Combination therapy

Abstract

The present invention relates to combination therapies 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) a chemotherapy. The 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 treating cancer, such as chemotherapy-resistant cancer. Background Art

[0002] Introduction to Pancreatic Ductal Adenocarcinoma

[0003] Pancreatic ductal adenocarcinoma (PDAC) is a type of exocrine pancreatic cancer. It is the most common type of pancreatic cancer, accounting for 95 of 100 (95%) cases of all pancreatic cancers. In the United States and the European Union, pancreatic cancer is the fourth leading cause of cancer death [1]. The incidence of pancreatic cancer is highest in Europe (7.7 per 100,000 people) and North America (7.6 per 100,000 people), followed by Oceania (6.4 per 100,000 people) [2]. In the United States, there are an estimated 57,600 new cases and an estimated 47,050 deaths in 2020 (Cancer facts and figures, American Cancer Society, 2020). The incidence rate is slightly higher in men than in women.

[0004] The five-year life expectancy for pancreatic cancer is approximately 5%, a figure that has not changed in the past two decades. Surgical resection is the only treatment option, but at most one in five patients is considered operable, and even in these cases, the five-year survival rate is approximately 20%. More than three-quarters of patients present with advanced disease, approximately half have distant metastases at diagnosis, and the remaining quarter have inoperable localized disease. Over time, most of these locally advanced cancers also metastasize[3].

[0005] Therefore, there is a need for effective treatments for metastatic pancreatic cancer. Until the last decade, the standard first-line treatment for metastatic pancreatic cancer was gemcitabine, which produced a median overall survival (mOS) of approximately 6 months. In 2011, a major advance was made with the introduction of the FOLFIRINOX regimen, which produced an objective response rate (ORR) of 31% and an mOS of 11.1 months in the randomized PRODIGE 4 study of 342 patients, compared with an ORR of 9.4% and an mOS of 6.8 months for gemcitabine monotherapy [4]. Two years later, the MPACT study, which compared gemcitabine plus nab-paclitaxel to gemcitabine, also showed that this combination, with an ORR of 23% and an mOS of 8.5 months, had superior activity relative to gemcitabine monotherapy, with an ORR of 7% and an mOS of 6.7 months [5].

[0006] Mitazalimab, a CD40 agonist

[0007] Mitazalimab (also known as JNJ-64457107 and ADC-1013) is an agonistic human monoclonal (IgG1) antibody that targets CD40. The agent has been investigated in two Phase 1 studies for the treatment of advanced solid tumors: A-14-1013-C-01 (EudraCT No. 2014-004556-56) and JNJ-64457107CAN1001 (EudraCT No. 2016-000969-23).

[0008] Introduction to CD40

[0009] CD40 is a co-stimulatory receptor that belongs to the tumor necrosis factor receptor (TNFR) superfamily [6]. CD40 is expressed in many 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 on granulocytes, endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells [6-8]. CD40 is also present on the membranes of a variety of malignant cells, including non-Hodgkin's and Hodgkin's lymphomas, myelomas, and some cancers, including nasopharyngeal carcinoma, bladder cancer, cervical cancer, renal cancer, and ovarian cancer [6,9]. CD40 interacts with a single ligand, CD40L (or CD154), which 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) [6,7].

[0010] The molecular outcome of CD40 signaling depends on the cell type expressing CD40 and the microenvironment providing the CD40 signal

[10] . CD40 ligation and cross-linking are necessary for adaptive immune responses by inducing the upregulation of co-stimulatory receptors and major histocompatibility complex molecules and the production of proinflammatory cytokines, through the "permission" of APCs, and especially DCs. Therefore, CD40 participates in the functional maturation of APCs and thereby in the activation of antigen-specific T lymphocytes [11-13]. CD40 also plays a role in humoral immunity by activating resting B lymphocytes and by increasing their antigen presenting function [10, 14]. In addition, CD40 participates in the induction of innate immunity by stimulating cytotoxic myeloid cells (such as NK cells, macrophages and granulocytes) [10, 14, 15].

[0011] Summary of Nonclinical Data

[0012] The CD40 agonistic properties of mitazalimab have been validated in vitro in human monocyte-derived DC cultures. Ligation of CD40 by mitazalimab results in upregulation of activation markers (such as CD80 and CD86) on the surface of DCs and 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 by upregulating, for example, CD83.

[0013] The agonistic effect of mitazalimab depends mainly on the binding of the Fc part of the antibody to the Fcγ receptor (FcγR). When a non-glycosylated variant of mitazalimab containing the N297Q mutation in the γ chain and thus not binding to FcγR was used, the ability of mitazalimab to activate DC (i.e., upregulate CD86) was significantly reduced. After cross-linking of this non-glycosylated mitazalimab variant with anti-human IgG, the CD40 agonistic effect was restored again, conclusively demonstrating that mitazalimab depends on FcγR cross-linking for optimal activity. Mitazalimab showed immune-mediated and antibody-dependent cell-mediated cytotoxicity (ADCC)-dependent in vivo antitumor efficacy in human tumors transplanted into NSG mice and human CD40 transgenic (hCD40tg) mice. In addition, mitazalimab induced T cell-dependent and tumor-specific immune memory against bladder cancer cells in hCD40tg mice, and immunity to tumor re-challenge lasted for at least 5 months. In experimental tumor models in hCD40tg mice, mitazalimumab also showed synergistic enhancement of tumor growth and survival when combined with other immunotherapies, such as immune checkpoint inhibitors (e.g., PD-1), vaccination, or chemotherapy (e.g., mFOLFIRINOX). For additional and detailed information on nonclinical data, see the mitazalimumab Investigator's Brochure.

[0014] Summary of clinical data

[0015] Mitazalimab single-agent study

[0016] First-in-human study using intratumoral administration of mitazalimab

[0017] Study A-14-1013-C-01 (NCT02379741) is a first-in-human, multicenter, non-randomized, open-label, multiple-dose-escalation, Phase 1 study of mirtazalimumab (ADC-1013) in patients with advanced solid malignancies.

[0018] Mitazalimab was administered as an intratumoral bolus injection (intratumoral administration) into 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.

[0019] The maximum tolerated dose (MTD) was not determined. Nine of the 18 patients given intratumoral administration observed an increase in liver enzymes or bilirubin, and three of the five patients given IV administration observed an increase in liver enzymes or bilirubin. Only two patients had normal liver enzyme and bilirubin values ​​at baseline. Two dose-limiting toxicities (DLTs) were observed, one patient had grade 3 abdominal pain, and one patient had grade 3 cholecystitis, both of which were administered intratumorally. The best response for a patient with renal cell carcinoma was stable disease (SD) (400 μg / kg intratumorally). Patients with pancreatic cancer were not enrolled in this study.

[0020] Dose escalation study with intravenous administration of mitazalimab

[0021] Study 64457107CAN1001 (NCT02829099) is a multicenter, nonrandomized, open-label, dose-escalation Phase 1 study of mitazalimumab (JNJ-64457107) in patients with advanced solid tumors. Mitazalimumab was administered as an IV infusion every 14 days at doses ranging from 75 μg / kg to 2000 μg / kg, with corticosteroids included in premedication, and up to 1200 μg / kg in the absence of corticosteroids.

[0022] A total of 95 patients were exposed to mitazalimab. One patient is still receiving treatment. MTD was not identified. Two patients experienced DLT, one patient had a headache lasting 5 days, and one patient had grade 3 liver enzyme elevation and grade 2 bilirubin elevation. Seven pancreatic cancer patients were enrolled in this study, one of whom experienced SD for more than 6 months. Partial responses (PR) were observed in patients with renal cell carcinoma who received fourteen cycles (i.e., 28 doses) at a dose level of 1200 μg / kg, and the study lasted 9.2 months

[16] .

[0023] Pharmacodynamic activity

[0024] Efficacy biomarker data in Study 644577107CAN1001 were investigated following the first administration of mitazalimumab and were consistent with CD40-mediated immune cell activation. Following IV administration of mitazalimumab, marginalization of B cells was observed at all doses tested, with B cell recovery being dose-dependent. NK cells and T cells were also reduced in the peripheral blood following infusion of all doses of mitazalimumab tested except the lowest dose (75 μg / kg). Levels of both cell types were fully restored by study day 8.

[0025] CD40 receptor occupancy was assessed on B cells and was shown to be dose-dependent. CD40 remained engaged on the 8th day after mitazalimab administration. Peripheral levels of MCP-1, IP-10, and MIP-1B chemokines peaked 1 to 4 hours after infusion, which is 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 infusion of mitazalimab. The pattern of biomarker changes is consistent with the proposed mechanism of mitazalimab as a CD40 agonist.

[0026] CD40 agonists administered to patients with pancreatic cancer

[0027] Selumetumab

[0028] Seluzumab (CP-870.893, RO7009789) is a CD40 agonist monoclonal antibody in an IgG2 format that activates CD40 independently of cross-linking to Fcγ receptors

[17] . For agonist CD40 antibodies, it is generally believed that systemic delivery of cross-linking-independent antibodies may increase the risk of toxicity, which may reduce the therapeutic window

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

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

[0029] APX005M

[0030] APX005M is a CD40 agonistic monoclonal antibody with a mutated IgG1 format for improved binding to FcγR2b, whose function is dependent on cross-linking with FcγR. APX005M has a similar profile to mitazalimumab and is designed to improve efficacy and safety compared to prior CD40 antibodies. APX-005M is in clinical development for a variety of malignancies.

[0031] Early clinical data of APX005M in combination with gemcitabine plus nab-paclitaxel with or without nivolumab in patients with previously untreated metastatic pancreatic cancer were presented in 2019, see Table 1. In each 28-day cycle, gemcitabine plus nab-paclitaxel was given three times, nivolumab was given twice, and APX005M was given once. Immune profiles of PBMCs showed remodeling of the bone marrow compartment in response to treatment with rapid activation of DCs in most patients. Analysis of circulating mutant KRAS DNA showed a significant and rapid decline with therapy. Preliminary efficacy data from 24 patients showed encouraging data for these combinations, and randomized studies are continuing

[20] .

[0032] Table 1: APX005M in combination with gemcitabine plus nab-paclitaxel (Gem-NabPac) with or without nivolumab Preliminary efficacy data for Nivolumab

[0033]

[0034] The listing or discussion of an apparently prior-published document in this specification is not necessarily considered an admission that the document is prior art or part of the common general knowledge. Summary of the invention

[0035] Combination chemotherapy for metastatic pancreatic cancer has been pushed to the edge of tolerability, but survival outcomes remain poor. Pancreatic cancer is immunologically classified as a “cold” tumor compared to other tumors characterized by immune infiltration. The desmoplastic stroma that forms around pancreatic cancer, in addition to serving as a physical barrier to chemotherapy, is a host of tumor fibroblasts and suppressive myeloid cells that inhibit immune responses in the tumor microenvironment. In addition, because pancreatic cancer contains relatively few non-synonymous mutations compared to other cancers, tumors are characterized by low expression of tumor neoantigens. These two factors contribute to the lack of activity of checkpoint inhibitors in pancreatic cancer.

