2D) of mitazalimab in combination with mFOLFIRINOX. In Part 2 of the trial, the RP2D of mitazalimab was administered to all patients in combination with mFOLFIRINOX.

[0232] Chemotherapy was administered as background treatment since it is the standard of care for first-line treatment of advanced pancreatic cancer recommended by the European Society for Medical Oncology (ESMO) and the National Comprehensive Cancer Network (NCCN). Therefore, mFOLFIRINOX and gemcitabine + nab-paclitaxel were classified as non-investigational agents.

[0233] mFOLFIRINOX: The mFOLFIRINOX treatment consisted of oxaliplatin (85 mg / m 2 ), leucovorin (400 mg / m 2 ), irinotecan (150 mg / m 2 ), and 5-fluorouracil (5-FU, 2400 mg / m 2 ). If there are problems with the supply of leucovorin, or to comply with local standard of care practices, other approved similar folinates may be used under the supervision of an experienced clinician. If mFOLFIRINOX is not found to be safe and tolerable in combination with the lowest dose of mitazalimab, mFOLFIRINOX is replaced by gemcitabine + nab-paclitaxel.

[0234] Gemcitabine + nab-paclitaxel (if applicable): Gemcitabine in combination with nab-paclitaxel is approved in Europe for the first-line treatment of adult patients with metastatic adenocarcinoma of the pancreas. Gemcitabine + nab-paclitaxel was administered in this trial only if mFOLFIRINOX was not found to be safe and tolerable in combination with mitazalimab at the lowest dose level.

[0235] Trial Design A phase 1b / 2 open-label, multi-center trial designed to evaluate the safety, tolerability, and efficacy of the combination of mitazalimab with chemotherapy in patients with metastatic pancreatic ductal adenocarcinoma.

[0236] This trial consisted of two parts (as shown in Figure 1). Part 1 (Phase 1b): Two dose levels of mitazalimab in combination with mFOLFIRINOX were evaluated to determine the recommended phase 2 dose (RP2D) of mitazalimab for part 2 of the trial. Part 1 followed a Bayesian optimal interval (BOIN) design with at least 3 patients enrolled at each dose level. At least 6 patients were evaluated at the RP2D of part 1. If the lowest dose of mitazalimab (450 μg / kg) in combination with mFOLFIRINOX was found to be unsafe and intolerable, mFOLFIRINOX treatment could be switched to gemcitabine + nab-paclitaxel.

[0237] Part 2 (Phase 2): Up to 60 enrolled patients were administered mitazalimab and mFOLFIRINOX (or gemcitabine + nab-paclitaxel) to investigate the clinical efficacy of mitazalimab in combination with chemotherapy in part 2 of the trial. A Simon's two-stage design with an interim analysis for futility or efficacy stopping was conducted when a total of 23 patients (including patients from both part 1 and part 2) were evaluable for objective response rate (ORR).

[0238] In part 1, there was at least an 11-day crossover between the first dose of mitazalimab administered to the first patient and the first dose of mitazalimab administered to the second patient at each dose level.

[0239] All patients were monitored for at least 4 hours after the end of the first infusion of mitazalimab and at least 2 hours after the second infusion of mitazalimab. If no infusion-related reactions were observed in the last infusion (subsequent infusions), patient monitoring could be shortened to 1 hour for subsequent infusions. Based on new safety data, the monitoring period could be extended for all patients by the data review committee (DRC).

[0240] Referring to Figure 2, if the lowest planned dose level of mitazalimab (450 μg / kg) in combination with mFOLFIRINOX in Part 1 was found to be not safe or tolerable, mFOLFIRINOX was replaced with gemcitabine + nab-paclitaxel, and the same mitazalimab dose escalation schedule was continued. If the lowest planned dose level of mitazalimab in combination with gemcitabine + nab-paclitaxel in Part 1 was found to be not safe or tolerable, the trial was terminated.

[0241] When the last patient in Part 1 completed the DLT evaluation period, data were collected and reviewed by the DRC, a decision was made to continue to Part 2. It was planned to enroll up to 60 patients in Part 2.

[0242] An interim analysis to stop for futility or efficacy was conducted when efficacy data became available for a total of 23 patients at the RP2D (Parts 1 and 2). The interim analysis was based on the objective response rate (ORR) observed for all patients treated at the mitazalimab RP2D. The interim analysis included efficacy data for up to 17 weeks of study treatment (8 cycles for mFOLFIRINOX and 4 cycles for gemcitabine + nab-paclitaxel). Depending on the results of the interim analysis, a decision was made whether to stop further enrollment in the trial or to continue to enroll an additional 37 patients.

[0243] The dosing schedule and the visit schedule were the same in Parts 1 and 2 of the trial for each respective combination of chemotherapy.

[0244] Treatment Period Patients could receive treatment with mitazalimab and mFOLFIRINOX for up to 12 treatment cycles (approximately 6 months; 1 cycle of 21-day treatment period / dose-limiting toxicity (DLT) evaluation period (Cycle 1), followed by 11 cycles of 14-day treatment cycles).

[0245] Patients may receive treatment with mitazalimab and gemcitabine + nab-paclitaxel for up to 6 treatment cycles (approximately 6 months; the first 28 days is the DLT evaluation period, followed by 5 treatment cycles of 28 days each for a total treatment period of 35 days (Cycle 1)) (if applicable).

[0246] Patients may continue test treatment within the limits defined above until progressive disease (PD) or obvious clinical deterioration, at the discretion of the principal investigator of the clinical trial, as long as the patient tolerates the treatment and consents to continue. After completion of the test treatment, the patient will attend a post-treatment visit and continue a treatment follow-up period to evaluate the disease and survival status.

[0247] Dose, dosing schedule, and dose escalation Both mFOLFIRINOX and gemcitabine + nab-paclitaxel are well-established and effective chemotherapy regimens for the treatment of advanced pancreatic cancer. The central principle in the dose-determination part of the trial is not to compromise the intensity of this standard chemotherapy. If an adverse event is considered to be related to chemotherapy based on the clinical judgment of the principal investigator of the clinical trial, treatment can be adjusted in a standard manner as summarized in the dose adjustment sections of Example 4 (for mFOLFIRINOX) and Example 5 (for gemcitabine + nab-paclitaxel) of this protocol. However, if unexpected toxicity or toxicity of unexpected intensity occurs during the escalation part of this trial with respect to these chemotherapies and the principal investigator of the clinical trial believes that this toxicity may compromise the effectiveness of the chemotherapy, the principal investigator of the clinical trial may discontinue mitazalimab. If this occurs during the DLT period of the trial and the adverse event is not considered a DLT, the patient may be replaced.

[0248] In Part 1, it was planned to administer mitazalimab at two dose levels of 450 μg / kg and 900 μg / kg in combination with mFOLFIRINOX. During the first treatment cycle (the first 21 days) for each patient, mitazalimab was administered on days 1 and 10, and the mFOLFIRINOX infusion was started on day 8. During the subsequent 14-day treatment cycles, mFOLFIRINOX was administered on day 1 of each cycle, and mitazalimab was administered on day 3 (see Figure 3). If the number of DLTs for a certain dose level met the stopping criteria (see the escalation rule in Table 3 based on the number of patients with DLTs), as shown in Figure 2, chemotherapy mFOLFIRINOX could be replaced with chemotherapy gemcitabine + nab-paclitaxel at the lowest dose level of mitazalimab. When changing chemotherapy from mFOLFIRINOX to gemcitabine + nab-paclitaxel, refer to the dosing schedule of Example 5.

[0249] The dose escalation in Part 1 follows the BOIN design with at least 3 evaluable patients per dose level. According to the BOIN design, each time a patient cohort is completed, the following decision rules shown in Table 3 are followed to determine whether the next cohort should remain at the same dose, escalate to the next higher dose level, or de-escalate to the next lower dose level. A range of 3 to 9 patients was included at each dose level. In this study, de-escalation from 450 μg / kg of mitazalimab in combination with mFOLFIRINOX was, as described above, a change in chemotherapy to gemcitabine + nab-paclitaxel.

Table 9

[0250] Dose escalation was determined by the DRC based on a review of safety data obtained during the DLT evaluation period and data available beyond the DLT evaluation period from all patients in the trial. Dose escalation was permitted when the DRC defined the dose and dosing schedule for the next dose level. Based on the available data, the DRC could determine to introduce intermediate dose levels between pre-planned dose levels and to change the dosing frequency.

[0251] Mitazalimab in combination with mFOLFIRINOX: The DLT evaluation period was defined as the time from the first dose of mitazalimab (day 1) to day 21 in the first treatment cycle (cycle 1). Mitazalimab in combination with gemcitabine + nab-paclitaxel: The DLT evaluation period was defined as the time from the first dose of mitazalimab (day 1) to day 28 within the 35-day first treatment cycle (cycle 1). This period allowed the DRC to evaluate the early safety of the combination of mitazalimab and chemotherapy.

[0252] DLT was defined as one of the following toxicities (i.e., drug-related AEs) classified by CTCAE version 5.0: · Grade 4 neutropenia lasting more than 7 days · Grade 4 thrombocytopenia lasting more than 7 days · Grade 4 infusion-related reaction · Grade 3 infusion-related reaction that does not resolve to a lower grade within 24 hours after onset · Grade 4 AST, ALT, and / or bilirubin · Grade 3 AST, ALT, and / or bilirubin that does not resolve to a lower grade within 7 days · Any grade ≥ 3 non-hematological toxicity: o Laboratory abnormalities that do not have clinical consequences and resolve to grade ≤ 2 within 14 days (including electrolyte abnormalities that respond to medical intervention) o Fatigue o Nausea and / or vomiting lasting less than 48 hours Diarrhea that resolves to less than grade 3 by the end of the oDLT period

[0253] The same DLT criteria were applied to mitazalimab in combination with mFOLFIRINOX and gemcitabine + nab-paclitaxel. DLTs were considered related to the investigational treatment unless there was a clear and well-documented alternative explanation for the adverse event (AE). AEs that met the above criteria but occurred after the DLT assessment period were not defined as DLTs unless the onset of the event was during the DLT assessment period. Events could be reported as AEs / serious adverse events (SAEs) if applicable.

[0254] The RP2D of mitazalimab in combination with mFOLFIRINOX or gemcitabine + nab-paclitaxel obtained in Part 1 was administered to all patients in Part 2. Changes in the administration of mFOLFIRINOX or gemcitabine + nab-paclitaxel were permitted as described in Example 4 and Example 5, respectively. The same dosing schedule of mitazalimab in combination with each respective chemotherapy was applied to both Part 1 and Part 2 of the trial.

[0255] Objectives and evaluation items

Table 10

Table 11

[0256] Evaluation The evaluations included demographics, medical history (including previous anti-cancer treatments), height and weight, vital signs (blood pressure, pulse rate, oxygen saturation, and body temperature), physical examination, ECG, ECOG performance status, and clinical laboratory tests (clinical chemistry, hematology, urine tests), concomitant medications, and collection of AEs. Blood samples were taken for pharmacokinetic, pharmacodynamic, and immunogenicity analyses. Anti-tumor activity was evaluated by assessing CT scans according to the RECIST v.1.1 guidelines. During the post-treatment follow-up period, evaluation of disease / survival status, subsequent cancer-related therapies, and anti-tumor activity (CT scan) were included. The post-treatment follow-up period continued for up to 2 years from the last patient enrollment (LPI).

[0257] Statistics Statistical methodology For Part 1 (Phase 1b), no formal statistical hypotheses were defined. In Part 2, the primary evaluation item ORR between mitazalimab and the existing control was compared using an exact one-sided binomial test conducted at the 10% significance level. All secondary and exploratory evaluation items were summarized using appropriate descriptive statistics. Patients in Part 1 receiving the same dose regimen as patients in Part 2 were pooled with patients enrolled in Part 2 for statistical analysis and data summary.