[0036] It is therefore an object of the present invention to provide improved combination therapies for the treatment of cancer (eg pancreatic cancer), optionally a chemotherapy-resistant cancer.

[0037] A first aspect of the invention provides a combination therapy for use in treating cancer, optionally a chemotherapy-resistant cancer, in a subject, the combination therapy comprising: an antibody, or antigen-binding portion thereof, that specifically binds to CD40, and chemotherapy.

[0038] A second aspect of the invention provides a combination therapy comprising an antibody, or an antigen binding portion thereof, and chemotherapy, for use in a dosage regimen for treating cancer, optionally a chemotherapy-resistant cancer, wherein the dosage regimen comprises the steps of: (a) administering an antibody, or an antigen binding portion thereof, that specifically binds to CD40, and (b) administering chemotherapy.

[0039] A third aspect of the invention provides an antibody, or antigen binding portion thereof, which specifically binds to CD40 for use in treating cancer, optionally a chemotherapy-resistant cancer, in a subject, wherein the antibody, or antigen binding portion thereof, is for use in combination with chemotherapy.

[0040] In an alternative version of the third aspect, the invention provides a chemotherapy for use in treating cancer, optionally a chemotherapy-resistant cancer, in a subject, wherein the chemotherapy is for use in combination with an antibody, or antigen binding portion thereof, that specifically binds to CD40.

[0041] In some embodiments, the subject may undergo treatment with an antibody, or antigen binding portion thereof, and then be treated with chemotherapy to achieve a combination therapy. In one embodiment, the subject may undergo treatment with chemotherapy, and then be treated with an antibody, or antigen binding portion thereof, to achieve a combination therapy.

[0042] A fourth aspect of the invention provides 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 a chemotherapy-resistant cancer, wherein the antibody or antigen binding portion thereof is for use in combination with chemotherapy.

[0043] In an alternative version of the fourth aspect, the invention provides the use of an antibody, or antigen binding portion thereof, which specifically binds to CD40 in the preparation of a medicament for use in a combination therapy according to the first or second aspect of the invention.

[0044] A fifth aspect of the invention provides a method of treating cancer, optionally a 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.

[0045] In an alternative version of the fifth aspect, the invention provides a method of treating cancer, optionally a 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.

[0046] In a further alternative version of the fifth aspect, the invention provides a method of treating cancer, optionally a chemotherapy-resistant cancer, in a subject, the method comprising administering to the subject a combination therapy according to the second aspect.

[0047] 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 of the foregoing aspects of the invention. The pharmaceutical composition may comprise one or more components of chemotherapy (e.g., 1, 2, 3 or all 4 components of the FOLFIRINOX regimen), and optionally further comprises an antibody or antigen binding portion thereof.

[0048] A seventh aspect of the invention provides a kit comprising an antibody or antigen binding portion thereof that specifically binds to CD40 and a chemotherapy. The kit may comprise the antibody or antigen binding portion thereof and / or the chemotherapy according to any of the preceding aspects of the invention.

[0049] The following paragraphs may relate to any of the aforementioned aspects. Unless the context dictates otherwise, preferences and options for a given aspect, feature or parameter of the invention should be considered to have been disclosed in conjunction with any and all preferences and options for all other aspects, features and parameters of the invention.

[0050] In some embodiments, cancer, optionally chemotherapy-resistant cancer, is pancreatic cancer. In some embodiments, pancreatic cancer is an exocrine tumor, such as exocrine gland cancer or pancreatic ductal adenocarcinoma (PDAC), or an endocrine tumor. In some embodiments, cancer is a solid tumor. Solid tumors can be selected from the group consisting of pancreatic tumors, adenomas, blastomas, carcinomas, desmoid tumors, desmoplastic small round cell tumors, endocrine tumors, germ cell tumors, lymphomas, sarcomas, Wilms tumors, lung tumors, colon tumors, lymphomas, breast tumors, and melanomas.

[0051] In some embodiments, the chemotherapy is selected from the group consisting of FOLFIRINOX or variants thereof (such as mFOLFIRINOX), gemcitabine, nab-paclitaxel, and combinations thereof. In some embodiments, the chemotherapy is FOLFIRINOX or variants thereof, and the antibody, or antigen-binding portion thereof, is mitazalimab.

[0052] In some embodiments, FOLFIRINOX or variants thereof include oxaliplatin (such as or generic drug) infusion, optionally with oxaliplatin at 85 mg / m 2 In some embodiments, FOLFIRINOX or its variants include a folinic acid (such as leucovorin, calcium folinate, calcium leucovorin, disodium folinate, and disodium leucovorin) infusion, wherein folinic acid is administered at 400 mg / m 2In some embodiments, FOLFIRINOX or a variant thereof includes irinotecan (such as ) infusion, with irinotecan at 150 mg / m 2 In some embodiments, FOLFIRINOX or variants thereof include an infusion of 5-fluorouracil, optionally wherein the 5-fluorouracil is administered at 2400 mg / m 2 dose, intravenously, and / or administered for a duration of 46 to 48 hours (e.g., 2.4 g / m 2 / sky).

[0053] In some embodiments, the subject receives at least one treatment cycle of FOLFIRINOX (e.g., mFOLFIRINOX). The treatment cycles may correspond to the OPTIMIZE-1 study (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 receives no more than 12 treatment cycles. In some embodiments, the subject receives 4 treatment cycles. In some embodiments, the subject receives 8 treatment cycles. In some embodiments, the subject receives more than 12 treatment cycles, for example, the subject may receive 13, 14, 15, 16, 17, 18, 19, 20 or more treatment cycles. The number of treatment cycles for the patient can be adjusted based on observations of the responsiveness to the treatment and / or the patient's level of toxicity. Such adjustments are within the physician's remit for the patient, and the readings of responsiveness and / or toxicity are routine matters (such as described herein).

[0054] In some embodiments, oxaliplatin is administered within 2 hours, followed immediately by folinate (eg, leucovorin) within 2 hours; and irinotecan is administered within 90 minutes, starting 30 minutes after starting leucovorin; followed by 5-fluorouracil over 46 to 48 hours.

[0055] In some embodiments, the subject receives premedication. In some embodiments, the subject receives postoperative medication. In some embodiments, the subject receives premedication and postoperative medication. Premedication may include: (i) NK1-receptor antagonists, such as Aprepritant, 125mg PO, 60 minutes before infusion and / or continuously during the days of chemotherapy, (ii) 5-HT3 receptor antagonists, such as Ondansetron, 8mg PO, 30 minutes before infusion and / or continuously during the days of chemotherapy, and / or (iii) corticosteroids, such as dexamethasone, 8mg IV or PO, 30 minutes before infusion. Postoperative medication may include G-CSF, such as Neulasta, 6mg SC, on the fourth day after the start of the FOLFORINOX regimen or its variants, and / or at least 24 hours after the end of the continuous 5-fluorouracil infusion.

[0056] 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 its 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 its 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.

[0057] In some embodiments, the antibody or antigen-binding portion thereof is administered more than once. For example, the antibody or antigen-binding portion thereof can be administered twice, three times, four times, five times, six times, seven times or more in any one or more treatment cycles.

[0058] In some embodiments, the antibody, or antigen-binding portion thereof, comprises the following CDRs:

[0059]

[0060] In some embodiments, the antibody or antigen-binding portion thereof comprises: (a) a light chain variable region of SEQ ID NO:7 and / or a heavy chain variable region of SEQ ID NO:8; (b) a light chain constant region of SEQ ID NO:11 and / or a heavy chain constant region of SEQ ID NO:12; or (c) a light chain of SEQ ID NO:7 plus SEQ ID NO:11, and / or a heavy chain of SEQ ID NO:8 plus SEQ ID NO:12.

[0061] In some embodiments, the antibody, or antigen-binding portion thereof, that specifically binds to CD40 is mitazalimab.

[0062] 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.

[0063] 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-linked Fv fragment) and a Fab-like fragment (such as a Fab fragment; a Fab' fragment or a F(ab)2 fragment).

[0064] In some embodiments, the antibody, or antigen-binding portion thereof, is human or humanized.

[0065] In some embodiments, the antibody, or antigen-binding portion thereof, and the chemotherapy are administered simultaneously, sequentially, or sequentially.

[0066] In some embodiments, the antibody or its antigen binding portion thereof and / or chemotherapy is administered topically to the tumor site. In some embodiments, the antibody or its antigen binding portion thereof and / or chemotherapy is administered systemically. In some embodiments, the antibody or its antigen binding portion thereof is administered topically, and chemotherapy is administered systemically. In some embodiments, chemotherapy is administered topically, and the antibody or its antigen binding portion thereof is administered systemically. In some embodiments, a part of chemotherapy can be administered systemically, and another part is administered topically. Systemic administration can be, for example, intravenous and / or subcutaneous.

[0067] In some embodiments, the antibody, or antigen-binding portion thereof, is administered on multiple different occasions and the chemotherapy is administered continuously for the duration of the method.

[0068] Those skilled in the art will appreciate that the presence of an antibody or antigen binding portion thereof and chemotherapy can provide a synergistic benefit in the treatment of a subject's cancer, optionally a chemotherapy-resistant cancer. By "synergistic" we mean that the therapeutic effect of the combination (e.g., as determined by reference to the growth rate or size of the tumor) is greater than the additive therapeutic effect of the two treatments administered alone. Such synergistic effects can be identified by testing antibodies or antigen binding portions thereof and chemotherapy alone or in combination in relevant cell line models of cancer.

[0069] Optionally, in addition to the antibody, or antigen-binding portion thereof, and the chemotherapy, the combination therapy further includes an additional agent having efficacy in the treatment of cancer.

[0070] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : Study design of a phase 1b / 2, open-label, multicenter study designed to evaluate the safety, tolerability, and efficacy of mitazalimab in combination with chemotherapy in patients with metastatic pancreatic ductal adenocarcinoma.

[0072] Figure 2 : Exemplary dose escalation schedule.

[0073] Figure 3 : Exemplary dosing regimens of mitazalimab and mFOLFIRINOX.

[0074] Figure 4 : Exemplary dosing regimens of mitazalimab and gemcitabine plus nab-paclitaxel.

[0075] Figure 5 : Exemplary dosing regimens of mitazalimab and FOLFIRINOX in mouse studies.

[0076] Figure 6 : Study of tumor volume (A) and survival rate (B) of hCD40tg carrying MB49-FFX-ACQ. Mitazalimab (mita), FOLFIRINOX (FFX).

[0077] Figure 7: A. MB49 tumor-bearing hCD40tg mice treated with FOLFIRINOX (oxaliplatin, irinotecan, folinic acid and 5-fluorouracil) on days 7-8, 14-15 and 21-22 had reduced tumor growth compared to vehicle treatment. B. Chemotherapy-resistant MB49 cells generated by continuous low-dose treatment with FOLFIRINOX in vitro were inoculated in tumor-bearing hCD40tg mice that received treatment with B. FOLFIRINOX alone on days 7-8, 14-15 and 21-22, or C. combined treatment with 100 μg mitazalimab on days 10, 17 and 24 (**, p<0.01 mitazalimab + FOLFIRINOX vs. vehicle, Mann-Whitney, non-parametric, 2-tailed).