[0258] Sample size In Part 1, at least 9 patients were enrolled at 2 planned dose levels, with 3 patients at the lower level and 3 + 3 patients at the higher dose level being estimated. The actual number of patients in Part 1 depended on the data obtained during the trial.

[0259] In Part 2, the sample size was based on an extended Simon's two-stage design that included an interim analysis of futility and efficacy, assuming a dropout rate of 15%. A total of 23 patients with evaluable data for ORR in RP2D (Part 1 + Part 2) were included in the futility and efficacy analyses. To achieve this number, an estimated 27 patients needed to be enrolled. Depending on the results of the interim analysis, if the trial continues to include a total of 54 evaluable patients, an additional 37 patients could be enrolled.

Table 12-1

Table 12-2

[0260] Clinic visit schedule The following clinic visit tables are applicable when mitazalimab is administered in combination with mFOLFIRINOX and include all evaluations conducted during the trial. Table 4 lists the trial evaluations conducted during screening and Cycle 1 of treatment. Table 5 lists the evaluations conducted during Cycle 2 and subsequent cycles of treatment, as well as at the end of treatment. Table 6 lists the evaluations conducted during the follow-up period after treatment and at the end of the trial. The timing of the different evaluations is indicated by "X".

[0261] Some premedications and postmedications can be administered at specific times in relation to the administration of mitazalimab and mFOLFIRINOX (up to 3 days before administration), respectively.

[0262] The evaluation schedule is applicable to both Part 1 and Part 2 of the trial.

[0263] The clinic visit schedule used when mitazalimab is administered in combination with gemcitabine + nab-paclitaxel is found in Example 5.

Table 13-1

Table 13-2

Table 14-1

Table 14-2

Table 14-3

Table 15-1

Table 15-2

Table 15-3

Table 15-4

Table 16

[0264] Inclusion Criteria Patients are eligible for inclusion in the study if all of the following criteria apply. 1. Have provided written informed consent 2. Are ≥ 18 years old at the time of signing the informed consent form (ICF) 3. Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 4. Have received a diagnosis of previously untreated metastatic pancreatic ductal adenocarcinoma (histologically documented) 5. Have measurable disease according to RECIST v.1.1 6. Have not received prior chemotherapy for pancreatic ductal adenocarcinoma 7. Have not received prior abdominal radiotherapy (except palliative radiotherapy for non-target lesions) 8. Have a life expectancy of ≥ 3 months 9. Have acceptable blood test values defined as follows. a. Neutrophils without growth factor stimulation within 3 weeks before blood test ≧ 1.5×10 9 / L b. Platelets ≧ 100×10 9 / L c. Hemoglobin ≧ 6.2 mmol / L (about 100 g / L) (even after injection) 10. Have acceptable clinical chemistry test values defined as follows. a. Bilirubin ≦ 1.5×ULN (biliary drainage is permitted) b. AST ≦ 3×ULN (regardless of liver metastasis) c. ALT ≦ 3×ULN (regardless of liver metastasis) d. Creatinine ≦ 1.5×ULN or glomerular filtration rate (GFR) ≧ 45 mL / min (see Example 6 for the calculation of GFR) e. INR ≦ 1.5×ULN f. Albumin ≧ 28 g / L 11. In the case of women who are capable of pregnancy 1 : a. The high-sensitivity serum (β-human chorionic gonadotropin [β-hCG]) pregnancy test at the time of screening is negative b. Have the intention to use a highly effective contraceptive method during and for at least 6 months after the test treatment 12. Reproductive men must practice an effective contraceptive method (i.e., surgical contraception or a condom used together with a spermicide) during and for at least 6 months after the test. 13. Have the intention to follow all test procedures Women are considered to have the potential for pregnancy (WOCBP), i.e., reproductive capacity, from menarche until menopause, unless permanently infertile. Permanent contraceptive methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. The postmenopausal state is defined as the absence of menstruation for 12 months without alternative medical causes. High follicle-stimulating hormone (FSH) levels in the postmenopausal range can be used to confirm the postmenopausal state in women not using hormonal contraception or hormone replacement therapy. However, a single FSH measurement is insufficient if there has not been 12 months of amenorrhea.

[0265] Exclusion Criteria Patients are excluded if any of the following criteria apply. 1. Having other types of non-ductal tumors of the pancreas, including endocrine tumors or acinar cell adenocarcinoma, cystadenocarcinoma, and papillary carcinoma of the duodenal papilla 2. Having a current cancer or cancer history other than intraepithelial cervical cancer or basal cell or squamous cell carcinoma treated only with local excision within the 3 years prior to signing the ICF 3. Known CNS metastasis or carcinomatous meningitis 4. Having a contraindication to any component of the investigational drug (mitazalimab and corresponding chemotherapy) 5. Having a history of chronic diarrhea, inflammatory diseases of the colon or rectum, or unresolved partial or complete intestinal obstruction 6. Having a history of myocardial infarction, uncontrolled angina, unstable arrhythmia, or congestive heart failure of New York Heart Association class II or higher within 12 months after the first dose of mitazalimab 7. Having a QTc > 450 msec 8. Having uncontrolled comorbidities including active infections 9. Having a known history of HIV, hepatitis B, or active hepatitis C infection 10. Female patients who are pregnant or breastfeeding 11. Having received a live attenuated vaccine within 28 days before the first dose of the test treatment 12. Any condition that, in the opinion of the responsible investigator, exposes the patient to an increased risk or interferes with the patient's compliance with the study 13. Participating in another investigational drug or device trial with any intervention within 4 weeks before the first dose of mitazalimab Additional exclusion criteria applicable only to mFOLFIRINOX treatment: 14. Having received previous treatment with irinotecan or platinum-containing chemotherapy 15. Having existing peripheral neuropathy greater than Grade 1 16. Having known Gilbert's disease 17. Having the known genotype UGT1A1*28 / *28 18. Having known fructose intolerance (malabsorption) 19. Having complete dihydropyrimidine dehydrogenase (DPD) deficiency Additional exclusion criteria applicable only to gemcitabine + nab-paclitaxel treatment: 14. Having a history of slowly progressive dyspnea and cough without sputum, or a history of conditions such as sarcoidosis, silicosis, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, pneumonia, or multiple allergies 15. Having a history of peripheral arterial disease (e.g., claudication, Buerger's disease) 16. Having a history of connective tissue disorders (e.g., lupus, scleroderma, polyarteritis nodosa, etc.)

[0266] Treatment Investigational Medicinal Product (IMP) - Mitazalimab IMP Mitazalimab is a human monoclonal (IgG1) antibody, an agonist targeting CD40, developed for cancer immunotherapy.

[0267] Manufacture Mitazalimab is manufactured by Biogen (USA) in accordance with GMP. This product is manufactured using a stable Chinese hamster ovary (CHO) cell line in a fed-batch bioreactor. This product does not contain any components of animal origin and has been confirmed to be safe from the perspective of exogenous infectious agents.

[0268] Formulation, Packaging, and Labeling Mithazalimab can be supplied as a lyophilized cake in a disposable glass vial, in which case it can be reconstituted using sterile water for injection (WFI) before use. Each vial requires the addition of 2 mL of sterile WFI to completely dissolve the cake. Each vial contains 40 mg of mithazalimab, and the resulting concentration after reconstitution is 20 mg / mL. The exact concentration is stated on the primary vial. Packaging, labeling, and distribution to clinical facilities are performed by Fisher Clinical Services (FCS). Packaging and labeling are performed at an FCS facility (Allentown, Pennsylvania, USA), and distribution to facilities is performed by FCS in Germany under the responsibility of the sponsor in accordance with Good Manufacturing Practice (GMP). For further details regarding formulation, packaging, and labeling, refer to the Investigational Medicinal Product Dossier (IMPD) for mithazalimab.

[0269] Handling, Preparation, and Storage Vials of mithazalimab can be refrigerated at a temperature of 2 - 8°C in a local pharmacy and protected from light in a safe place in accordance with local regulations. Vials must not be used after the expiration date. Preparation of the mithazalimab solution for injection is performed at the local pharmacy according to the instructions in the pharmacy manual. After reconstitution, mithazalimab is diluted with physiological saline or 5% dextrose in water. The mithazalimab solution prepared for injection is preferably stored at room temperature protected from light in a safe place in accordance with local regulations. The mithazalimab solution for injection can be injected within 16 hours from the reconstitution of the lyophilized cake. For detailed guidance regarding the preparation, handling, and storage of investigational medicinal products, refer to the pharmacy manual.

[0270] Administration of Mithazalimab Mitazalimab is administered intravenously during a 2-hour rate-controlled infusion. The baseline body weight is used to calculate the dose of mitazalimab throughout the study. If the pre-dose assessment of body weight evaluated at the start of each treatment cycle changes by >10% from baseline, the mitazalimab dose can be recalculated and adjusted accordingly. All patients are monitored for at least 4 hours after the end of the first infusion of mitazalimab and at least 2 hours after the second infusion of mitazalimab. If no infusion-related reactions are observed during the last infusion (second and subsequent infusions), patient monitoring can be shortened to 1 hour for subsequent infusions.

[0271] Dose modification of mitazalimab The dose and dosing frequency of mitazalimab for individual patients can be modified in the following ways: · The dose can be reduced due to AE: · If an adverse event is possibly, probably, or definitely related to mitazalimab and a treatment delay of more than 2 weeks occurs, the dose of mitazalimab is reduced by 50%. If the dose has already been reduced once, mitazalimab treatment is stopped and / or · If the adverse event is severe or greater than grade 2 and is possibly, probably, or definitely related to mitazalimab, mitazalimab is held until the AE resolves to either the baseline grade or the higher of grade 1 or lower. At that point, mitazalimab can be restarted at the previous dose or, if the dose has not been reduced, at a 50% dose reduction, at the discretion of the principal investigator of the clinical trial. The principal investigator of the clinical trial may also choose to refrain from rechallenge. · Prior to dosing, it can be introduced due to AE, such as infusion-related AE.

[0272] Handling of infusion-related reactions If clinically significant symptoms of infusion-related reactions occur, the infusion can be temporarily stopped (interrupted). When recovered, the infusion can be restarted at 50% of the rate at which the reaction occurred for at least 30 minutes. If clinically significant symptoms do not recur after 30 minutes, the infusion rate can be increased according to the original infusion plan. For example, a lower infusion rate can be selected if clinically indicated.

[0273] Depending on the time of onset and severity of the reaction, the principal investigator of the clinical trial may consider administering supportive medications, such as antihistamines, acetaminophen, or corticosteroids, in addition to the mandatory premedication and postmedication described herein.

[0274] If a severe infusion-related reaction is observed, the DRC may introduce a change to the mitazalimab infusion.

[0275] If an AE considered to be related to the infusion, such as an infusion-related reaction, is not adequately controlled by the above schedule, the DRC can determine to introduce a pre-dose of mitazalimab the day before the first administration. The pre-dose will be up to 10% of the full dose. The pre-dose can be set for all patients enrolled after the determination by the DRC.

[0276] Cytokine release syndrome may not be distinguishable from infusion-related reactions when symptoms occur in relation to the infusion. Cytokine release syndrome may also have many symptoms similar to infectious diseases or even sepsis. Cytokine release syndrome reported using CD3-targeted antibodies or CAR T cell infusions has not been observed with mitazalimab.

[0277] The preferred condition for reporting a reaction considered to be related to the infusion is an infusion-related reaction rather than cytokine release syndrome. Grade 2 or higher infusion-related reactions should be reported as particularly notable adverse events (AESIs).

[0278] Handling of liver function impairment parameters Liver injury is defined by changes in the following liver parameters: · For patients with normal liver function at baseline (ALT, AST, and bilirubin within normal limits), an increase in AST and / or ALT > 3 times ULN and / or an increase in bilirubin > 2 times ULN measured in the same blood sampling sample, combined with an increase in bilirubin > 2 times ULN, · For patients with liver dysfunction at baseline, i.e., having AST or ALT ULN as described above, an increase in AST and / or ALT > 5 times ULN combined with an increase in total bilirubin > 2 times ULN measured in the same blood sampling sample.

[0279] If laboratory values consistent with the liver injury defined above are observed, the following laboratory tests can be repeated within 48 - 72 hours: ALT, AST, and bilirubin (total and direct). If hepatobiliary toxicity is observed, treatment can be withheld to allow evaluation of alternative causes, e.g., biliary obstruction / stent dysfunction. Other causes may need to be followed up according to drug-induced liver injury (DILI) in Example 7. Increases in AST or ALT of grade 3 or higher, and bilirubin of grade 2 or higher should be reported as AESI.

[0280] Non-study drug Premedication and postmedication of mitazalimab Premedication and postmedication can be administered in relation to each mitazalimab administration. Premedication can be started 3 days before mitazalimab infusion. Advantageous premedication and postmedication are listed in Table 7 below and include the type of drug and the timing of intake related to mitazalimab infusion.

[0281] Antihistamine H1 and leukotriene inhibitors can be administered as premedication, for example, starting 3 days before mitazalimab administration.

Table 17

[0282] mFOLFIRINOX The mFOLFIRINOX used in this trial consists of the components oxaliplatin, leucovorin, irinotecan, and 5-fluorouracil (5-FU) and is considered standard of care for first-line treatment of metastatic pancreatic cancer by the European Society for Medical Oncology (ESMO) and the National Comprehensive Cancer Network (NCCN) based on evidence from large-scale phase 3 trials.

[0283] The original "complete" FOLFIRINOX regimen as defined in the phase 3 trial ACCORD 11 / 0402 of 342 patients consisted of a 400 mg bolus of 5-FU, followed by 2400 mg / m 2 of 5-FU over 46 hours, 400 mg / m 2 of leucovorin, 85 mg / m 2 of oxaliplatin, and 180 mg / m 2 of irinotecan. Over the past 10 years, it has been common practice to administer a less intensive version of this regimen, collectively known as mFOLFIRINOX, to reduce side effects. Current clinical guidelines consider the mFOLFIRINOX regimen equivalent to complete FOLFIRINOX as a preferred regimen for first-line treatment of patients with metastatic pancreatic cancer and a good performance status (NCCN Clinical Practice Guidelines in Oncology. Pancreatic Adenocarcinoma. Version 1.2021. Available from the following web page: https: / / www.nccn.org / professionals / physician_gls / pdf / pancreatic.pdf).

[0284] Based on phase 2 trials, other modifications of FOLFIRINOX are recognized as acceptable alternatives that maintain comparable clinical efficacy with an improved side effect profile. These include, for example, regimens that reduce all components by 20%

[30] , or regimens that reduce the 5-FU bolus to 300 mg, and regimens that reduce irinotecan to 135 mg / m 2

[31] .

[0285] Other clinical trials attempting to combine a novel agent with FOLFIRINOX have selected similar modifications to FOLFIRINOX. For example, in a trial combining FOLFIRINOX with the hedgehog inhibitor IPI-926, the 5-FU bolus was not administered

[32] . In the AVENGER 500 trial combining FOLFIRINOX with CPI-613, the 5-FU component was maintained at full strength including the bolus, but oxaliplatin was reduced to 65 mg / m 2 and irinotecan was reduced to 140 mg / m 2

[33] .

[0286] The mFOLFIRINOX regimen used in the current trial is identical to the regimen used in the adjuvant pancreatic trial of the Phase 3 PRODIGE 24 trial, and is different from the original FOLFIRINOX regimen in that the 5-FU bolus is omitted and the dose of irinotecan is 150 mg / m 2

[34] .

[0287] Administration of mFOLFIRINOX Mitazalimab can be administered as a first choice in combination with the chemotherapy regimen mFOLFIRINOX. mFOLFIRINOX can be composed of the components oxaliplatin, leucovorin, irinotecan, and / or 5-FU. The recommended parameters regarding the timing and order of infusion of the components of mFOLFIRINOX are described in Table 8 below.

[0288] Leucovorin is one of several folinates that can be used in combination with 5-FU in cytotoxic therapy. Leucovorin enhances the inhibition of thymidylate synthase by 5-FU and increases the antimetabolic activity of 5-FU. Alternative folinates to leucovorin, including but not limited to calcium folinate, calcium levofolinate, disodium folinate, and disodium levofolinate, can be utilized based on local availability. Folic acid-based administration as defined in the relevant SmPC can be carried out. When leucovorin is referenced within this application, it is recognized that similar approved folinates can be used.

[0289] Variations in administration may be permitted, achievable based on drug administration and modification guidelines such as those described in Example 4.

[0290] Using the baseline body weight and height, the dose of the mFOLFIRNOX components can be calculated. If the patient's body surface area (BSA) changes by >10% from the baseline, the dose can be readjusted. If the patient's BSA changes by ≦10%, no adjustment is necessary unless the facility does not have a standard procedure for adjusting the dose based on the current BSA according to the facility's guidelines.

Table 18

[0291] Recommended injection timing and sequence: · Oxaliplatin IV over 2 hours, immediately followed by; · Leucovorin or a similar approved folinate over 2 hours; · Irinotecan over 90 minutes (starting 30 minutes after the start of the leucovorin injection), followed by; · 5-FU infusion over 46 - 48 hours.

[0292] If one of the mFOLFIRINOX components is discontinued, one or more of the other components may be administered. If mFOLFIRINOX is not considered safe and tolerable in combination with the minimum planned dose level of mitazalimab (450 μg / kg), mFOLFIRINOX may be replaced with gemcitabine + nab-paclitaxel and / or a lower dose of mitazalimab may be used. The approved dosing schedules and dosing modifications for gemcitabine + nab-paclitaxel are described in Example 5.

[0293] mFOLFIRINOX premedication and postmedication. Premedication and postmedication may be administered in connection with each mFOLFIRINOX administration. The recommended premedication starts 1 hour before the administration of oxaliplatin, the first component of mFOLFIRINOX. The recommended premedication and postmedication are listed below.

[0294] The recommended premedication may include one or more of the following pharmaceuticals administered 30 - 60 minutes before infusion: · NK1 receptor antagonist, for example, aprepitant, 125 mg PO, 60 minutes before infusion, and can be continued for several days using chemotherapy. Aprepitant is a cytochrome 2C9 inducer and can inactivate some oral contraceptives. Therefore, women of childbearing potential may use alternative contraceptive means when aprepitant is administered. · 5-HT3 receptor antagonist, for example, ondansetron, 8 mg PO, 30 minutes before infusion, and can be continued for several days using chemotherapy. · Corticosteroid, for example, dexamethasone 8 mg IV or PO, 30 minutes before infusion. The postmedication may include the following pharmaceuticals: · G-CSF, for example, Neulasta, 6 mg SC, on the 4th day after the start of the mFOLFIRINOX regimen (at least 24 hours after the end of the continuous 5-FU iv infusion).

[0295] Alternative premedication and postmedication for mFOLFIRINOX are permitted.

[0296] Gemcitabine and nab-paclitaxel (where applicable) Gemcitabine and nab-paclitaxel are considered standard of care for first-line treatment of metastatic pancreatic cancer by the European Society for Medical Oncology (ESMO) and the National Comprehensive Cancer Network (NCCN), based on evidence from large randomized phase III trials.

[0297] Participants should be informed to seek advice on germline donation and cryopreservation prior to study treatment, as there is a potential for irreversible infertility caused by treatment with gemcitabine or nab-paclitaxel.

[0298] Gemcitabine + nab-paclitaxel can be supplied / obtained in accordance with the clinical trial agreement and local guidelines. Gemcitabine and nab-paclitaxel are stored and handled in accordance with the package insert and stored in a safe place under appropriate storage conditions.

[0299] Administration of Gemcitabine and nab-paclitaxel If mFOLFIRINOX is found to be not viable for a particular subject, gemcitabine + nab-paclitaxel can be administered in combination with mitazalimab. The recommended (exemplary) parameters for the timing and sequence of each infusion of gemcitabine and nab-paclitaxel are listed in Table 9 below. Variations in administration are permitted, subject to compliance with the drug administration and change guidelines. See Example 5. [Table 19]

[0300] Recommended timing and sequence of infusion: · Nab-paclitaxel infused over 30 - 40 minutes · Gemcitabine is infused over 30 minutes immediately after the completion of nab-paclitaxel infusion.

[0301] Test evaluation Demographics and medical history The age, gender, race, and ethnic origin of the trial participants were recorded. Clinically significant abnormal findings observed during the physical examination and non-serious AEs that occurred before treatment (i.e., after signing the ICF but before the first dose) can be recorded as part of the medical history.

[0302] The status of cancer diseases, including other cancers other than the test disease The date of the first pathological histological diagnosis of pancreatic adenocarcinoma, as well as the disease stage at diagnosis and the current disease stage were recorded. Surgery for cancer, including information on whether the surgery was intended to be curative, can be recorded. Radiation therapy can also be recorded if it was administered for palliative purposes.

[0303] Any previous anti-cancer treatment for other cancer diseases other than pancreatic adenocarcinoma (if applicable), including the following, can be recorded: · If applicable, the treatment identity, including surgery, radiation therapy, and chemotherapy, · The start date and end date of the treatment, · The best response (CR, PR, SD, PD, and NE), · The reason for discontinuation (in the case of treatment stopped before it was initially planned).

[0304] Weight and height Weight (without overcoat and shoes) was measured at screening and during treatment and rounded to the nearest kilogram. The dose calculation of mitazalimab is based on weight. If the weight changes by more than 10% from the baseline measurement, the mitazalimab dose can be recalculated. The dose calculations of the components of mFOLFIRINOX, gemcitabine, and nab-paclitaxel are based on BSA. If the BSA changes by more than 10% based on the calculation using the baseline measurement of weight, the doses of the components of mFOLFIRINOX, gemcitabine, and nab-paclitaxel can be recalculated. Additional weight evaluations can be performed based on the judgment of the study physician at regular visits or additional (unscheduled) visits. Height (without shoes) was measured at screening and rounded to the nearest centimeter.

[0305] Vital Signs The measurement of vital signs included systolic and diastolic blood pressure, pulse rate, oxygen saturation, and body temperature. The determination of blood pressure can preferably be performed using the same device during each visit after the patient has rested for 10 minutes. New or worsened clinically significant abnormal findings compared to the pre-treatment evaluation are reported as AEs. Additional vital sign evaluations can be performed based on the judgment of the study physician at regular visits or additional (unscheduled) visits.

[0306] Physical Examination The physical examination may include the following body systems: eyes, mouth and throat, lymph node regions, respiratory, cardiovascular, abdomen, extremities, and general appearance and examination of the skin. The examination of the nervous system can be performed based on the judgment of the study physician. New or worsened clinically significant abnormal medical findings compared to the pre-treatment evaluation can be reported as AEs. Additional evaluations of the physical examination can be performed based on the judgment of the study physician at regular visits or additional (unscheduled) visits.

[0307] Electrocardiogram (ECG) A standard 12-lead ECG was recorded, and the overall interpretation of the ECG was performed by the principal investigator of the clinical trial, or, if applicable, the principal investigator delegated this task to a cardiologist. The ECG printout was signed and dated after review and interpretation. For ECG recording, the patient had to rest for at least 10 minutes and be in a horizontal or semi-recumbent position. The same evaluation method could be used throughout the study. Any irregularities (e.g., vomiting, coughing) observed or occurring during the ECG could induce repetition of the ECG. New or worsened clinically significant abnormal ECG findings, compared to the pre-treatment evaluation, could be reported as AEs. Additional ECG recordings could be performed based on the judgment of the principal investigator at regular visits or additional (unscheduled) visits.