[0078] Figure 8 : Duration of treatment for some patients (denoted as SC-##). Patient SC-13 withdrew from the trial for administration reasons after the first infusion of mitazalimab but before receiving mFOLFIRINOX and was not included in the RP2D determination.

[0079] Fig. 9 and Fig.10 : Peripheral blood assessment of IFN-γ (A), MCP-1 (B), B cell marginalization (C), and B cell activation (D). Square dots correspond to 900 μg / kg and round dots correspond to 450 μg / kg.

[0080] Fig.11 : Dosing regimen of the combination of mitazalimab (Mita) and folfirinox (FFX / F) in MB49–hCD40 mice.

[0081] Fig.12 : Antitumor efficacy (A) and survival (B) of mitazalimab given on days 10, 17, and 24. Mice were treated with vehicle (dextrose), FOLFIRINOX (administered on days 7-8, 14-15, and 21-22), and / or mitazalimab (days 10, 17, and 24). Differences in tumor volume were calculated on day 17 using the Mann-Whitney test. *p<0.05, **p<0.01, and ***p<0.005.

[0082] Fig.13 : Principal component analysis (PCA) plot showing the complete transcriptome of whole blood samples collected on day 5 from the MB49 hCD40 mouse tumor model.

[0083] Fig.14 : Heat map showing 3452 differentially expressed genes (DEGs) from multiple group comparisons (p value < 0.05, fold change > 2).

[0084] Fig.15 : Gene expression targets associated with mode of action (MoA).

[0085] Fig.16 : Radar chart showing the gene set variation analysis (GSVA) ​​scores of selected pathways from pathway enrichment analysis.

[0086] Figure 17: (A) Key showing the classification of points in a volcano plot according to their position as determined by the dotted line in the plot. Volcano plots for: Mita vs. untreated (B); Folfirinox vs. untreated (C); Mita vs. Folfirinox (D); Mita+Folfirinox vs. Folfirinox (E); Mita vs. Mita+Folfirinox (F); and Mita+Folfirinox vs. untreated (G).

[0087] Figure 18: Top ten enriched pathways for: Folfirinox vs. untreated (A); Mita vs. Folfirinox (B); Mita+Folfirinox vs. Folfirinox (C); Mita vs. untreated (D); Mita+Folfirinox vs. untreated (E); and Mita vs. Mita+Folfirinox (F).

[0088] Fig.19 : Describes the treatment schedule of KPCY tumor cell inoculation and FOLFIRINOX and mitazalimab administration. D = day; hCD40tg = human CD40 transgenic; sc = subcutaneous.

[0089] Fig. 20 : Antitumor efficacy of the combination of mitazalimab and FOLFIRINOX KPCY pancreatic tumor model. A) Tumor growth curve and B) Change in tumor volume on day 31 after tumor inoculation. The change in tumor volume was calculated as the percentage change of the baseline tumor volume on day 6 (one day before the start of treatment) to the tumor volume on day 31. *** indicates p<0.001; ** indicates p<0.01.

[0090] Figure 21: Swimmer's plot of patients from Example 15. Symbols show complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), not evaluable (NE), death, and ongoing treatment at cutoff date. 900 μg / kg mitazalimab, N=57

[0091] Fig. 22: Waterfall plot of percent change in phloem from baseline for patients used in Example 15. 900 μg / kg mitazalimab, N=56.

[0092] Fig.23 : Waterfall plot of percent change from baseline at the last available CT scan for patients used in Example 15. 900 μg / kg mitazalimab, N=56.

[0093] Fig.24 : Spider plot of the percent change in target lesions (sum of longest diameters) over time for patients used in Example 15. 900 μg / kg mitazalimab, N=57.

[0094] Fig.25 : Kaplan-Meier plot of duration of response (FAS), 900 μg / kg mitazalimab, N=18

[0095] Fig.26 : Kaplan-Meier plot of time to progression (FAS), 900 μg / kg mitazalimab, N=57

[0096] Fig. 27 : Kaplan-Meier plot of progression-free survival (FAS), 900 μg / kg mitazalimab, N=57

[0097] Fig.28 : Kaplan-Meier plot of overall survival (FAS), 900 μg / kg mitazalimab, N=57 DETAILED DESCRIPTION

[0098] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0099] It should be understood that different applications of the disclosed combination therapy, uses, methods, pharmaceutical compositions and kits can be adjusted according to the specific needs of the art. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments of the present invention only and is not intended to be limiting.

[0100] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes "antibodies", reference to "an antigen" includes two or more such antigens, reference to "a subject" includes two or more such subjects, and so forth.

[0101] As used herein, the term "combination therapy" or "combined treatment" or "in combination" refers to any form of simultaneous or parallel treatment using at least two different treatment groups. For example, a first treatment group can use an antibody or antigen-binding portion thereof as described herein, and a second treatment group can use chemotherapy.

[0102] "Antibodies or antigen-binding portions thereof" may be referred to as immunotherapies or immunotherapeutics. Therefore, combination therapy may be considered as a combination of immunotherapy and chemotherapy. The term "antibody" as referred to herein includes complete antibodies and any antigen-binding portions thereof (i.e., "antigen-binding fragments") or single chains thereof. "Antibodies" refers to glycoproteins or antigen-binding portions thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of heavy and light chains contain binding domains that interact with antigens. The VH region and the VL region may be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0103] 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 to a tumor or cancer in a subject. The term "immune response" includes a T cell-mediated immune response and / or a B cell-mediated immune response. Exemplary immune responses include T cell responses, such as the production of cytokines and cellular cytotoxicity. In addition, the term "immune response" includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine responsive cells (e.g., macrophages).

[0104] The heavy chain can be of any isotype, including IgG (IgGl, IgG2, IgG3, and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM, and IgE.

[0105] Light chains include kappa chains and lambda chains.

[0106] Of interest are antibodies and antigen-binding fragments thereof that have been "isolated" so as to exist in a physical environment that is different from that in which it may be found in nature, or that have been modified so as to differ from the naturally occurring antibody in amino acid sequence.

[0107] The antibody or its antigen binding portion may be a polyclonal antibody or a monoclonal antibody. The antibody or its antigen binding portion may 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 may also be used.

[0108] 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 performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementary determining regions (CDRs). Single-chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of these fragments can be screened in the same manner as intact antibodies.

[0109] The antibody used in the method of the present invention can be a human antibody. As used herein, the term "human antibody" is intended to include antibodies with variable regions, in which both framework regions and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include following antibodies, in which the CDR sequences derived from the germline of another mammalian species (such as mice) have been transplanted onto human framework region sequences - such antibodies are typically referred to as chimeric or humanized.

[0110] The human antibodies used in the methods of the present invention are typically human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas, which include B cells obtained from transgenic non-human animals such as animals, which have a genome comprising a human heavy chain transgene and a light chain transgene fused with immortalized cells. Human antibodies can also be prepared by in vitro immunization of human lymphocytes followed by transformation of lymphocytes with Epstein-Barr virus. The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody and another agent or antibody.

[0111] The antibody or antigen binding portion thereof according to the invention may alternatively be a humanized antibody.

[0112] The term "humanization" refers to an antibody molecule that is usually prepared using recombinant technology, which has an antigen binding site derived from an immunoglobulin from a non-human species and a residual immunoglobulin structure based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may include a complete non-human antibody variable domain fused to a human constant domain, or only include a complementary determining region (CDR) of such variable domains of a suitable human framework region transplanted to a human variable domain. 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 the human antibody whose sequence has been used as a humanization basis. Humanization reduces or eliminates the possibility that the constant region of the molecule will serve as an immunogen in a human individual, but the possibility of an immune response to an exogenous variable region still exists (LoBuglio, AF et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86: 4220-4224).

[0113] Another approach focuses not only on providing human-derived constant regions, but also on modifying variable regions so as to reshape them as close to human form as possible. It is known that the variable regions of both heavy and light chains contain three complementary determining regions (CDRs), which vary in response to the antigen in question and determine the binding capacity, flanked by four framework regions (FRs), which are relatively conserved in a given species and are considered to be the scaffolds of the CDRs. When non-human antibodies are prepared for a specific antigen, the variable regions can be "reshaped" or "humanized" by transplanting CDRs derived from non-human antibodies onto FRs present in the human antibody to be modified. The application of this method to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856; Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534-1536; Kettleborough, CA 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, SD et al. (1991) "Reshaping A Therapeutic CD4 Antibody," Proc. vivo,” Bio / Technology9:266-271; Co, MSet al. (1991) "Humanized Antibodies For Antiviral Therapy," Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al. (1992) "Humanization OfAn Anti-p185her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al. (1992) "Chimeric And Humanized AntibodiesWith Specificity For The CD33 Antigen," J. Immunol. 148:1149-1154. .

[0114] In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are changed with respect to the original antibody, which is also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanize an antigen is well known (see, e.g., U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

[0115] Any antibody mentioned herein may be provided in isolated form or may optionally be provided in a form linked (directly or indirectly) to another moiety. The other moiety may be a therapeutic molecule, such as a cytotoxic moiety or a drug.

[0116] The therapeutic molecule can be directly connected to the antibody 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 a variety of agents (including doxorubicin) to antibodies or peptides. Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly suitable for conjugating functional moieties to binding moieties.

[0117] Other methods for conjugating moieties to antibodies may also be used. For example, sodium periodate oxidation followed by reductive alkylation with appropriate reactants may be used, as may glutaraldehyde cross-linking. However, it should be appreciated that no matter which method is chosen to produce the conjugates of the invention, it must be ensured that the antibody retains its targeting ability and that the functional moiety retains its relevant function.

[0118] The cytotoxic moiety may be directly and / or indirectly cytotoxic. "Directly cytotoxic" means that the moiety is a moiety that is itself cytotoxic. "Indirectly cytotoxic" means that the moiety is a moiety that, while not itself cytotoxic, can induce cytotoxicity, for example, through its effect on another molecule or through another effect thereon. The cytotoxic moiety may be cytotoxic only when intracellular, and is preferably non-cytotoxic when extracellular.

[0119] The antibody or its antigen binding portion thereof can be linked to a cytotoxic moiety that is a direct cytotoxic chemotherapeutic agent. Optionally, the cytotoxic moiety is a direct cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art. In this context of the present invention, the antibody or its antigen binding portion thereof is used together with the cytotoxic chemotherapeutic agent for use in combination with different chemotherapy. In some embodiments, the antibody or its antigen binding portion thereof is not conjugated to a cytotoxic chemotherapeutic agent.