[0308] ECOG Performance Status The performance status of the patient in activities of daily living was graded using the Performance Status Scale of the Eastern Cooperative Oncology Group (ECOG) provided in Table 10 below

[35] .

Table 20

[0309] Clinical Examinations The timing of clinical laboratory evaluations was specified in the Section 0 Visit Schedule. However, more frequent tests could be performed if indicated by the patient's clinical condition or abnormalities that required more frequent monitoring as determined by the principal investigator. Screening test results had to be obtained and reviewed by the principal investigator prior to the first dose of mitazalimab. The results of clinical examinations were summarized in the clinical study report. Therefore, worsening compared to pre-treatment in these parameters was reported as an AE only if it met any of the SAE criteria or was a reason to modify the study treatment. Worsening of test values clearly attributable to disease progression was not reported as an AE.

[0310] Procedures for the collection, preparation, and handling of blood and urine samples were conducted according to local procedures. The DPD test performed during screening can be analyzed in a central laboratory or according to local procedures. Clinical tests to be performed are listed in Table 11. All tests can be performed by a local laboratory, but urine analysis tests can be performed and evaluated by the facility staff. If abnormal urine test results (urine dipstick) are considered clinically significant, a microscope can be used to measure the precipitate, namely, cultures of red blood cells, white blood cells, epithelial cells, crystals, cylinders, and bacteria.

Table 21

[0311] Immunogenicity (anti-drug antibody) Blood samples (serum) were collected for immunogenicity testing according to the Section 0 visit schedule for mitazalimab in combination with mFOLFIRINOX and according to the visit schedule in Example 5 for mitazalimab in combination with gemcitabine and nab-paclitaxel. Also, based on the judgment of the principal investigator, samples may be collected at additional time points, regular visits, or additional (unscheduled) visits. If the infusion of mitazalimab is interrupted due to an AE, samples for immunogenicity can be collected at the time of interruption or as soon as practicable considering patient safety together with PK samples (except during the first infusion). Samples for immunogenicity testing can be used for anti-drug antibody (ADA) analysis (i.e., antibodies against mitazalimab). Samples analyzed for immunogenicity and confirmed positives can be tested for neutralizing antibodies. Other analyses may be performed to further characterize the immunogenicity of mitazalimab. If deemed necessary, an immune response analysis may be performed on PK samples collected at other time points described in the visit schedule. Details regarding sample collection and processing are provided in the laboratory manual.

[0312] Pharmacokinetics (PK) For the section on mitazalimab in combination with mFOLFIRINOX, the visit assessment schedule in section 0, and for mitazalimab in combination with gemcitabine and nab-paclitaxel, in accordance with Example 5, blood samples (serum) were collected for analysis of mitazalimab concentration and PK analysis. Based on the judgment of the principal investigator of the clinical trial, PK samples may be collected at additional time points, regular visits, or additional (unscheduled) visits. Samples for PK analysis are preferably collected from a peripheral vein on the opposite side of the arm where mitazalimab is infused. If the infusion of mitazalimab is interrupted due to an AE, PK samples and samples for immunogenicity can be collected at the time of interruption or as soon as practicable considering the safety of the patient (except during the first infusion if there is no need to collect samples for immunogenicity).

[0313] The following PK parameters are derived for mitazalimab: ·C max ·T max ·AUC (0-T) If the following data permits, other PK parameters may be derived: ·AUC 0-∞ ·AUC0 · Elimination half-life (T 1 / 2 ) · Total serum clearance (CL) · Volume of distribution (V d )

[0314] Evaluation of antitumor activity Computed tomography (CT) scan Chest / abdomen / pelvic CT scans can be performed according to local practice. If necessary to evaluate the tumor(s), other body areas can also be CT scanned (e.g., patients with neck nodules or head and neck tumors will require a CT scan of the neck). Additional CT scans can be performed based on the judgment of the principal investigator of the clinical trial during regular visits or additional (unscheduled) visits. The use of intravenous contrast agent is at the discretion of the radiologist performing the scan, but the imaging should be consistent for each patient throughout the trial.

[0315] If the principal investigator of the clinical trial determines that a CT scan cannot be performed, magnetic resonance imaging (MRI) can be done. It is preferred that the same scan modality be used throughout the trial. The principal investigator and / or radiologist identified the tumors to be followed throughout the trial.

[0316] CT scans are performed at screening (within 28 days from the first dose), 9 weeks after the start of the trial treatment (at the end of cycle 4 for mFOLFIRINOX combination and at the end of cycle 2 for gemcitabine + nab-paclitaxel treatment), and then every 8 weeks assuming no treatment delay. Additional CT scans can be performed at the discretion of the principal investigator of the clinical trial during unscheduled visits. CT scans are evaluated according to RECIST v.1.1. Patients with a response (PR or CR) can have a confirmatory CT scan at least 4 weeks later to confirm the response. If a patient has progressive disease, the patient should discontinue treatment.

[0317] Evaluation of Tumor Response The evaluation of tumor response was performed according to the RECIST v.1.1 guidelines of Example 3 by the clinical trial facility using CT scans according to RECIST v.1.1. Patients with PD can discontinue the trial treatment. However, patients in whom progression is suspected can continue treatment if they are considered clinically stable according to the opinion of the principal investigator of the clinical trial until clinical or radiological progression is documented.

[0318] Pharmacodynamics The objective of pharmacodynamic biomarkers is to characterize changes in intratumoral and systemic immune activation related to the mechanism of action of mitazalimab and to correlate the pharmacodynamic effects with clinical response. Disease progression and treatment response also follow liquid biopsies that analyze biomarkers such as carbohydrate antigen 19-9 (CA19-9) and circulating tumor DNA (ctDNA). All biomarker analyses in blood and tumor biopsies can be essentially exploratory. Since the data from the analyses are intended only for scientific use and decision-making, all exploratory analyses were performed in a purpose-fit laboratory. The data may be included in scientific publications but not in clinical study reports.

[0319] Blood Blood samples are taken according to the section 1 hospital evaluation schedule for mitazalimab in combination with mFOLFIRINOX and according to Example 5 for mitazalimab in combination with gemcitabine + nab-paclitaxel.

[0320] Different types of blood samples are taken and the following pharmacodynamic biomarkers can be evaluated: Cytokines and chemokines: Serum samples are analyzed for the levels of cytokines and chemokines involved in the immune activation of mitazalimab, including but not limited to IFN-γ, TNF-α, IL12p70, IL-6, MCP-1, IP-10, MIP-1α, MIP-1β, and IL-8, using, for example, a 30-plex kit with Luminex, MSD, or similar.

[0321] Immunophenotyping: Whole blood samples are used for whole blood immunophenotyping for the quantification of immune cell populations and immune cell activation directly or indirectly involved in the immune activation of mitazalimab using flow cytometry. The following biomarker panels can be analyzed: · T / NK / NKT cells (e.g., CD45, CD3, CD8, CD4, CD16, CD56, CCR7, CD45RA + activation marker CD25, Ki67), ·B cells (e.g., CD45, CD19, CD27, IgD+ activation marker CD86, CD83, CD54, HL-DR), and / or ·Monocytes and DCs (CD45, CD14, CD16, CD11c, CD123+ activation marker CD54, CD86, HLA-DR, CD83).

[0322] Whole blood RNA: Whole blood RNA samples are collected for the analysis of gene signatures involved in immunomodulation by mitazalimab (including but not limited to immune scores, IFNγ gene profiles, APC gene profiles, and T cell gene profiles) and prognostic tumor gene profiles (e.g., TMB and high MSI), and can be analyzed, for example, by Nanostring IO 360, RNAseq, or the like.

[0323] Whole blood DNA: Cell-free whole blood DNA samples are collected for the evaluation of circulating tumor DNA (ctDNA). Changes in tumor-specific genes, e.g., KRAS, are tracked as biomarkers for monitoring treatment response. Whole blood DNA can be collected for the evaluation of TCRb clonality.

[0324] CA19-9: Changes in the levels of the pancreatic serum protein CA19-9 are tracked as biomarkers for monitoring disease progression and treatment response.

[0325] Tumor biopsy Tumor biopsies are collected according to the section 1 visit schedule for mitazalimab in combination with mFOLFIRINOX and according to Example 5 for mitazalimab in combination with gemcitabine + nab-paclitaxel. Biopsy collection may be omitted if the treating investigator determines that access to the tumor is not possible or the patient would be at increased risk by the biopsy procedure. If a biopsy cannot be obtained at screening, archived biopsy material (i.e., tissue slides or tissue blocks, preferably from the most recently collected tumor biopsy prior to enrollment) may be collected if available. If no biopsy (fresh or archived) is obtained at screening, no further biopsies will be collected during the study. Biopsies (core biopsies, 18-gauge needles) are obtained under local anesthesia. Tumor biopsies are preferably taken from the same tumor throughout the study. Imaging may be used to guide the biopsy. Tumor tissue collected by punch biopsy or excisional biopsy is an acceptable alternative for cutaneous tumors. Anticoagulants may be discontinued once medically indicated prior to the performance of the tumor biopsy. Biopsies may be formalin-fixed and paraffin-embedded.

[0326] Tumor biopsy analysis may include the following: Immunohistochemistry: Archived or newly collected tumor biopsies may be analyzed for baseline CD40 target expression and immune cell infiltration, which may correlate with clinical response. Biopsies during treatment are compared to baseline biopsies for analysis of immune cell infiltration and immune activation induced by mitazalimab for proof of mechanism. The following immunohistochemistry (IHC) panel may be included: · CD40 target expression, · T cell infiltration and activation status, · Macrophage infiltration with M1 / M2 polarization and activation status, · Cell surface markers associated with immune regulation, e.g., PD-L1, and / or · Other markers that describe proof of mechanism, e.g., fibrosis markers and stromal (e.g., fibronectin and type I collagen) macrophage functionality associated with macrophage functionality.

[0327] Genetic profiling: Baseline and on-treatment tumor biopsies are collected for analysis of gene signatures involved in immunomodulation by mitazalimab (including but not limited to immune scores, IFNγ gene profiles, APC gene profiles, and T cell gene profiles) and prognostic tumor gene profiles (e.g., high TMB and MSI), and can be analyzed, for example, by Nanostring IO 360, RNAseq, or the like.

[0328] Tumor biopsy material remaining after the described analysis was stored in a biobank for possible future analysis.

[0329] Disease / survival status (post-treatment follow-up) During post-treatment follow-up, disease and survival status were collected. For mitazalimab combined with mFOLFIRINOX, please refer to Table 4 of the Outpatient Evaluation Schedule in Section 0, and for mitazalimab combined with gemcitabine + nab-paclitaxel, please refer to Table 22 in Example 5.

[0330] The following were evaluated: · Survival, and / or · Current disease stage.

[0331] If the patient died, the date and cause of death were collected and documented. Disease / survival status can be followed up through the patient's medical records (permitted by local regulations) or by telephone contact. If permitted by local law, public records can be used to record death for the purpose of obtaining survival status.

[0332] Subsequent cancer-related therapies (post-treatment follow-up) During post-treatment follow-up, all subsequent cancer-related therapies were collected. Subsequent cancer-related therapies can be followed up through the patient's medical records (permitted by local regulations) or by telephone contact.

[0333] Statistics Test hypothesis For Part 1 (Phase 1b), no formal statistical hypothesis is defined.

[0334] For Part 2 (Phase 2), the null hypothesis for the primary endpoint ORR is as follows: H0: ORR = 30% The null hypothesis is tested against the alternative: H1: ORR > 30% Considerations for sample size

[0335] In Part 1, at least 9 patients were included at two dose levels, with at least 3 patients at each dose and 6 patients at the RP2D. For Part 2, the sample size calculation was based on the assumption of a 30% ORR for mFOLFIRINOX [4]. Adding mitazalimab was assumed to increase the ORR to approximately 45%. Gemcitabine + nab-paclitaxel was assumed to have the same ORR (29%) as mFOLFIRINOX [5], and since all other assumptions were the same, the sample size estimate was considered valid for both treatment options. The sample size estimate was based on an extended Simon's two-stage design with breaks for futility and efficacy

[36] . The optimal design was selected to minimize the expected sample size. In the interim analysis, there were two cut-offs for the number of responders, one for stopping for futility and one for stopping for efficacy. If the number of responders was between these cut-offs, the trial continued. Table 12 presents the sample size.