[0120] Cytotoxic chemotherapeutic agents, whether conjugated to an antibody or antigen-binding portion thereof or used alone as a chemotherapeutic (such as an anticancer agent), include: alkylating agents, including nitrogen mustards, such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-myosin), and chlorambucil; ethyleneimines and methylmelamines, such as hexamethylmelamine, thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (B CNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin (streptozotocin); and triazenes, such as dacarbazine (DTIC; dimethyltriazaimidazole-carboxamide); antimetabolites, including folate analogs, such as methotrexate (methotrexate); pyrimidine analogs, such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside); and purine analogs and related inhibitors, such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin). Natural products, including vinca alkaloids, such as vinca alkaloids (VLB) and vincristine; epipodophyllotoxins, such as etoposide and teniposide; antibiotics, such as dactinomycin (actinomycin D), daunorubicin (daunorubicin; erythromycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes, such as L-asparaginase; and biological response modifiers, such as interferon alpha type. Other agents, including platinum coordination complexes, such as cisplatin (cis-DDP) and carboplatin; anthracenediones, such as mitoxantrone and anthracyclines; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressants, such as mitotane (o, p'-DDD) and aminoglutethimide; paclitaxel and analogs / derivatives; and hormone agonists / antagonists, such as flutamide and tamoxifen.

[0121] The cytotoxic moiety can be a cytotoxic peptide or polypeptide moiety that causes cell death. Cytotoxic peptides and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, etc. Methods for connecting them to targeting moieties such as antibodies are also known in the art. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96 / 06641. Pseudomonas exotoxins can also be used as cytotoxic polypeptides. Some cytokines, such as TNF α and IL-2, can also be used as cytotoxic agents.

[0122] If delivered with enough dosages, some radioactive atoms may also be cytotoxic. Therefore, the cytotoxic part may include radioactive atoms, which deliver sufficient amounts of radioactivity to the target site in use so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188 or yttrium-90, or emit enough energy to destroy any other isotope of adjacent cells, organelles or nucleic acids. Preferably, the isotope and density of the radioactive atoms in the medicament of the present invention make the dosage of more than 4000cGy (preferably at least 6000, 8000 or 10000cGy) be delivered to the target site, and preferably delivered to the cell and organelle thereof, particularly the nucleus, in the target site.

[0123] The radioactive atom can be attached to the antibody, antigen-binding fragment, variant, fusion or derivative thereof in a known manner. For example, EDTA or another chelating agent can be attached to the binding moiety and used to attach 111In or 90Y. Tyrosine residues can be directly labeled with 125I or 131I.

[0124] The cytotoxic moiety can be a suitable indirect cytotoxic polypeptide. The indirect cytotoxic polypeptide can be a polypeptide with enzymatic activity and can convert nontoxic and / or relatively nontoxic prodrugs into cytotoxic drugs. For antibodies, this type of system is generally referred to as ADEPT (antibody-directed enzyme prodrug therapy). The system requires that the antibody locates the enzymatic moiety to the desired site in the patient's body, and after the enzyme is allowed to locate at the site for a period of time, the prodrug as the substrate of the enzyme is administered, i.e., the final product of catalysis is a cytotoxic compound. The purpose of the method is to maximize the concentration of the drug at the desired site and minimize the concentration of the drug in normal tissues. The cytotoxic moiety can convert non-cytotoxic prodrugs into cytotoxic drugs.

[0125] The enzymes and prodrugs using the system of targeted enzymes as described herein can be any of those previously proposed. The cytotoxic substance can be any existing anticancer drug, such as an alkylating agent; an agent inserted into DNA; an agent that inhibits any key enzyme such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinase or topoisomerase; or an agent that affects cell death by interacting with any other cellular component. Etoposide is an example of a topoisomerase inhibitor.

[0126] Reported prodrug systems include those listed in Table 2.

[0127] Table 2

[0128]

[0129] Suitable enzymes for forming part of the enzymatic portion include: exopeptidases, such as carboxypeptidases G, G1 and G2 (for glutamylated nitrogen mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs), and aminopeptidases (for 2-α-aminoacyl MTC prodrugs); endopeptidases, such as, for example, thrombolytics (for thrombin prodrugs); hydrolases, such as phosphatases (e.g., alkaline phosphatase) or sulfatases (e.g., arylsulfatase) (for phosphorylated or sulfated prodrugs); amidases, such as penicillin amidase and aroyl amidase; lactamase, such as β-lactamase; glycosidase, 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 nitrogen mustard) and DT-diaphorase (for CB1954); oxidases such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemase, catalytic antibodies and cyclodextrins.

[0130] Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% toxicity, preferably less than 1% toxicity, as measured in a suitable in vitro cytotoxicity assay.

[0131] The portion capable of converting a prodrug into a cytotoxic drug will likely be active when isolated from the remainder of the agent of the invention, but it need only be active when: (a) it is combined with the remainder of the agent of the invention; and (b) the agent of the invention is attached to, adjacent to, or internalized in a target cell.

[0132] When each part is a polypeptide, the two parts can be linked together by any conventional means for cross-linking polypeptides. For example, the antibody or antigen-binding portion thereof can be rich in thiol groups, and the other part is reacted with a bifunctional agent capable of reacting with these thiol groups, such as N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Amide and thioether bonds, such as those achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulfide bonds.

[0133] The cytotoxic moiety can be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanixadole, pimonidazole and misonidazole. In addition, gene delivery into cells can make them radiosensitized, such as delivering p53 genes or cyclin D. The other part can be a part that becomes cytotoxic or releases the cytotoxic part after radiation. For example, boron-10 isotopes can release alpha particles with cytotoxicity when properly radiated. Similarly, the cytotoxic part can be a part that can be used for photodynamic therapy, such as photofrin.

[0134] "Therapeutically effective amount", "effective amount" or "therapeutically effective" means that a given substance is administered to a subject suffering from a condition in an amount sufficient to cure, respond to or partially inhibit the condition or one or more symptoms thereof. Such therapeutic treatment may result in a reduction in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. The effective amount for a given purpose and a given medicament will depend on the severity of the disease or injury and the weight and general condition of the subject. This is a predetermined amount of active antibody calculated to produce the desired therapeutic effect together with the required additives and diluents (i.e., carriers or administration vehicles). In addition, it is intended to mean an amount sufficient to reduce or prevent clinically significant defects in the activity, function and response of the host. Alternatively, the therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. A suitable dosage may contain a predetermined amount of an active composition calculated to produce the desired therapeutic effect together with the required diluent. The therapeutically effective amount can be determined by an ordinarily skilled medical or veterinary practitioner based on patient characteristics (such as age, weight, sex, condition, comorbidities, other diseases, etc.), as is well known in the art.

[0135] When we refer to "cancer," we include both solid tumors and blood-based cancers (e.g., leukemias). Cancers may be malignant and / or metastatic. Solid tumors are often defined by the tissue in which they originate (e.g., pancreatic, breast, colon, etc.). However, because immunotherapy acts on the immune system, not the tumor itself, the immune status of the tumor may be a better predictor of response than the tumor's origin.

[0136] Cancers may 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, a more immunogenic cancer profile, has been shown to be associated with a good prognosis after therapy, for example in colon cancer (Galon et al., 2014, J. Pathol. 232(2): 199-209).

[0137] Cancer may be non-immunogenic or poorly immunogenic. Poorly immunogenic tumors typically have low class I MHC expression or absence of class I MHC expression, and are characterized by a low number of infiltrating immune cells (such as T cells and bone marrow-derived cells) (Lechner et al., 2013, J Immunotherapy 36 (9): 477-89). Tumors may be pancreatic tumors, adenomas, adenocarcinomas, blastomas, carcinomas, desmoid tumors, desmoid small round cell tumors, endocrine tumors, germ cell tumors, lymphomas, sarcomas, Wilms tumors, lung tumors, colon tumors, lymphomas, breast tumors or melanomas.

[0138] In some embodiments, the cancer is a CD40-positive cancer. By "CD40-positive cancer" we include any cancer that expresses CD40, albeit at varying levels.

[0139] The type of pancreatic tumor includes exocrine tumor, such as exocrine gland cancer or pancreatic ductal adenocarcinoma (PDAC), or endocrine tumor. The type of blastoma includes hepatoblastoma, glioblastoma, neuroblastoma or retinoblastoma. The type of cancer includes colorectal cancer or hepatocellular carcinoma, pancreatic cancer, prostate cancer, gastric cancer, esophageal cancer, cervical cancer and head and neck cancer and adenocarcinoma. The type of sarcoma includes Ewing sarcoma, osteosarcoma, rhabdomyosarcoma or any other soft tissue sarcoma. The type of melanoma includes lentigo maligna, lentigo maligna melanoma, superficial diffuse melanoma, acral lentigo melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma, melanoma with small nevus-like cells, melanoma with the characteristics of spitz nevus and uveal melanoma. Types of lymphoma include precursor T-cell leukemia / lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma, Burkitt lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosis form of Hodgkin lymphoma, mixed cell subtype of Hodgkin lymphoma. Types of lung tumors include non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) and small cell lung cancer tumors.

[0140] Each of the above cancers is well known, and symptoms and cancer diagnostic markers are well described, as are therapeutic agents for treating those cancers. Thus, symptoms, cancer diagnostic markers, and therapeutic agents for treating the above cancer types will be known to those skilled in the medical arts.

[0141] The clinical definitions of diagnosis, prognosis, and progression of a large number of cancers rely on certain classifications called staging. These staging systems are used to collate many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of cancer. Those skilled in the art of oncology will know how to use the staging system to assess the diagnosis, and / or prognosis, and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms should be used for assessment.

[0142] “Cancer stage” includes Rai staging, which includes stage 0, stage I, stage II, stage III, and stage IV, and / or Binet staging, which includes stage A, stage B, and stage C, and / or Ann Arbour staging, which includes stage I, stage II, stage III, and stage IV.

[0143] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur repeatedly in certain cancers, which means that examining these morphological changes (also known as histological examination) can be used for diagnosis or prognosis of cancer. Techniques for visualizing samples to examine cell morphology and techniques for preparing samples for visualization are well known in the art; for example, optical microscopy or confocal microscopy.

[0144] By "chemotherapy-resistant cancer" we mean cancer that is unresponsive to chemotherapy. For example, despite treatment with chemotherapy, the cancer may continue to grow and / or metastasize. The selection of a particular chemotherapy can be made by a physician after a subject or patient is diagnosed with cancer. A cancer may initially respond to chemotherapy and subsequently develop resistance to chemotherapy. Thus, a subject may be undergoing treatment with chemotherapy and subsequently develop resistance to chemotherapy, and the subject may then be treated with an antibody or antigen-binding portion thereof as described herein, resulting in a combination therapy for treating a chemotherapy-resistant cancer.

[0145] In some embodiments, cancer is a cancer that is resistant to treatment with therapeutic anti-cancer antibodies. Such resistant cancers can be recurrent and / or refractory cancers. Recurrent cancers are cancers that have been previously treated, and as a result of the treatment, the subject recovers completely or partially (i.e., the subject is said to be in remission), but after stopping treatment, the cancer relapses or worsens. In other words, recurrent cancer is a cancer that becomes resistant to treatment after a period of time when it is effective and the subject recovers completely or partially. Refractory cancers are cancers that have been treated but do not respond to the treatment, and / or cancers that have been treated but have progressed during treatment. In other words, refractory cancers are cancers that are resistant to treatment. It should be understood that cancer may be refractory cancer due to inherent resistance. "Intrinsic resistance" includes that cancer and / or subjects and / or target cells are resistant to a particular treatment from the time of the first administration, or before full administration. Recurrent cancers and / or refractory cancers can be easily diagnosed by technicians in the medical field.

[0146] The term "subject" (which is used interchangeably with "patient" herein) includes any animal, including humans, in need of treatment with an antibody or antigen-binding portion thereof that specifically binds to CD40 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.

[0147] Preferably, the subject is a subject who has been diagnosed as having cancer, optionally a chemotherapy-resistant cancer, or a subject who has been identified as being likely to have cancer, optionally a chemotherapy-resistant cancer, and / or a subject who exhibits symptoms of cancer, optionally a chemotherapy-resistant cancer. "Expression" includes that the subject exhibits symptoms of cancer and / or cancer diagnostic markers, and / or that the symptoms of cancer and / or cancer diagnostic markers can be measured, and / or assessed, and / or quantified. For a person skilled in the medical field, what would be symptoms of cancer and cancer diagnostic markers, and how to measure and / or assess and / or quantify whether there is a decrease or increase in the severity of cancer symptoms or whether there is a decrease or increase in cancer diagnostic markers; and how these cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0148] As used herein, the term "regimen" is synonymous with a regime or regimen. By "dosage regimen" we include the meaning of step-wise administration of the antibody, or antigen-binding portion thereof, and chemotherapy, wherein multiple steps form a regimen.

[0149] In some embodiments, the dosage regimens described herein can be repeated multiple times as needed for a particular patient. For example, each time an antibody or antigen-binding portion thereof that specifically binds to CD40 is administered to a subject, such a dosage regimen can be employed. In some embodiments, the exact form of the dosage regimen (in terms of the timing and amount of the dose) can be varied between repeated administrations to a patient. The advantage of repeatedly using the dosage regimens described herein is that it enhances the anti-cancer effect.

[0150] However, as will be appreciated by those skilled in the art, repeated dosing may also use higher or lower total doses as directed by patient tolerance. Similar dosing regimens based on flat dosing or receptor occupancy guidance may be used.

[0151] It will be understood that the dosage and dosage regimen of each therapeutic antibody discussed and contemplated herein will depend on the approved dosages / regimens for these therapeutic antibodies, and will also vary depending on the indication (e.g., cancer type / stage) and / or the subject (e.g., BMI or age).

[0152] The term "chemotherapy" includes the meaning of chemotherapy regimens (which may include treatment periods, treatment cycles, and total treatment time), chemotherapeutic agents (and combinations thereof), chemotherapeutic drugs (and combinations thereof), chemotherapeutic agents (and combinations thereof), and chemotherapeutic drugs (and combinations thereof).

[0153] As used herein, a "treatment period" of a particular formulation or treatment is intended to be a period of time during which the particular formulation or treatment is administered to a patient. For example, if chemotherapy (e.g., a chemotherapy drug) is administered for 8 consecutive days, followed by 2 days without chemotherapy, the treatment period for chemotherapy is 8 days.

[0154] As used herein, the term "treatment cycle" means a course of one or more treatments or a treatment period repeated on a regular schedule, and may include a rest period. For example, a treatment lasting 8 days, followed by a 2-day rest period, is 1 treatment cycle. Treatment cycles may be repeated in the same or modified form, such as with different doses and / or schedules, or with different additional treatments. A "treatment interval" is the interval between starting and completing a treatment cycle.

[0155] "Total treatment time" means the time period including all treatment cycles. As described above, a treatment cycle may include a time period without treatment (an interval in which treatment is not applied to the patient, i.e., chemotherapy is not applied and antibodies are not applied, and other drugs are optionally not applied). Therefore, as used herein, the total treatment time may also include the interval in which treatment is not applied within the treatment cycle. For example, if a patient receives 8 10-day treatment cycles, 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.

[0156] In a given treatment cycle, the antibody or its antigen binding portion thereof and chemotherapy can be applied simultaneously, sequentially or individually. As used herein, "simultaneously" includes the following meanings: the medicament is taken together on at least one treatment day, and may or may not be formulated as a single composition. "Simultaneously" also covers the partial overlap of the number of days for the treatment of the medicament. For example, chemotherapy can be applied for one day or multiple consecutive days, and then both chemotherapy and antibody or its antigen binding portion thereof can be applied on subsequent consecutive days. "Sequentially" includes the meaning of applying drugs on consecutive treatment days rather than the same treatment day. For example, chemotherapy can be applied for one day or multiple consecutive days, and antibody or its antigen binding portion thereof can be applied for the next day or multiple consecutive days. As used herein, "single" application means that antibody or its antigen binding portion thereof and chemotherapy are applied as part of the same overall dosing regimen, but they are not applied on the same day. For example, chemotherapy can be applied for one day or multiple consecutive days, and then there can be one or more days where neither chemotherapy nor antibody or its antigen binding portion thereof is applied, and then on the subsequent day or multiple days, antibody or its antigen binding portion thereof can be applied. Typically, the antibody, or antigen-binding portion thereof, is administered concurrently with the chemotherapy (or a portion thereof), and more typically, the treatment periods of the antibody, or antigen-binding portion thereof, and the chemotherapy partially overlap.

[0157] In certain embodiments, chemotherapy is in the form of FOLFIRINOX. FOLFIRINOX is a chemotherapy regimen comprising oxaliplatin, a folinate (also referred to as folinic acid; e.g., leucovorin), irinotecan, and 5-fluorouracil, each of which can be administered as an infusion (e.g., IV infusion). The FOLFIRINOX regimen is known in the art, and any one or more components can be adjusted to produce an adjusted FOLFIRINOX (mFOLFIRINOX) regimen (also referred to herein as FOLFIRINOX variants). These adjustments can be before starting the FOLFIRINOX regimen, during the FOLFIRINOX regimen (e.g., to mitigate the side effects of specific components of the FOLFIRINOX regimen), and / or after the treatment cycle of the FOLFIRINOX regimen (e.g., if repeated treatment cycles, to mitigate the side effects of specific components of the FOLFIRINOX regimen).

[0158] As used herein, the term "FOLFIRINOX" may be replaced by the term "mFOLFIRINOX."

[0159] Exemplary and preferred mFOLFIRINOX corresponds to the following (as shown in Example 2):

[0160]

[0161]

[0162] Adjusted forms of FOLFIRINOX are known in the art. For example, as follows (full details of the authors can be found in the references section of this article, see references 41-51; as derived from Tong et al., 2018):

[0163]

[0164] 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, a treatment cycle may begin with a particular regimen but then switch to an alternative regimen.

[0165] Additional variations of FOLFIRINOX include the following (LV=leucovorin, an exemplary folinic acid); OX=oxaliplatin; Irino=irinotecan; Freq=frequency of cycle; q2W=2 weeks (i.e., FOLFIRINOX is repeated every 2 weeks); mCyc=number of cycles of FOLFIRINOX; Met=cohort of subjects with metastatic pancreatic cancer; LA=cohort of subjects with locally advanced pancreatic cancer):

[0166]

[0167]

[0168] In some embodiments, FOLFIRINOX comprises or consists of: oxaliplatin at a dose of 85 mg per square meter given as a 2-hour intravenous infusion; immediately followed by leucovorin at a dose of 400 mg per square meter given as a 2-hour intravenous infusion; irinotecan is added 30 minutes later at a dose of 180 mg per square meter given as a 90-minute intravenous infusion via a Y-connector. This treatment is immediately followed by fluorouracil at a dose of 400 mg per square meter administered by intravenous push; followed by continuous intravenous infusion of 2400 mg per square meter over a 46-hour period every 2 weeks.

[0169] In some embodiments, patients may receive palonosetron, aprepitant, and dexamethasone for emesis prevention. 12 cycs are planned in met and 8 in LAPC; and / or pegylated filgrastim is used on day 3 or 4 of each cycle in the absence of severe leukocytosis.

[0170] In some embodiments, FOLFIRINOX or variants thereof comprise oxaliplatin (such as or generic drug) infusion, optionally wherein oxaliplatin is administered at a dose of at least 85 mg / m 2 In some embodiments, oxaliplatin is administered at a dose of 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 In some embodiments, oxaliplatin is administered at a dose of 40 mg / m 2 Up to 85 mg / m 2The dose of oxaliplatin may be varied between treatment cycles and / or adjusted during a treatment cycle.

[0171] In some embodiments, FOLFIRINOX or variants thereof comprises a folinic acid (such as leucovorin, calcium folinate, calcium leucovorin, disodium folinate, and disodium leucovorin) infusion, optionally wherein the folinic acid (e.g., leucovorin) is administered at a dose of at least 400 mg / m 2 The dosage of folinate may be varied between treatment cycles and / or adjusted during a treatment cycle.

[0172] In some embodiments, FOLFIRINOX or variants thereof comprise irinotecan (such as ) infusion, optionally wherein irinotecan is administered at a dose of at least 150 mg / m 2 In some embodiments, irinotecan is administered intravenously at a dose of at least 80 mg / m 2 , for example 90 mg / m 2 , 100mg / m 2 , 110mg / m 2 , 120mg / m 2 , 130mg / m 2 , 135mg / m 2 , 140mg / m 2 , 150mg / m 2 , 160mg / m 2 , 170mg / m 2 or 180 mg / m 2 In some embodiments, irinotecan is administered at 80 mg / m 2 Up to 180 mg / m 2 The dose of irinotecan may be varied between treatment cycles and / or adjusted during a treatment cycle.

[0173] In some embodiments, FOLFIRINOX or variants thereof comprise 5-fluorouracil ("5-FU") infusion, optionally wherein 5-fluorouracil is administered at a dose of at least 2400 mg / m 2 dose, intravenously and / or for a duration of 46-48 hours (e.g., 2.4 g / m 2 In some embodiments, 5-FU is administered at a dose of at least 300 mg / m 2 , for example 400 mg / m 2 , 500mg / m 2 , 600mg / m 2 , 700mg / m 2, 800mg / m 2 , 900mg / m 2 , 1000mg / m 2 , 1100mg / m 2 , 1200mg / m 2 , 1300mg / m 2 , 1360mg / m 2 , 1400mg / m 2 , 1500mg / m 2 , 1600mg / m 2 , 1700mg / m 2 , 1800mg / m 2 , 1900mg / m 2 or 1920 mg / m 2 In some embodiments, 5-FU is administered at a bolus dose of 1360 mg / m 2 Up to 2400 mg / m 2 The dose of 5-FU can be varied between treatment cycles and / or adjusted during a treatment cycle.