Table 22

[0336] Assuming a dropout rate of 15% (not evaluable for efficacy), the interim analysis required a total of 27 patients to be enrolled at the RP2D to reach 23 evaluable patients. Patient enrollment continued during the interim analysis. If the trial continued after the interim analysis, assuming the same dropout rate as above, a total of 64 patients needed to be enrolled at the RP2D to reach 54 evaluable patients.

[0337] Statistical methods Primary efficacy The primary endpoint is defined as the objective response rate (ORR), which is the proportion of patients who achieved a complete response (CR) or partial response (PR) confirmed by RECIST v.1.1 at any time point during the trial. For the interim analysis, the ORR was based on response evaluations after 8 cycles for mitazalimab in combination with mFOLFIRINOX, or after 4 cycles for mitazalimab in combination with gemcitabine + nab-paclitaxel (i.e., approximately 4 months after the first dose). For the interim analysis, it was not necessary to confirm the response according to RECIST v.1.1. A one-sided exact binomial test was performed at the 10% significance level to compare the primary endpoint ORR between mitazalimab and the existing control. The two-sided exact binomial 90% confidence interval was also presented. In addition, the ORR was presented for each trial visit. Patients for whom the ORR was not evaluable and who completed at least 2 cycles were considered non-responders.

[0338] Secondary efficacy All secondary efficacy evaluation items were presented using descriptive statistics. The best overall response (BOR) for each patient was defined as the best effect from among CR, PR, SD, and PD in the RECIST v.1.1 categories at any point during the trial. The disease control rate was defined as either CR, PR, or SD at each visit, as determined by RECIST v.1.1. The duration of response (DoR) was defined as the number of days from the first response of CR or PR to either the earlier of progressive disease or death due to the underlying disease. The duration of stable disease (SD) was defined as the number of days from the first dose of mitazalimab to either the earlier of progressive disease or death. The time to the next anticancer therapy was defined as the number of days from the first dose of mitazalimab until the next treatment was initiated. Kaplan–Meier curves were presented for DoR, duration of SD, and time to the next anticancer therapy. The progression-free survival (PFS) was defined as the number of days from the first dose of mitazalimab to either the earlier of progressive disease or death from any cause. The overall survival (OS) was defined as the number of days from the first dose of mitazalimab to the date of death from any cause. Kaplan–Meier curves were presented for PFS and OS. In addition, the detection and characterization of anti-drug antibody (ADA) titers in serum were summarized.

[0339] Safety analysis The number of patients experiencing AEs and the number of events were summarized by SOC and preferred terms. In addition, the following AEs were presented: · Incidence of DLT (Part 1), · Grade 3 or higher AEs, · Serious AEs, · AEs leading to discontinuation of the trial treatment, and · AEs leading to death.

[0340] Analysis population Patients in Part 1 receiving the same dosing regimen as patients in Part 2 were pooled with patients in Part 2 for statistical analysis and data summary.

[0341] Full analysis set (FAS) The FAS included all patients who had at least one post-baseline efficacy measurement. The FAS population was used for all efficacy and exploratory evaluation items.

[0342] Safety set The safety set included all patients who received any study treatment (mitazalimab) and was used for all evaluation items related to safety objectives.

[0343] Protocol compliance set (PP) The PP population included all patients who completed at least one treatment cycle, had at least one post-baseline efficacy measurement, and had no major protocol violations. In addition to the FAS, the PP was used for all evaluation items related to efficacy objectives.

[0344] Dose escalation in Part 1 Part 1 followed a Bayesian optimal interval (BOIN) design with at least three evaluable patients per dose level. The BOIN is described in statistical detail in the clinical applications of reference

[37] and reference

[38] . It can be considered a generalization of the 3+3, accelerated titration, and 3+3+3 designs and is very similar to these designs. According to the BOIN design, each time a patient cohort is completed, according to the decision rules shown in Table 3, it is evaluated whether the next cohort should remain at the same dose, escalate to the next higher dose level above, or de-escalate to the next lower dose level. The BOIN design shares the simplicity of the 3+3 design, which is 0 / 3, 1 / 3, 2 / 3, 0 / 6, 1 / 6, and 2 / 6 and

Number

Number

[0345] Interim analysis of Part 2 An interim analysis was conducted when 23 evaluable patients for RP2D completed 17 weeks of treatment (8 treatment cycles for the combination of mitazalimab and mFOLFIRINOX, or 4 cycles for the combination of mitazalimab and gemcitabine + nab-paclitaxel) according to the extended Simon's two-stage design. The cut-off limits for stopping for futility or efficacy are ≤7 or ≥12 responders (ORR), i.e., if there are 8 - 11 responders, the trial continues.

[0346] Example 3: Appendix 1 Response Evaluation Criteria in Solid Tumors - RECIST v.1.1 Guidelines The revised RECIST v.1.1 guidelines are available at: https: / / ctep.cancer.gov / protocoldevelopment / docs / recist_guideline.pdf

[0347] Definition Response and progression are evaluated in this trial using the international criteria (version 1.1) proposed by the Response Evaluation Criteria in Solid Tumours (RECIST) Committee [Eur J Cancer. 45(2009)228-247]. Changes in the maximum diameter (one-dimensional measurement) of tumour lesions only are used in the RECIST v.1.1 criteria. Note: Lesions are either measurable or non-measurable using the criteria provided below. The term "evaluable" regarding measurability is not used as it does not provide additional meaning or accuracy.

[0348] Measurable disease Measurable disease is defined by the presence of at least one measurable lesion. Measurable lesions are defined as those that can be accurately measured with at least one dimension [the longest diameter (LD) of the measurement plane is recorded] at the following minimum sizes. · 10 mm by CT scan (CT scan slice thickness is 5 mm or less), · 10 mm caliper measurement by clinical examination (lesions that cannot be accurately measured with calipers need to be recorded as non-measurable), and / or · 20 mm by chest X-ray.

[0349] Malignant lymph nodes: To be considered pathologically enlarged and measurable, lymph nodes must be ≥ 15 mm in short axis when evaluated by CT scan (CT scan slice thickness is 5 mm or less).

[0350] Non-measurable disease All other lesions (or sites of disease) including small lesions (< 10 mm longest diameter or pathological lymph nodes with ≥ 10 - < 15 mm short axis) are considered non-measurable disease. Lesions considered truly non-measurable include leptomeningeal disease, ascites, pleural / pericardial effusions, cutaneous lymphomas / pulmonary lymphomas, inflammatory breast disease, abdominal tumours / abdominal enlargement identified on physical examination and not followed by CT or MRI.

[0351] Bone lesions, cystic lesions, and lesions previously treated with local therapy may be considered as follows: Bone lesions: · Bone scans, positron emission tomography (PET) scans, or plain films are not considered appropriate imaging techniques for measuring bone lesions. However, these techniques can be used to confirm the presence or absence of bone lesions. · Lytic bone lesions or lytic blastemic mixed lesions with distinguishable soft tissue components that can be evaluated by cross-sectional imaging techniques (i.e., CT or MRI) can be considered measurable lesions if the soft tissue components meet the above definition of measurability. · Blastemic bone lesions are not measurable. Cystic lesions: · Lesions that meet the criteria for radiologically defined simple cysts should not be considered malignant lesions (neither measurable nor non-measurable) as they are, by definition, simple cysts. · "Cystic lesions" that are thought to represent cystic metastases can be considered measurable lesions if they meet the above definition of measurability. However, if non-cystic lesions are present in the same subject, they are preferred for selection as target lesions. Lesions with previous local treatment: · Tumor lesions in areas previously irradiated or treated with other local regional therapies are usually not considered measurable unless progression of the lesion has been demonstrated.

[0352] Target lesions All measurable lesions can be identified as target lesions up to a maximum of two lesions per organ and a total of five lesions representing all involved organs, and can be recorded and measured at baseline. Target lesions need to be selected based on their suitability for size (the lesion with the longest diameter) and accurate repeated measurements (either imaging techniques or clinical methods). The sum of the diameters of all target lesions (longest for non-nodular lesions, short axis for nodular lesions) was calculated and reported as the baseline sum diameter. The baseline sum diameter was used as a criterion for characterizing objective tumor response.

[0353] Lymph node evaluation For lymph nodes, the short axis, defined as perpendicular to the LD of the lymph node evaluated on the measurement plane, can be measured. · If the short axis ≥ 15 mm, it is a target lesion. · If the short axis ≥ 10 but < 15 mm, it is a non-target lesion. · If the short axis < 10 mm, it is normal.

[0354] For baseline, the actual short axis measurement value was added to the sum of the LD of non-nodular lesions.

[0355] Non-target lesions All other lesions (or disease sites) including pathological lymph nodes can be identified as non-target lesions and recorded at baseline. Measurement of these lesions is not required, and these lesions can be followed as "present", "absent", or in rare cases "definite progression". In addition, it is possible to record multiple non-target lesions associated with the same organ as a single item in the case report form (e.g., "multiple enlarged pelvic lymph nodes" or "multiple liver metastases").

[0356] Guidelines for the evaluation of measurable diseases All measurements can be obtained and recorded in metric notation using a ruler or calipers. All baseline evaluations can be performed as close as possible to the start of treatment and not exceeding 4 weeks from the start of treatment. Using the same evaluation method and the same technique, preferably characterize each lesion identified and reported during baseline and follow-up. When both methods are used to evaluate the antitumor effect of treatment, imaging-based evaluation is preferred over clinical examination-based evaluation.

[0357] Clinical lesions. Clinical lesions are considered measurable only if they are on the surface and have a diameter of ≥ 10 mm, as evaluated using calipers (e.g., skin nodules). For skin lesions, documentation by color photography including a ruler to estimate the size of the lesion is recommended. If lesions can be evaluated by both clinical examination and imaging, imaging evaluation should be performed as it is more objective and can be reviewed at the end of the clinical trial.

[0358] Chest X-ray. Chest CT is preferred over chest X-ray, especially when progression is an important evaluation item. Lesions on chest X-ray can be considered measurable if they are clearly defined and surrounded by aerated lung.

[0359] Conventional CT and MRI. This guideline defines the measurability of lesions in CT scans based on the assumption that the CT slice thickness is 5 mm or less. If the CT scan has a slice thickness > 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is acceptable in certain situations (e.g., for body scans).

[0360] Ultrasound (US). Tumor lesions should not be measured using US. Since US examinations are operator-dependent, they cannot be reproduced in their entirety for independent review at a later date. If new lesions are identified by US, confirmation by CT or MRI is recommended. If there are concerns about radiation exposure with CT, MRI can be used instead of CT.

[0361] Endoscopy, laparoscopy. These techniques are not recommended for use in objective tumor assessment. However, such techniques may be useful for confirming a complete pathologic response when a biopsy is obtained or for determining recurrence in a clinical trial where complete response or recurrence after surgical resection is an evaluation item.

[0362] Tumor markers. Objective tumor response cannot be evaluated using tumor markers alone. If the marker is initially above the normal upper limit, it must normalize in order for the subject to be considered a complete clinical response.

[0363] Cytology, histology. These techniques can be used to distinguish partial response (PR) and complete response (CR) in rare cases (e.g., in residual lesions of tumor types such as germ cell tumors, known residual benign tumors can be left).