[0174] The side effects of chemotherapy (including each component associated with FOLFIRINOX) are well known. It is a routine matter for physicians to recognize the onset of side effects associated with chemotherapy (or its components) and 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. In addition, additional agents that compensate for the side effects of specific chemotherapy (or its components) are known and may be included as premedication and / or postoperative medication.

[0175] By "premedication" we include the meaning of administering some form of medication prior to treatment or surgery. For example, premedication may be prior to treatment with the antibody or antigen-binding portion thereof, prior to chemotherapy or a portion thereof, or prior to both. The premedication selected may be one that counteracts or mitigates a known side effect of the antibody or antigen-binding portion thereof or chemotherapy before symptoms of the side effect appear in the subject.

[0176] By "postoperative medication" we include the meaning of administering some form of medication after treatment or surgery. For example, postoperative medication can be after treatment with the antibody or antigen-binding portion thereof, after chemotherapy or a portion thereof, or after both. The postoperative medication selected can be to counteract or alleviate a known side effect of the antibody or antigen-binding portion thereof or chemotherapy after symptoms of the side effect are observed in the subject.

[0177] The additional drug can be before one agent of the combination therapy (or its dosage regimen) and after a different agent, in which case the drug can be considered as premedication and postmedication. The presence of the drug in the subject can be considered as being present during the combination therapy (or its dosage regimen). Thus, premedication and / or postmedication can be considered as being administered during a particular step of the combination therapy (or its dosage regimen).

[0178] Treatment with chemotherapy is often associated with cytopenias. Depending on the cell lineage, affected patients may experience different symptoms. Neutropenia is associated with an increased risk of infection, which may be severe and even fatal. Thrombocytopenia may result in an increased risk of bleeding. Anemia can lead to fatigue, dyspnea, and tachycardia. Subjects can be monitored by regular blood tests to assess these laboratory parameters, and chemotherapy dose adjustments can be made accordingly. Patients treated with mFOLFIRINOX can receive primary prophylaxis with G-CSF as a postoperative medication. G-CSF is allowed if a gemcitabine plus nab-paclitaxel chemotherapy regimen is used, but its use is at the discretion of the investigator. Severe neutropenia and febrile neutropenia should be managed according to institutional standards, such as regarding coverage of antibiotics. Blood component transfusions and the administration of erythropoietin are alternative postoperative medications approved at the discretion of the investigator. Red blood cell transfusions may be considered for patients with hemoglobin <9.5 g / dL or significant symptoms of anemia.

[0179] Subjects treated with gemcitabine plus nab-paclitaxel are at increased risk for non-neutropenic sepsis. In the Phase 3 studies, patients with fluoroquinolone allergy could be started on ciprofloxacin (500 mg orally twice daily) or amoxicillin / clavulanate ( Oral administration: 500 mg, 2 to 3 times a day) as postoperative medication.

[0180] In general, chemotherapy-induced diarrhea is caused by the death of rapidly dividing intestinal cells and the subsequent loss of absorptive function, as well as inflammation and changes in the gastrointestinal osmotic gradient (which leads to the secretion loss of fluid and electrolytes). Treatment is usually supportive, and hospitalization may be required in severe or persistent cases, and maternal fluids are administered as postoperative medication. When co-occurring with neutropenia, patients are at increased risk of infectious complications, and the use of broad-spectrum antibiotics may be considered according to institutional standard practice. In particular, the irinotecan component of mFOLFIRINOX can cause both acute (within 24 hours) diarrhea and delayed (2 to 14 days after administration) diarrhea.

[0181] Cytotoxic chemotherapy targets rapidly dividing cells, such as the gastrointestinal epithelium including the oral mucosa, resulting in tissue damage and inflammation, mucositis. This may be severe enough to restrict intake and compromise nutrition, so symptomatic supportive care and nutritional monitoring may be required as postoperative medications. Of the components in mFOLFIRINOX, 5-fluorouracil (5-FU) is the most prominent contributor to mucositis.

[0182] Both taxane and platinum-containing chemotherapy are associated with nerve damage, which is mainly attributed to the altered microtubule transport that interferes with axonal function. Typically, this affects peripheral neurons, which are farther than proximal neurons, and more commonly sensory neurons rather than motor neurons. Sensory neuropathy may begin with paresthesia, but may progress to activities that impair daily life. Toxicity accumulates over time and may persist or even worsen after dose adjustment. Predisposing factors include a history of peripheral neuropathy, diabetes, advanced age, or previous exposure to neurotoxic drugs. The main factor for neuropathy in the gemcitabine / albumin-bound paclitaxel regimen is the paclitaxel component, while oxaliplatin is the cause of most neurotoxicity in the mFOLFIRINOX regimen. In addition to this systemic peripheral neuropathy, a special acute neuropathy, i.e., paresthesia of the throat, rarely occurs in patients treated with oxaliplatin within a few hours. This may be manifested as a feeling of discomfort in the laryngopharyngeal area, and the patient may experience dyspnea and anxiety. For patients with a history of oxaliplatin-associated laryngospasm, sedation is recommended prior to oxaliplatin infusion and over six hours.

[0183] mFOLFIRINOX is considered a moderately emetogenic regimen, and antiemetic prophylaxis is recommended based on the investigator's judgment and institutional standards. Gemcitabine plus nab-paclitaxel is considered to have a lower emetogenic potential. Note that corticosteroid use should be limited when given as prophylaxis. Nausea has been observed as a symptom of an infusion-related reaction with mitazarimab therapy, and antiemetics may be part of the premedication and / or postoperative medications associated with mitazarimab administration.

[0184] If chemotherapy leaks into the tissue surrounding the intended vessels, severe local tissue necrosis may occur; this has been reported for irinotecan and oxaliplatin. If this occurs, the infusion should be stopped immediately. Leaving the needle or catheter in place, as much of the agent as possible can be aspirated as postoperative medication. Ice can be applied as an alternative or additional postoperative medications can be applied to the infiltrated area, for example every 4 to 6 hours for 15 to 20 minutes over a 72 hour period. Alternatively or in addition, local cortical therapy can be applied.

[0185] In some embodiments, the subject receives at least one treatment cycle of FOLFIRINOX. A FOLFIRINOX treatment cycle can 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 receives no more than 12 FOLFIRINOX treatment cycles. The number of FOLFIRINOX cycles may vary between individuals, and therefore the need for additional iterations may be assessed on a case-by-case basis. Therefore, the physician in charge of administering a FOLFIRINOX cycle may determine based on responsiveness (e.g., assessed by reducing / preventing 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, then a decision may be made to stop additional treatment cycles).

[0186] In some embodiments, FOLFIRINOX treatment cycles may be as follows:

[0187] ●IV oxaliplatin within 2 hours, immediately thereafter;

[0188] ●Leucovorin or a similar approved folinate within 2 hours;

[0189] ●Administer irinotecan over 90 minutes (starting 30 minutes after the start of the leucovorin infusion), followed by;

[0190] - 5-FU infusion within 46 to 48 hours, such as within 46 hours, within 47 hours, or within 48 hours.

[0191] Local administration to the tumor site is preferred and includes peritumoral, paratumoral, intratumoral, intralesional, perilesional, intracranial and intravesicular administration by any suitable means such as injection. Local administration may also include intracavitary infusion and inhalation, depending on the site of the tumor.

[0192] Systemic administration of any agent described herein (such as 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 carried out by any suitable route, but is typically parenteral.

[0193] As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, and is typically achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, intracerebral, intrathecal, intraosseous or other parenteral administration routes.

[0194] In some embodiments, the antibody, or antigen-binding portion thereof, is formulated and / or suitable for delivery by a route selected from the group consisting of: intravenous; intramuscular; and subcutaneous. Preferably, the antibody, or antigen-binding portion thereof, is formulated and / or suitable for intravenous (i.e., "iv" or "IV") delivery.

[0195] In some embodiments, the antibody, or antigen-binding portion thereof, is delivered to the subject by a route selected from the group consisting of: intravenous; intramuscular; and subcutaneous. Preferably, the antibody, or antigen-binding portion thereof, is delivered intravenously.

[0196] Thus, in preferred embodiments, the first and / or second and / or additional doses of the antibody or antigen-binding portion thereof are formulated for and / or delivered to the subject by intravenous delivery.

[0197] Methods and formulations for intravenous administration of antibodies or antigen-binding portions thereof are well known in the art.In the present invention, any type of intravenous administration, such as injection or infusion, can be used.

[0198] In some embodiments, the chemotherapy is formulated and / or adapted for delivery by a route selected from the group consisting of: intravenous; intramuscular; and subcutaneous.

[0199] In an embodiment of the invention, chemotherapy is delivered to the subject by a route selected from the group consisting of: intravenous; intramuscular; and subcutaneous.

[0200] Thus, in preferred embodiments, the first and / or second and / or additional doses of chemotherapy are formulated for and / or delivered to the subject by intravenous delivery.

[0201] Depending on the route of administration, the antibody or its antigen binding portion thereof and / or chemotherapy can be coated in a material to protect the medicament from the effects of acid and other natural conditions that may inactivate or denature the antibody or its antigen binding portion thereof and / or chemotherapy. Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used for the pharmaceutical composition of the present invention include water, buffered water, and 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). Suitable fluidity can be maintained, for example, using coating materials such as lecithin, by maintaining the desired particle size in the case of a dispersion, and using a surfactant. In many cases, it will be preferred to include an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol, or sodium chloride.

[0202] Methods and formulations for various routes of administration are well known in the art.

[0203] The combination therapies and methods of the present invention utilize antibodies that specifically bind to CD40, i.e., "anti-CD40 antibodies". In one embodiment, the antibody is retained at the tumor site after administration to a subject. The antibody preferably binds specifically to CD40, i.e., it binds to CD40 but not to other molecules, or binds to other molecules with lower affinity (e.g., 10-fold lower affinity). Unless otherwise indicated, the term CD40 as used herein refers to human CD40. The sequence of human CD40 is set forth in SEQ ID NO: 13. The anti-CD40 antibodies of the present invention may have a certain binding affinity for CD40 from other mammals, such as primates or murine CD40. The antibody preferably binds to human CD40 when located on the surface of a cell.

[0204] In particular, the anti-CD40 antibodies used in the combination therapy of the present invention compete for binding to human CD40 with a "reference antibody" comprising a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 8 (optionally together with a light chain constant region and a heavy chain constant region of SEQ ID NO: 11 and SEQ ID NO: 12, respectively). Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using a BIAcore chip with immobilized human CD40 and incubating in the presence of the reference antibody with and without the antibody polypeptide to be tested. Alternatively, a paired mapping method can be used, in which a reference antibody is immobilized to the surface of a BIAcore chip, human CD40 is bound to the immobilized antibody, and then a second antibody is tested for simultaneous binding ability to human CD40 (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00AA 05 / 2012; the disclosure of which is incorporated herein by reference).

[0205] Exemplary anti-CD40 antibodies are disclosed in WO 2013 / 034904 and WO 2016 / 023960 to Alligator Bioscience AB (the disclosures of which are incorporated herein by reference).

[0206] The antibody preferably has the ability to bind to CD40 in its native state, and in particular the ability to bind to CD40 located on the surface of a cell. Preferably, the antibody will bind specifically to CD40. That is, the antibody used in the method of the present invention will preferably bind to CD40 with a greater binding affinity than it binds to another molecule.

[0207] "Located on the surface of a cell" means that CD40 is associated with the cell such that one or more regions of CD40 are present on the outside of the cell surface. For example, CD40 may be inserted into the plasma membrane of a cell (i.e., oriented as a transmembrane protein) with one or more regions presented on the extracellular surface. This may occur during the process of CD40 being expressed by the cell. Thus, in one embodiment, "located on the surface of a cell" may mean "expressed on the surface of a cell". Alternatively, CD40 may be located outside the cell, localized to one or more specific regions of the cell surface by covalent and / or ionic interactions.

[0208] 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. "Enhancement" means that the number of cells that are lysed or induced to undergo apoptosis in the presence of the antibodies of the present invention is increased relative to the number of cells that are lysed or induced to undergo apoptosis in the presence of an appropriate control substance. Methods for determining the level of ADCC-mediated lysis or apoptosis in a sample of cells are well known in the art. For example, chromium-51 release assays, europium release assays, or sulfur-35 release assays can be used. In such assays, a previously labeled target cell line expressing an antigen (in this case CD40) is incubated with the antibody to be tested. After washing, effector cells (typically expressing Fc receptor CD16) are co-incubated with antibody-labeled target cells. Target cell lysis is then measured by releasing intracellular markers via a scintillation counter or spectrophotometry.

[0209] Preferably, the antibody, or antigen-binding portion thereof, comprises an antibody Fc region. It will be appreciated by the skilled artisan that the Fc portion may be from an IgG antibody, or from a different class of antibodies (such as IgM, IgA, IgD or IgE). For example, the Fc region may be from an IgG1, IgG2, IgG3 or IgG4 antibody. However, advantageously, the Fc region is from an IgG1 antibody.

[0210] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or may be artificial (e.g., comprising one or more point mutations relative to a naturally occurring Fc region). An Fc region with point mutations that improves its ability to bind to an FcR may be advantageous, for example by altering serum half-life or improving binding to Fcγ receptors (FcγRs) involved in ADCC and CDC. In particular, given the link between FcγRIIB binding and the functional activity of the CD40 antibody, mutations that enhance binding to FcγRIIB, such as S267E (Strohl et al., 2009, Curr Opin Biotechnol, 20:685-691) may be advantageous for the present invention (Li et al., 2011, Science, 333:1030-1034).

[0211] As an alternative to labeling with radioisotopes required in such assays, a method for detecting cleavage by measuring the release of naturally occurring enzymes in target cells can be used. This can be achieved by detection of the product of the enzyme catalyzed reaction (e.g., bioluminescence detection). In such assays, cells do not need to be previously labeled. A typical cellular enzyme detected with such assays is GAPDH.

[0212] The anti-CD40 antibodies described herein can modulate the activity of cells expressing CD40, wherein the modulation is an increase or decrease in the activity of the cells. The cells are typically dendritic cells or B cells.

[0213] When signaling occurs via CD40, professional APCs (such as dendritic cells) are activated, which triggers several biological events, including immune cell activation, proliferation, and production of cytokines and chemokines. Methods for determining dendritic cell activation associated with CD40 are known in the art (e.g., 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.

[0214] Stimulation of human B cells with recombinant CD40L or anti-CD40 antibodies induces upregulation of surface markers such as CD23, CD30, CD80, CD86, Fas, and MHC II, as well as 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.

[0215] Methods and assays for determining the ability of antibodies to regulate the activity of dendritic cells and B cells are well known in the art. For example, the activation of dendritic cells can be assessed by measuring the level of cell surface markers such as CD86 and CD80 and / or by measuring the IFNγ secretion from T cells induced by anti-CD40 antibodies, wherein the increase of any one of these parameters indicates increased activation, while the reduction indicates reduced activation. Similarly, the ability of antibodies to regulate the activity of B cells can be assessed by measuring the level of cell surface markers (such as CD86) and / or by measuring the B cell proliferation induced by anti-CD40 antibodies (see Example 3 of WO 2016 / 023960), wherein the increase of any one of these parameters indicates increased activation, while the reduction indicates reduced activation.

[0216] Preferably, the anti-CD40 antibodies described herein that increase activation of dendritic cells or B cells have potency for dendritic cell or B cell activation. Cell activation can typically be measured at an EC50 level in an assay involving incubation of isolated dendritic cells or B cells with a test stimulus and then detecting cell proliferation as a measure of activation.

[0217] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of an antibody molecule to bind to a target or not to bind to a target. Binding affinity can be quantified by determining the dissociation constant (Kd) for the antibody and its target. Similarly, the specificity of an antibody binding to its target can be defined based on the comparative dissociation constant (Kd) for the antibody and its target compared to the dissociation constant for the antibody and another non-target molecule.

[0218] Typically, the Kd of an antibody relative to a target will be 2 times smaller, preferably 5 times smaller, more preferably 10 times smaller than the Kd relative to other non-target molecules (such as unrelated materials or accompanying materials in the environment). More preferably, the Kd will be 50 times smaller, even more preferably 100 times smaller, and still more preferably 200 times smaller.

[0219] The value of the dissociation constant can be determined directly by well-known methods, and even for complex mixtures can be calculated by methods such as those described in, for example, Caceci et al. (Byte 9:340-362, 1984). For example, a dual filter nitrocellulose filter binding assay can be used to establish Kd, such as those disclosed by Wong and Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays for assessing the binding ability of a ligand (such as an antibody) to a target are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of an antibody can also be assessed by standard assays known in the art, such as by BIAcore. TM System analysis to assess.

[0220] Competitive binding assays can be performed in which the binding of an antibody to a target is compared to the binding of that target by another known ligand for that target, such as another antibody. The concentration at which 50% inhibition occurs is called the Ki. Ideally, the Ki is equal to the Kd. The Ki value will never be less than the Kd, so the measurement of the Ki can be conveniently substituted to provide an upper limit for the Kd.

[0221] The anti-CD40 antibodies described herein are preferably capable of binding to their target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than their affinity for binding to another non-target molecule.

[0222] Antibodies used in the combination therapies and methods of the invention will typically exhibit the following abilities:

[0223] (i) specifically binds to CD40 when located on the surface of a cell;

[0224] (ii) enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis of CD40-expressing cells;

[0225] (iii) enhancing apoptosis of cells expressing CD40; and / or

[0226] (iv) modulating the activity of cells expressing CD40, wherein the modulation is an increase or decrease in the activity of the cells.

[0227] In some embodiments, the combination therapies and methods of the invention will demonstrate an improvement in recipient survival and / or control of tumor volume compared to a relevant control. By "relevant control" we include the meaning of individuals who: (i) are not undergoing treatment with a combination therapy as described herein; (ii) are undergoing treatment with a chemotherapy as described herein (e.g., mFOLFIRINOX) but are not administered an anti-CD40 antibody (e.g., mitazalimumab), preferably wherein the chemotherapy is the same as that used in the combination therapy; and / or (iii) are undergoing treatment with an anti-CD40 antibody (e.g., mitazalimumab) but are not undergoing treatment with a chemotherapy as described herein (e.g., mFOLFIRINOX), preferably wherein the anti-CD40 antibody is the same as that used in the combination therapy.

[0228] The antibody may be or may comprise a variant or fragment of one of the specific anti-CD40 antibodies disclosed herein, provided that the variant or fragment retains specificity for CD40 and at least one of the functional characteristics (i) to (iv).

[0229] The fragment is preferably the antigen binding portion of the antibody. Fragments can be produced by truncation, for example by removing 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 can be removed from the N-terminus and / or C-terminus. Fragments can also be produced by one or more internal deletions.

[0230] Variants may comprise one or more substitutions, deletions or additions relative to the sequence of a specific anti-CD40 antibody disclosed herein. Variants may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from a specific sequence disclosed herein. "Deletion" variants may comprise the deletion of a single amino acid, the deletion of a small amino acid group such as 2, 3, 4 or 5 amino acids, or the deletion of a larger amino acid region, such as the deletion of a specific amino acid domain or other characteristics. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid.

[0231] Some properties of the 20 main amino acids that can be used to select suitable substitutions are as follows:

[0232]

[0233] Preferred "derivatives" include those in which the amino acids appearing in the sequence are not naturally occurring amino acids but structural analogs thereof. The amino acids used in the sequence may also be derivatized or modified, for example, labeled, provided that the function of the antibody is not significantly adversely affected.

[0234] Variants may be prepared during antibody synthesis or by post-production modifications, or when the antibody is in recombinant form, using known techniques of site-directed mutagenesis, random mutagenesis or enzymatic cleavage and / or ligation of nucleic acids.

[0235] Preferably, the variant sequence has an amino acid sequence with more than 60%, or more than 70%, such as 75% or 80%, preferably more than 85%, such as more than 90% or 95% amino acid identity with the VL or VH domain of the antibodies disclosed herein. This level of amino acid identity can be seen across the entire length of the relevant SEQ ID NO sequence or on a portion of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0236] With respect to amino acid sequences, "sequence identity" refers to sequences having the stated value when assessed using ClustalW (Thompson et al., 1994, supra) using the following parameters:

[0237] Pairwise alignment parameters—method: accurate, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10;

[0239] Multiple Alignment Parameters - Matrix: PAM, Gap Open Penalty: 10.00, Percent Delayed Identity: 30, Penalize Terminal Gaps: On, Gap Separation Distance: 0, Negative Matrix: No, Gap Extension Penalty: 0.20, Residue Specific Gap Penalty: On, Hydrophilicity Gap Penalty: On, Hydrophilic Residues: GPSNDQEKR. Sequence identities at specific residues are intended to include identical residues that have been simply derivatized.

[0240] The anti-CD40 antibodies of the present invention may bind to the same epitope as the specific antibodies disclosed herein, because such antibodies may mimic the effects of the disclosed antibodies. Whether an antibody binds to the same epitope as another antibody can be determined by conventional methods. For example, the binding of each antibody to the target can be determined using a competitive binding assay. Methods for implementing competitive binding assays 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. The antibody / target complex can then be contacted with a second (test) antibody, and the extent to which the test antibody can displace the first antibody from the antibody / target complex can be assessed. Such assessments can 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 proves that the test antibody can compete with the first antibody for binding to the target, and therefore the test antibody binds to the same epitope or region on the target as the first antibody, and therefore can mimic the effects of the first antibody.

[0241] The anti-CD40 antibody of the present invention may be an antibody comprising one, two or all three of the CDR sequences of SEQ ID NOs: 1 to 3 and / or one, two or all three of the CDR sequences of SEQ ID NOs: 4 to 6. The antibody may comprise all six CDR sequences of SEQ ID NOs: 1 to 6.

[0242] The antibody may 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.