[0364] Response Criteria

Table 23

[0365] Evaluation of Target Lymph Nodes For lymph nodes identified as target lesions, the actual short-axis measurement can be recorded even if the lymph node regresses to less than 10 mm during the clinical trial (measured in the same anatomical plane as the baseline examination). To qualify for CR, each node can achieve a short axis of <10 mm. For PR, SD, and PD, the actual short-axis measurements of the nodes are included in the sum of the target lesions.

[0366] Target lesions that are "too small to measure" All lesions (nodular and non-nodular) recorded at baseline can have their actual measurements recorded at each subsequent evaluation, even if they are very small (e.g., 2 mm). If the radiologist opines that a lesion has disappeared, the measurement can be recorded as 0 mm. If a lesion is thought to be present but is only faintly visible and too small to measure, a default value of 5 mm can be assigned.

[0367] Lesions that split or coalesce during treatment If a non-nodular lesion fragments, the longest diameters of the fragmented parts can be added together to calculate the total of the target lesions. Similarly, when lesions coalesce, the planes between them that were useful for obtaining diameter measurements of each individual lesion can be maintained. If the lesions are truly coalesced such that they can no longer be separated, the vector of the longest diameter can be the maximum longest diameter of the "coalesced lesion". [Table 24]

[0368] New lesions Findings of new lesions can be clear (i.e., not due to differences in scan techniques, changes in imaging modalities, or findings that are thought to represent something other than a tumor such as a "new" healing bone lesion). Lesions identified in follow-up trials at anatomical locations not scanned at baseline are considered new lesions and indicate disease progression. If a new lesion was ambiguous, continued therapy and follow-up evaluations clarified whether it represented a truly new disease. If this is confirmed to be an unmistakably new lesion on repeated scans, the date of the first scan can be used to declare progression.

[0369] Best overall response The best overall response is the best response recorded from the start of treatment until disease progression / recurrence (for progressive diseases, based on the minimum measurements recorded after the start of treatment). The best overall response assignment for a subject depends on the findings for both the target and non-target diseases, taking into account the appearance of new lesions. Additionally, depending on the nature of the clinical trial, confirmatory measurements may be required. Specifically, in non-randomized trials where response is the primary endpoint, confirmation of PR or CR is preferred for any response to be considered a "best overall response". Table 13 provides a summary of the calculation of the overall response status at each time point for subjects with baseline measurable disease.

Table 25

[0370] Best response determination for trials requiring confirmation of CR or PR: Complete or partial response can only be claimed if each criterion is confirmed by repeated assessment at least 4 weeks later. In this situation, the best overall response can be interpreted as shown in Table 14.

Table 26

[0371] Confirmatory measurement / response duration Confirmation To assign a PR or CR status, changes in tumor measurements can be confirmed by repeated assessment preferably performed 4 weeks after the response criteria were first met. In the case of SD, follow-up measurements are preferably at least 1 SD criterion after study entry at a minimum interval of 7 weeks.

[0372] Duration of overall response The duration of the overall response was measured from the time (whichever was first recorded) that the criteria for CR or PR were met until the first date that recurrent or progressive disease was objectively documented (using the smallest measurement recorded since treatment initiation as the criterion for progressive disease). The duration of overall CR was measured from the time the criteria for CR were first met until the first date that recurrent disease was objectively documented.

[0373] Duration of stable disease Stable disease was measured from treatment initiation until the criteria for progression were met, using the smallest measurement recorded since treatment initiation as the criterion.

[0374] Example 4: Appendix 2 mFOLFIRINOX and Approved Dose Modifications The guidelines for approved dose modifications of mFOLFIRINOX during the trial are described in this appendix. All other information related to mFOLFIRINOX treatment is described through the protocol.

[0375] Rules for Dose Omissions and Modified Schedules Dose modifications for mFOLFIRINOX treatment are outlined below. Toxicity is graded according to CTCAE v5.0. Dose adjustments are made according to the system showing the highest degree of toxicity. For hematologic and non-hematologic toxicities, the dose is reduced by one level at a time.

[0376] · For each component of mFOLFIRINOX, three levels of dose modification are permitted according to the following criteria (Table 15). · If toxicity requiring a dose change occurs after the third dose reduction of any component, no additional dose reduction is permitted. However, further treatment can be discussed with the medical monitor.

Table 27

[0377] If treatment is continued continuously for more than 4 weeks due to treatment-related toxicity, the patient may discontinue mFOLFIRINOX and mitazalimab treatment. However, if the patient is clinically benefiting at the end of the 4-week hold, the principal investigator of the clinical trial may contact the medical monitor to potentially continue protocol-based therapy. Patients who discontinue the trial treatment may come to the hospital for the end-of-treatment visit and proceed to the post-treatment follow-up period. After the end-of-treatment visit, the patient will receive recommendations regarding standard care treatment from the principal investigator of the clinical trial.

[0378] Decisions regarding the need for dose modification of 5-FU, irinotecan, oxaliplatin, and / or leucovorin can be made based on the guidelines by the system outlined below. Except as shown below, the management of toxicity and supportive therapy can be carried out according to the judgment of the principal investigator of the clinical trial.

Table 28

[0379] If there is a delay in treatment due to hematological toxicity including neutropenia, the use of prophylactic G-CSF is recommended. G-CSF can be initiated for the first time 24 hours after the end of cytotoxic chemotherapy. Other hematological toxicities do not require dose modification. However, red blood cell transfusion may be considered according to hemoglobin < 9.5 g / dL or significant symptoms of anemia or the facility's guidelines.

Table 29

Table 30

[0380] Regarding diarrhea (and / or abdominal cramps) that occurs at any time during the treatment cycle, patients should be instructed to take an antidiarrheal agent such as loperamide (2 mg every 2 hours until diarrhea resolves for 12 hours; 4 mg is permitted every 4 hours at night) or diphenoxylate / atropine (Lomotil) for the treatment of diarrhea. For the treatment of persistent diarrhea (i.e., lasting more than 48 hours) with broad-spectrum antibiotics, fluoroquinolones are administered orally for 7 days. In cases of severe diarrhea, hospitalization for parenteral rehydration and a change to intravenous antibiotics should be considered. Acute diarrhea and abdominal cramps that occur during or within 24 hours after irinotecan administration can occur as part of the cholinergic syndrome. For irinotecan-related cholinergic reactions, the infusion time can be increased to relieve these symptoms, and prophylactic atropine is permitted according to the facility's guidelines.

[0381] Dose Modification for Drug-Related Hepatotoxicity For all hepatobiliary toxicities, withhold treatment and evaluate for non-drug causes, such as biliary obstruction / stent malfunction (see also Example 7). Once the underlying etiology is corrected and improved, resume treatment (5-FU and oxaliplatin only without irinotecan) at the previous dose level and add irinotecan when the toxicity improves to ≤ Grade 1.

[0382] For hyperbilirubinemia, the following dose modification guidelines can be used. 1) Grade 2 and Grade 3 hyperbilirubinemia: Omit irinotecan until Grade ≤ 1 and resume at the same dose level; 2) Grade 4 hyperbilirubinemia: Hold therapy until ≤ Grade 1 and resume at the next dose level once the underlying etiology is corrected*.

[0383] *Note: If the etiology of hyperbilirubinemia is biliary obstruction (i.e., reversible and non-therapy related), discussion with the medical monitor may allow the option to continue irinotecan at the original level once liver function tests resolve to ≤ Grade 1.

[0384] Dose modification for mucositis Mucositis as toxicity is caused by 5-FU. When grade 3-4 toxicity occurs, continuous 5-FU IV infusion can be reduced by 25% for the remaining course.

[0385] Dose modification for peripheral neuropathy In the case of peripheral neuropathy, only oxaliplatin is modified: 1) Grade peripheral neuropathy: Continue monitoring at the same dose level; 2) Grade 2 peripheral neuropathy lasting more than 14 days: Reduce oxaliplatin by 1 dose level; 3) Grade 3 peripheral neuropathy: If G3 peripheral neuropathy lasts more than 14 days, reduce the oxaliplatin dose to 65 mg / m 2 and discontinue oxaliplatin; 4) Grade 4 peripheral neuropathy: Discontinue oxaliplatin, continue 5-FU, irinotecan, and leucovorin at the same dose level, and if resolved to grade 1, therapy can be restarted on a case-by-case basis after discussion with the medical monitor.

[0386] Hand-foot syndrome Hand-foot syndrome as toxicity is caused by 5-FU. When grade 3-4 toxicity occurs, continuous 5-FU IV infusion can be reduced by 25% for the remaining course.

[0387] Other clinically significant non-hematological * toxicities (excluding alopecia and grade 3 nausea and vomiting that respond to medical treatment within 72 hours)

Table 31

[0388] For peptic ulcers, regardless of whether there is bleeding, 5-FU is retained until the symptoms are resolved. Acute pharyngeal sensory disturbances are caused by oxaliplatin and can be managed according to the practice of facilities that may include extending the duration of oxaliplatin infusion to 6 hours and infusing 1 g of calcium gluconate and 1 g of magnesium sulfate over 15 minutes before all subsequent oxaliplatin infusions.

[0389] Dose Modifications for Infusion-Related Reactions In the case of infusion-related reactions to any chemotherapy component administered according to the protocol, it is possible to follow either the facility's guidelines or those described below. Infusion reactions are defined according to the National Cancer Institute CTCAE (version 5.0) definitions of allergic reactions or anaphylaxis as described below.

Table 32

[0390] Cardiotoxicity In the case of angina, such as angina pectoris or myocardial infarction, 5-FU treatment can be discontinued.

[0391] Extravasation Serious reactions due to extravasation of irinotecan or oxaliplatin have been reported

[39] .

[0392] General recommendations in case of extravasation are as follows: - Immediately stop the infusion, - Do not remove the needle or catheter, - Aspirate the maximum amount of infiltrated product through the needle, - Apply ice to the infiltration area for 15 - 20 minutes every 4 - 6 hours for a period of 72 hours, - Apply topical corticosteroid therapy.

[0393] Check the infiltration site regularly during the next day to confirm whether further treatment is necessary. If in doubt, do not hesitate to require a surgical consultation.

[0394] Example 5: Gemcitabine + nab-Paclitaxel Administration Including Administration Schedule, Clinic Visit Evaluation Form, and Potential Dose Modifications (if applicable) Note that gemcitabine + nab-paclitaxel treatment is applicable to the study only if the Data Review Committee (DRC) makes a formal decision to change chemotherapy mFOLFIRINOX to gemcitabine + nab-paclitaxel during Part 1 of the study.

[0395] Dose Modification Adjust the dose according to the system showing the highest degree of toxicity. For hematological and non-hematological toxicities, the dose may be reduced by one level at a time.

[0396] According to the following criteria, at least two levels of dose modification are allowed for each drug. See Table 16. If toxicity requiring dose modification occurs after the second or further dose reduction of either drug, gemcitabine and nab-paclitaxel, no additional dose reduction is permitted.

Table 33

[0397] If treatment is withheld for > 3 weeks consecutively due to treatment-related toxicity, the patient may discontinue all investigational treatment. However, if the patient is clinically benefiting at the end of the 3-week hold, the treating physician may contact the medical monitor to potentially extend the therapy. Patients who discontinue investigational treatment may proceed to end-of-treatment visits and the post-treatment follow-up period according to Tables 21 and 22, respectively. In situations where toxicity justifies discontinuation of a drug, it is only necessary to remove the individual causative drug from the regimen and treatment can continue according to the protocol. Decisions regarding the need for dose modification of nab-paclitaxel and / or gemcitabine can be made based on the following guidelines.

Table 34

[0398] In cases of delay in treatment due to hematologic toxicity including neutropenia, the use of prophylactic G-CSF is recommended. G-CSF can be initiated for the first time 24 hours after the end of cytotoxic chemotherapy. If the hematologic toxicity is limited to only platelet count, dose modification of only gemcitabine can be considered. Other hematologic toxicities do not necessarily require dose modification. However, red blood cell transfusions can be considered according to hemoglobin < 9.5 g / dL or significant symptoms of anemia or institutional guidelines.

Table 35

[0399] All other ≥ Grade 3 non-hematologic toxicities (*excluding nausea, vomiting, alopecia, and pulmonary embolism, and specific adverse events of special interest (AESI) described below).

[0400] · Hold the dose of one or both drugs until improvement to ≤ Grade 1 · Resume at the next lower dose level.

[0401] Hepatotoxicity and Dose Reduction of Gemcitabine + Nab-Paclitaxel · If the increase in AST or ALT or both is less than 5 × ULN, gemcitabine can be continued without dose reduction. · If the increase in AST or ALT or both is more than 5 × ULN but less than 20 × ULN, the gemcitabine dose can be reduced by 25%. · If the increase in AST or ALT or both exceeds 20 × ULN, gemcitabine can be discontinued.

[0402] Dosing Schedule The first treatment cycle with mitazalimab and gemcitabine + nab-paclitaxel lasts for 35 days. Mitazalimab is administered on days 1, 10, and 24, and gemcitabine + nab-paclitaxel is administered on days 8, 15, and 22. During the next 28-day treatment cycle, mitazalimab is administered on days 3 and 17, and gemcitabine + nab-paclitaxel is administered on days 1, 8, and 15. Up to 6 treatment cycles are permitted for the combination of mitazalimab and gemcitabine + nab-paclitaxel. See Figure 4.

Table 36-1

Table 36-2

Table 36-3

Table 36-4

Table 37-1

Table 37-2

Table 37-3

Table 38-1

Table 38-2

Table 38-3

Table 39

[0403] Example 6: Calculation of Glomerular Filtration Rate in Appendix 4 The glomerular filtration rate (GFR) can be estimated based on a commonly used and accepted formula, i.e., one of the following formulas.

[0404] Cockcroft Gault formula:

Table 40

[0405] Modification of Diet in Renal Disease (MDRD) formula:

Table 41

[0406] Modification of the MDRD formula:

Table 42

[0407] Example 7: Appendix 5 Drug-induced Liver Injury (DILI) The changes in liver test parameters can be further evaluated using the following procedure: Procedure Repeat the following clinical tests: ALT, AST, and bilirubin (total and direct) - within 48 to 72 hours. If an increase in total bilirubin > 2 times the ULN combined with an increase in ALT and / or AST > 3 times the ULN is confirmed (when normal values at baseline / screening), or if an increase in total bilirubin > 2 times the ULN combined with an increase in ALT and / or AST > 5 times the ULN is confirmed (when elevated values at baseline / screening), the results of the following test parameters should be made available to the principal investigator and the sponsor of the clinical trial as soon as possible.

[0408] In addition, the following are obtained: · Current symptoms and concurrent diagnoses, as well as a detailed history of the medical history; · History of concomitant medications used (including over-the-counter medications, herbal and nutritional supplements), alcohol use, use of recreational drugs, and special diets; · History of exposure to environmental chemicals (considering exposure at home and work).

[0409] Provide abdominal ultrasound or other appropriate imaging to rule out pathologies in the biliary tract, pancreas, or liver, such as bile duct stones or neoplasms.

[0410] Clinical Chemistry Alkaline phosphatase, albumin, PT or INR, CK, CK-MB, ceruloplasmin, α-1 antitrypsin, transferrin, amylase, lipase, fasting glucose, cholesterol, triglyceride, glutamate dehydrogenase, D-dimer, C-reactive protein, gamma-glutamyl transpeptidase.

[0411] Serology Hepatitis A (RNA), Hepatitis B (HbsAg, anti-HB, DNA), Hepatitis C (anti-HCV, RNA), Hepatitis D (anti-IgM, anti-IgG), Hepatitis E (anti-HEV, anti-HEV IgM, RNA if anti-HEV IgM is positive), Cytomegalovirus (repeated CMV DNA), anti-smooth muscle antibody (titer), anti-nuclear antibody (titer), anti-LKM (liver-kidney microsome) antibody, anti-mitochondrial antibody.

[0412] Hormone Thyroid-stimulating hormone.

[0413] Hematology White blood cell count + differential, hemoglobin, platelets.

[0414] If AST / ALT remains elevated and no cause likely to result in elevation in previous tests is indicated, the following tests can be performed. Epstein-Barr virus (VCA IgG, VCA IgM), Herpes simplex virus (IgG, IgM), Varicella (IgG, IgM), Parvovirus (IgG, IgM), Toxoplasmosis (IgG, IgM).

[0415] Perform tests for ALT, AST, and total bilirubin (fractionated by total and direct bilirubin) at least weekly until laboratory ALT and / or AST abnormalities stabilize or return to normal, and then initiate close observation of the patient according to the protocol. Follow-up can be based on medical judgment and the criteria for clinical trials of pharmaceuticals (GCP) according to further laboratory changes or specified additional parameters.

[0416] Example 8: Antitumor Efficacy of Mitazalimab and Chemotherapy (FOLFIRINOX) in the Preclinical Tumor Model MB-49 Resistant to Chemotherapy Summary The purpose of this study was to examine the antitumor efficacy of the CD40 antibody mitazalimab in a preclinical chemotherapy-resistant tumor model in addition to chemotherapy (FOLFIRINOX). The combination of mitazalimab and FOLFIRINOX demonstrated a potent antitumor response in mice bearing established FOLFIRINOX-resistant MB-49 tumors. These data support the fundamental concept that the combination of chemotherapy and immuno-oncology is well tolerated and has a very potent antitumor effect in vivo against chemotherapy-resistant cancer cells. The data further validate the potential of mitazalimab in combination with standard-of-care chemotherapy such as FOLFIRINOX.

[0417] Materials and Methods Drugs and Drug Candidates Evaluated Note: All mice received a uniform dose, and the mg / kg doses reported in this document are based on an average mouse weight of 20 g (0.02 kg). [Table 43] *Combined, these four components constitute the chemotherapy known as FOLFIRINOX. **Vehicle for mitazalimab and FOLFIRINOX.

[0418] Administration of FOLFIRINOX Since FOLFIRINOX is composed of four individual components, it was administered in a total of 3 cycles over a 2-day period. Mice were first administered oxaliplatin, followed by irinotecan, and the next day folic acid was administered, and 2 hours later, the mice were administered 5-fluorouracil.

[0419] Administration of Mitazalimab Three days after the start of FOLFIRINOX administration, mitazalimab was administered in 5% glucose (dextrose buffer) at 5 mg / kg.

[0420] Dosing Regimen Figure 5 outlines the dosing regimens applied to two efficacy trials that detail the number of doses and their relationships to each other.

[0421] Cell line Scandion Oncology generated an MB49 cell line resistant to the three cytostatic components of the combination chemotherapy FOLFIRINOX (irinotecan, oxaliplatin, and 5-fluorouracil). Two chemotherapy-resistant MB49 bladder cancer cell lines were produced, namely, MB49-FOLFIRINOX-Novel and MB49-FOLFIRNINOX-Acquired (MB49-FFX-ACQ). Methods for generating chemotherapy-resistant cell lines (such as MB49) are known to those skilled in the art (as described in Amaral et al., 2019, Establishment of Drug-resistant Cell Lines as a Model in Experimental Oncology: A Review, Anticancer Research, 36: 6443-6455). The novel cell line was later ignored for in vivo titration due to poor growth in culture, and the experiments described in this report were conducted on the MB49 cell line with acquired chemotherapy resistance.

[0422] The MB49 mouse bladder cancer cell line was used as the starting point for the FOLFIRINOX resistance model. Resistance can be established using the following approach: 1. Acquired resistance: Cancer cells were exposed to gradually increasing concentrations of the drug until a resistant cell population was generated. Based on previous experience (PMID: 26801902, 25759163, 25596703; Jandu et al., 2016, Jensen et al., 2015, and Hansen et al., 2015), starting concentrations below 50 times the IC50 value of each drug were used, and the difference in the IC50 of each drug was gradually increased 2-fold with each third passage of the cells (approximately every 3 weeks) until it increased by at least 5-fold compared to drug-sensitive cells. This process resulted in an MB49 cell line with increased resistance to FOLFIRINOX. 2. New resistance: Cancer cells were exposed to initial high drug concentrations that varied from 10-fold below to 10-fold above the IC50 value of each drug. The aim of this approach was to kill most cancer cells. However, some cancer cells with "new" resistance survived and were then cultured and expanded to establish a drug-resistant cell population.

[0423] Initially, only MB49-FFX-ACQ cultured without FOLFIRINOX before cell banking was titrated in vivo (ELN 148285), and later a small-scale follow-up titration was added to enable comparison with cell lines grown with FOLFIRINOX before master cell banking (ELN 148525).

[0424] Experiments in hCD40tg mice with tumors The hCD40tg mouse strain on a C57Bl / 6 background was previously generated by Alligator Bioscience (DOCID-1084249735-8226; Mangsbo et al., 2015, The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T-cell-dependent tumor immunity, Clin Cancer Res; 21(5):1115-26).

[0425] On day 0, female hCD40tg mice, 10 - 12 weeks old, were subcutaneously injected in the right posterior flank with 0.4×10 6 MB49-FOLFIRINOX-ACQ in a volume of 100 μl of PBS. The cells were cultured either with FOLFIRINOX (Experiment 1) or without FOLFIRINOX (Experiment 2) before inoculation into the mice. Controls or antibodies were administered as outlined in Table 24, and the mice were monitored immediately after each treatment to detect potential signs of toxicity. Tumor volume was measured three times a week with calipers and calculated as follows: ((width / 2 × length / 2 × height / 2) × 4π / 3). The mice were weighed weekly, and a weight loss of more than 25%, as well as a 3Tumor volume exceeding, tumor ulceration, or compromised health were regarded as ethical evaluation items.

Table 44

[0426] Statistical analysis For survival duration, Kaplan–Meier and log-rank (Mantel–Cox) were used, and for FACS data and tumor growth, Mann–Whitney, non-parametric, two-sided was used to analyze the data collected using the GraphPad Prism program (*, p < 0.05; **, p < 0.01).

[0427] Results Test using MB49-FFX-ACQ co-cultured with FOLFIRINOX MB49-FFX-ACQ cells were added with 0.3 μM of 5-fluorouracil, 4.5 μM of oxaliplatin, and 0.12 μM of irinotecan (SN38) to the culture medium and grown up to 3 passages before master cell banking.

[0428] Figure 7A shows that FOLFIRINOX reduces tumor growth in chemosensitive tumors. Figure 7B shows no effect of FOLFIRINOX in chemoresistant tumors. Figure 7C shows the synergistic effect of mitazalimab with FOLFIRINOX in chemoresistant tumors.

[0429] The combination of mitazalimab + FOLFIRINOX induced statistically significant tumor growth inhibition and tumor-bearing mice with healed tumors compared to the vehicle (complete responders: 5 / 10 for mitazalimab + FOLFIRINOX). See Figures 6 and 8.

[0430] Evaluation and conclusion In the conducted efficacy test, the combination of mitazalimab and FOLFIRINOX showed a strong anti-tumor response in mice with established FOLFIRINOX-resistant MB49 tumors. These data support the basic concept that the combination of chemotherapy and immuno-oncology is well-tolerated and has a very strong anti-tumor effect in vivo against chemotherapy-resistant cancer cells. The data further validate the potential of mitazalimab in combination with standard care chemotherapy such as FOLFIRINOX.

[0431] Example 9: Safety data from OPTIMIZE-1, a Phase 1b / 2 trial of mitazalimab in combination with mFOLFIRINOX in patients with metastatic pancreatic ductal adenocarcinoma (PDAC): Mitazalimab 900 μg / kg was determined to be the safe and recommended dose for the second phase (RP2D) part of the trial. Mitazalimab is an IgG1 antibody of a human CD40 agonist developed as a cancer immunotherapy. Targeting CD40 kick-starts the cancer immune cycle by licensing DCs that lead to tumor-specific T cell priming and activation. Furthermore, in PDAC, CD40 agonists activate myeloid cells, promote the degradation of the desmoplastic tumor stroma, and improve the influx of T cells and chemotherapeutic agents into the tumor.

[0432] In a Phase I trial (NCT02829099), mitazalimab has been shown to have signs of clinical activity, be safe, and well-tolerated (at doses up to 1200 μg / kg) in solid tumors. Most drug-related adverse events (AEs) were Grade 1 or 2.

[0433] OPTIMIZE-1 (NCT04888312) is a Phase 1b / 2 open-label, multi-center trial designed to evaluate the safety, tolerability, and efficacy of mitazalimab in combination with mFOLFIRINOX in adults diagnosed with previously untreated metastatic PDAC.

[0434] The objective of the first (Phase 1b) part of the trial was to determine the RP2D of mitazalimab + mFOLFIRINOX. Mitazalimab was escalated from 450 μg / kg to 900 μg / kg according to a Bayesian optimal interval design with at least 3 patients enrolled per dose level. In the first 21-day treatment cycle (dose-limiting toxicity assessment period), mitazalimab was administered intravenously on Days 1 and 10, and mFOLFIRINOX was initiated on Day 8. In the second and subsequent cycles, treatment followed a 14-day cycle schedule with mitazalimab administered 2 days after mFOLFIRINOX.

[0435] In Part 2 (Phase 2) of the trial, mitazalimab at the RP2D was administered in combination with mFOLFIRINOX. The primary endpoint was the overall response rate as defined by RECIST. Progression-free survival and overall survival were evaluated as secondary endpoints.

[0436] In this Phase 1b (dose escalation) part of the trial, 11 patients were treated with mitazalimab: 5 at 450 μg / kg and 6 at 900 μg / kg mitazalimab doses. One patient in the 900 μg / kg dose cohort dropped out of the trial for administrative reasons after the first mitazalimab infusion, prior to receiving mFOLFIRINOX, and was not included in the RP2D determination. Key baseline characteristics included: 7 females, 4 males; median age 63 (range 57–70); ECOG 0–1; median time from PDAC diagnosis 25 days.

[0437] Mitazalimab-related AEs were reported in 9 / 11 patients. Treatment-related AEs that occurred in >1 patient were fever (60%), myalgia (50%), and fatigue (20%). At a dose of 450 μg / kg, all mitazalimab-related AEs were grade 1-2. At a dose of 900 μg / kg, 4 patients (67%) experienced grade 1-2 mitazalimab-related AEs. One patient at the 900 μg / kg dose experienced grade 3 fatigue and grade 3 headache related to mitazalimab, which led to treatment discontinuation after the first cycle. There were no grade 4 or 5 mitazalimab-related AEs. 1 / 10 patients required a reduction in the mFOLFIRINOX dose, and at the cutoff date, the range of treatment duration was 1-4 weeks.

[0438] Mitazalimab in combination with mFOLFIRINOX was safe and well tolerated. As the RP2D, a dose of 900 μg / kg of mitazalimab was selected, and patient enrollment at the RP2D is ongoing.

[0439] OPTIMIZE-1 Safety Summary DLT: · SC-12(001-009): G3 headache (related to mitazalimab). 59-year-old female with h / o NSCLC; progression on C4 scan and new lung lesions. Outside of the trial.

[0440] SAE: · SC-01(002-001): SAE G3 new lung cancer (unrelated). 60-year-old female with h / o NSCLC; progression on C4 scan and new lung lesions. Outside of the trial.

[0441] · SC-05(103-001): SAE G3 anorexia (unrelated). 65-year-old male with h / o abdominal pain, ascites. Hospitalization for pain management. Resolved. Trial treatment is ongoing.

[0442] · SC-06(001-001): Inactivated SAE G2 change in general status. 66-year-old male. Another cause: progression.

[0443] · SC-14 (001 - 011): Supraventricular tachycardia (unrelated) on SAE G3. 60-year-old female. Resolved. Trial treatment is in progress.

[0444] AESI: · None (infusion-related reaction ≥ G2, cytokine release syndrome ≥ G2, LFT ≥ 5 × ULN, bilirubin ≥ 1.5 × ULN).

[0445] Related to mitazalimab (in 11 patients at 2 dose levels): · Grade 3: Fatigue (2), headache (1), hypokalemia (1).

[0446] · Grade 1 - 2 (the most common): Fever or similar symptoms (6), influenza / myalgia (5), fatigue (3).

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Claims

**Claim 1** A combination therapy for treating cancer, optionally chemotherapy-resistant cancer, in a subject, comprising: a. an antibody or antigen-binding portion thereof that specifically binds to CD40; and b. chemotherapy. **Claim 2** A combination therapy comprising an antibody or antigen-binding portion thereof and chemotherapy for use in a dosing regimen for treating cancer, optionally chemotherapy-resistant cancer, wherein the dosing regimen comprises: a. a step of administering an antibody or antigen-binding portion thereof that specifically binds to CD40; and b. a step of administering chemotherapy. **Claim 3** An antibody or antigen-binding portion thereof that specifically binds to CD40 for treating cancer, optionally chemotherapy-resistant cancer, in a subject, wherein the antibody or antigen-binding portion thereof is for use in combination with chemotherapy. **Claim 4** Use of an antibody or antigen-binding portion thereof that specifically binds to CD40, wherein: a. the antibody or antigen-binding portion thereof is for use in combination with chemotherapy for treating cancer, optionally chemotherapy-resistant cancer, or b. the use is in the preparation of a medicament for the combination therapy according to claim 1 or 2. **Claim 5** A method for treating cancer, optionally chemotherapy-resistant cancer, in a subject, comprising administering to the subject: a. a therapeutically effective amount of an antibody or antigen-binding portion thereof that specifically binds to CD40 and chemotherapy; b. a therapeutically effective amount of the combination therapy according to claim 1; or c. the combination therapy according to claim 2. **Claim 6** A pharmaceutical composition comprising an antibody or antigen-binding portion thereof that specifically binds to CD40 and chemotherapy. **Claim 7** A kit comprising an antibody or antigen-binding portion thereof that specifically binds to CD40 and chemotherapy. **Claim 8** Optionally, the cancer, optionally chemotherapy-resistant cancer, is pancreatic cancer, Optionally, the pancreatic cancer is an exocrine tumor such as exocrine gland cancer or pancreatic ductal adenocarcinoma (PDAC), or an endocrine tumor, and the combination therapy according to claim 1 or 2, the use according to claim 3 or 4, the treatment method according to claim 5, the pharmaceutical composition according to claim 6, or the kit according to claim 7. **Claim 9** The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, wherein the chemotherapy is selected from the group consisting of FOLFIRINOX or a variant thereof (such as mFOLFIRINOX), gemcitabine, nab-paclitaxel, and combinations thereof.

10. The combination therapy, use, method, pharmaceutical composition, or kit according to claim 9, wherein the chemotherapy is FOLFIRINOX or a variant thereof, and the antibody or antigen-binding portion thereof is mitazalimab.

11. The FOLFIRINOX or a variant thereof is a. Oxaliplatin (such as Elotatin (registered trademark) or a generic drug) infusion (optionally, the oxaliplatin is i. 85 mg / m ２ at a dose of ii. Intravenous and / or iii. Administered over 2 hours), b. Folinate (such as leucovorin, calcium folate, calcium levofolinate, disodium folate, and disodium levofolinate) infusion (optionally, the leucovorin is i. 400 mg / m ２ at a dose of ii. Intravenous and / or iii. Administered over 2 hours), c. Irinotecan (such as Camptosar (registered trademark)) infusion (optionally, the irinotecan is i. At a dose of 150 mg / m ２ , ii. Intravenous and / or iii. Administered 30 minutes after the end of the leucovorin infusion), and / or d. 5-Fluorouracil infusion (optionally, the 5-fluorouracil is i. 2400 mg / m ２ at a dose of ii. Intravenous and / or iii. A duration of 46 to 48 hours (e.g., 2.4 g / m ２ / day), administered), the combination therapy, use, method, pharmaceutical composition, or kit according to claim 10.

12. The combination therapy, use, method, pharmaceutical composition, or kit according to claim 11, wherein the oxaliplatin is administered over 2 hours, immediately followed by the folinate (such as leucovorin) administered over 2 hours, the irinotecan is administered starting 30 minutes after the start of the leucovorin and over 90 minutes, and then the 5-fluorouracil is administered over 46 to 48 hours.

13. a. Premedication (optionally, the premedication is i. An NK1 receptor antagonist, such as aprepitant, 125 mg PO, 60 minutes before infusion and / or continuously during the day of chemotherapy, ii. A 5-HT3 receptor antagonist, such as ondansetron, 8 mg PO, 30 minutes before infusion and / or continuously during the day of chemotherapy, and / or iii. A corticosteroid, such as dexamethasone, 8 mg IV or PO, 30 minutes before infusion), and / or b. Post - dosing (optionally, the post - dosing is i. G - CSF, such as Neulasta, 6 mg SC, on the 4th day after the start of the FOLFORINOXX regimen or its variant and / or at least 24 hours after the end of continuous infusion of the 5 - fluorouracil, and is further included in the combination therapy, use, method, pharmaceutical composition, or kit according to claim 11 or 12).

14. The antibody or its antigen - binding portion is a. 50 μg / kg to 1200 μg / kg, for example, 450 μg / kg to 900 μg / kg, b. 50 μg / kg, 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1000 μg / kg, 1050 μg / kg, 1100 μg / kg, 1150 μg / kg, 1200 μg / kg, or more, c. 450 μg / kg, and / or d. administered at a dose of 900 μg / kg, in the combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims.

15. The antibody or its antigen - binding portion is administered more than once, in the combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims.

16. The antibody or its antigen - binding portion has the following CDRs: V Ｌ CDR1: CTGSSSNIGAGYNVY [SEQ ID NO: 1], V Ｌ CDR2: GNINRPS [SEQ ID NO: 2], V Ｌ CDR3: CAAWDKSISGLV [SEQ ID NO: 3], V Ｈ CDR1: GFTFSTYGMH [SEQ ID NO: 4], V Ｈ CDR2: GKGLWLSYISGGSSYIFYADSVGR [SEQ ID NO: 5], and V Ｈ The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, comprising CDR3: CARILRGGSMDL [SEQ ID NO: 6].

17. The antibody or its antigen - binding portion is a. the light - chain variable region of SEQ ID NO: 7 and / or the heavy - chain variable region of SEQ ID NO: 8, b. the light - chain constant region of SEQ ID NO: 11 and / or the heavy - chain constant region of SEQ ID NO: 12, or c. the light - chain of SEQ ID NO: 7 and SEQ ID NO: 11, and / or the heavy - chain of SEQ ID NO: 8 and SEQ ID NO: 12, in the combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims.

18. The antibody or its antigen - binding portion that specifically binds to CD40 includes, or consists of, an intact antibody, such as an IgG1 antibody, in the combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims.

19. The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, wherein the antibody or antigen-binding portion thereof comprises or consists of an antigen-binding fragment selected from the group consisting of Fv fragments (such as single-chain Fv fragments or disulfide-bonded Fv fragments) and Fab-like fragments (such as Fab fragments, Fab' fragments, or F(ab)2 fragments).

20. The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, wherein the antibody or antigen-binding portion thereof is human or humanized.

21. The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, wherein the antibody or antigen-binding portion thereof and the chemotherapy are administered simultaneously, sequentially, or subsequently to each other.

22. The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, wherein the antibody or antigen-binding portion thereof and / or the chemotherapy is administered locally to the tumor site.

23. The combination therapy, use, method, pharmaceutical composition, or kit according to any one of the preceding claims, wherein the antibody or antigen-binding portion thereof is administered on multiple separate occasions and the chemotherapy is administered continuously for the duration of the method.

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