[0243] The antibody may be an antibody comprising a light chain variable region sequence of SEQ ID NO: 7 and a heavy chain variable region sequence of SEQ ID NO: 8, or may bind to the same epitope as an antibody comprising a light chain variable region sequence of SEQ ID NO: 7 and a heavy chain variable region sequence of SEQ ID NO: 8. In addition, the antibody may comprise a light chain constant region sequence of SEQ ID NO: 11 and / or a heavy chain constant region sequence of SEQ ID NO: 12.

[0244] The anti-CD40 antibody or any variant or fragment thereof used in the combination therapies and methods of the present invention preferably has a theoretical isoelectric point (pI) of 9.0 or above, preferably 9.1 or above, more preferably 9.2 or above or 9.25 or above, most preferably 9.3 or above.

[0245] According to certain embodiments, the antibody or its antigen binding portion thereof and chemotherapy are administered simultaneously, either in the same composition or in a separate composition. According to other embodiments, the antibody or its antigen binding portion thereof and chemotherapy are administered sequentially, i.e., the antibody or its antigen binding portion thereof is administered before, during and / or after the administration of chemotherapy. In certain embodiments, the administration of the antibody or its antigen binding portion thereof and chemotherapy is simultaneous, i.e., the administration period of the antibody or its antigen binding portion thereof overlaps with the administration period of chemotherapy. In certain embodiments, the administration of the antibody or its antigen binding portion thereof and chemotherapy is not simultaneous. For example, in certain embodiments, the administration of the antibody or its antigen binding portion thereof is terminated before the administration of chemotherapy. In certain embodiments, the administration of chemotherapy is terminated before the administration of the antibody or its antigen binding portion thereof.

[0246] In some embodiments, the antibody or its antigen-binding portion thereof and chemotherapy are administered in 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 its antigen-binding portion thereof is administered as a pharmaceutical composition, and chemotherapy is not administered as a pharmaceutical composition.

[0247] The present invention also provides a kit for treating cancer, optionally chemotherapy-resistant cancer, in a subject, the kit comprising a combination therapy as defined above. For example, the kit may comprise (a) a therapeutically effective amount of an antibody or antigen binding portion thereof that specifically binds to CD40 as described herein, and optionally (b) a therapeutically effective amount of a chemotherapy suitable for administration (e.g., systemic administration) to a subject. The antibody or antigen binding portion thereof is preferably provided in a form suitable for local administration to a tumor site.

[0248] The kit of the present invention may further comprise one or more other reagents or instruments that enable any of the embodiments mentioned above to be implemented. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution) and a device for administering anti-CD40 antibodies and / or chemotherapy (such as a container or instrument containing a needle). The kit may include instructions for performing a combination therapy or method as described herein.

[0249] The anti-CD40 antibodies and chemotherapy described herein or provided in the kits of the present invention can each be provided as a separate pharmaceutical composition formulated together with a pharmaceutically acceptable carrier. If the chemotherapy consists of multiple agents, each agent can 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 and absorption delaying agents, etc., which are physiologically compatible and also compatible with the desired route of administration.

[0250] The pharmaceutical composition may comprise a pharmaceutically acceptable antioxidant. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms may be prevented by the above-mentioned sterilization procedures and by comprising various antibacterial agents and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugar, sodium chloride, etc. in these compositions. In addition, the absorption of the extension of the injectable drug form may be achieved by comprising an agent that delays absorption (such as aluminum monostearate and gelatin).

[0251] Pharmaceutical compositions typically must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of active agent (e.g., antibody) into a suitable solvent as required with one or combination of the components listed above, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating active agents into a sterile vehicle containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (freeze drying), which produce active agents plus powders of any other required components from their previously sterile filtered solutions. Pharmaceutical compositions can include additional active ingredients and those mentioned above.

[0252] Suitable pharmaceutically acceptable buffers, diluents, carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR 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).

[0253] The term "buffer" is intended to include aqueous solutions containing acid-base mixtures for the purpose of stabilizing pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphates, carbonates, acetates, citrates, glycolates, lactates, borates, ACES, ADA, tartrates, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0254] The term "diluent" is intended to include aqueous or non-aqueous solutions for the purpose of diluting the agent in the pharmaceutical preparation. The diluent can be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0255] The term "adjuvant" is intended to include any compound added to a formulation to increase the biological effect of the agent of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, cyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetate of different acyl compositions. The adjuvant may also be a cationic polymer such as a cationic cellulose ether, a cationic cellulose ester, hyaluronic acid, chitosan, a cationic dendrimer, a cationic synthetic polymer such as poly(vinyl imidazole) and a cationic polypeptide such as polyhistidine, polylysine, polyarginine and peptides containing these amino acids.

[0256] Excipient can be one or more in carbohydrate, polymer, lipid and mineral.The example of carbohydrate includes lactose, glucose, sucrose, mannitol and cyclodextrin, and they are added in the composition, for example, for promoting freeze-drying.The example of polymer is starch, cellulose ether, cellulose carboxymethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate of different hydrolysis degree, and polyvinyl pyrrolidone, all of which have different molecular weights, and they are added in the composition, for example, for viscosity control, for realizing bioadhesion, or for protecting lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di- and triglycerides, ceramides, sphingolipids and glycolipids, varying in acyl chain length and degree of saturation, egg lecithin, soy lecithin, hydrogenated egg lecithin and soy lecithin, which are added to the composition for similar reasons as the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced fluid accumulation or favorable pigment properties.

[0257] The active antibody-based agents and / or chemotherapeutics of the present invention may be formulated into any type of pharmaceutical composition known in the art suitable for their delivery.

[0258] In one embodiment, the pharmaceutical composition of the present invention may be in the form of liposomes, wherein in addition to other pharmaceutically acceptable carriers, the medicament is also combined with an amphipathic agent (such as a lipid), which is present in aggregated form as micelles, insoluble monolayers, and liquid crystals. Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. Suitable lipids also include lipids modified by poly(ethylene glycol) in polar head groups to extend blood circulation time. The preparation of such liposome formulations can be found, for example, in US 4,235,871, the disclosure of which is incorporated herein by reference.

[0259] The pharmaceutical composition of the present invention can also be in the form of biodegradable microspheres. Aliphatic polyesters, such as polylactic acid (PLA), polyglycolic acid (PGA), copolymers (PLGA) of PLA and PGA or poly (caprolactone) (PCL) and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. The preparation of such microspheres can be found in US 5,851,451 and EP 0213303, the disclosure of which is incorporated herein by reference.

[0260] In another embodiment, the pharmaceutical composition of the present invention is provided in the form of nanoparticles, for example based on poly-gamma glutamic acid. The details of the preparation and use of such nanoparticles can be found in WO 2011 / 128642, the disclosure of which is incorporated herein by reference. It will be appreciated by those skilled in the art that one or more active components of the combination therapy of the present invention can be formulated in separate nanoparticles, or two active components can be formulated in the same nanoparticles.

[0261] In other embodiments, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, wherein polymers (such as starch, cellulose ethers, cellulose carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate of varying degrees of hydrolysis, and polyvinyl pyrrolidone) are used for thickening of solutions containing medicaments. The polymer may also include gelatin or collagen.

[0262] Alternatively, the agent may be simply dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, or oils (such as safflower, corn, peanut, cottonseed, or sesame oils), tragacanth, and / or various buffers.

[0263] It should be understood that the pharmaceutical composition of the present invention may include ions and defined pH for enhancing the effect of the active agent. 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, emulsifiers, buffers, fillers, etc.).

[0264] The pharmaceutical composition according to the present invention can be administered via any suitable route known to those skilled in the art. Therefore, possible routes of administration include parenteral (intravenous, subcutaneous and intramuscular), topical, ocular, nasal, pulmonary, oral, parenteral, vaginal and rectal. Similarly, administration by implant is also possible.

[0265] Advantageously, the pharmaceutical composition is suitable for administration at or near the tumor site, such as within or around the tumor.

[0266] In some embodiments, the pharmaceutical composition is suitable for parenteral administration.Methods for formulating antibodies into pharmaceutical compositions will be well known to those skilled in the medical and pharmaceutical arts.

[0267] The combination therapy of the present invention can be delivered using an injectable sustained-release drug delivery system. These are specifically designed to reduce the frequency of injections. An example of such a system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, slowly release rhGH over a sustained period of time. Preferably, delivery is carried out intramuscularly (im) and / or subcutaneously (sc) and / or intravenously (iv).

[0268] Combination therapy of the present invention can be used by surgically implanted devices, which release the drug directly to the desired position. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. This toxic agent is directly applied to the position of the disease to achieve effective therapy without the significant systemic side effects of the drug.

[0269] Electroporation therapy (EPT) systems can also be used to administer the combination therapies of the invention. Devices that deliver pulsed electric fields to cells increase the permeability of cell membranes to drugs, resulting in significant enhancement of intracellular drug delivery.

[0270] The combination therapy of the present invention can also be delivered by electroincorporation (EI). EI occurs when small particles up to 30 microns in diameter on the surface of the skin are subjected to an electrical pulse identical or similar to that used in electroporation. In EI, these particles are driven through the stratum corneum and into the deeper layers of the skin. These particles can be loaded or coated with drugs or genes, or can simply act as "bullets" that create holes in the skin through which drugs can enter.

[0271] An alternative combination therapy of the present invention is the thermosensitive ReGel injectable system. At below body temperature, ReGel is an injectable liquid, and at body temperature, it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. As the biopolymer dissolves, the active substance is released over time.

[0272] The combination therapy of the present invention can also be delivered orally. This process uses oral uptake of vitamin B 12 and / or vitamin D to co-deliver proteins and peptides. 12 and / or vitamin D uptake system, the agents, drugs and pharmaceutical compositions of the present invention can move across the intestinal wall. The complex is in the form of vitamin B 12 Analogs and / or vitamin D analogs synthesized between drugs that retain the vitamin B complex 12 Both the remarkable affinity for intrinsic factor (IF) in the part / vitamin D part and the remarkable biological activity of the active substance of the complex.

[0273] The combination therapy of the present invention can be introduced into cells through "Trojan peptides". These are a class of polypeptides called transmembrane peptides, which have translocation properties and can carry hydrophilic compounds across the plasma membrane. This system allows oligopeptides to be directly targeted to the cytoplasm and nucleus and may be non-cell type specific and highly effective. See Derossi et al. (1998), Trends Cell Biol.8, 84-87.

[0274] The combination therapies of the present invention may be presented as unit doses containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof of the active ingredients.

[0275] The combination therapy of the present invention will generally be in the form of a pharmaceutical composition comprising an active ingredient, optionally in the form of a non-toxic organic or inorganic, acid or base addition salt, in a pharmaceutically acceptable dosage form orally or by any parenteral route of administration. Depending on the condition to be treated and the patient and the route of administration, the composition can be administered in different doses.

[0276] In human therapy, the combination therapies of the invention may be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0277] For example, the combination therapy of the present invention can be orally, buccally or sublingually administered in the form of tablets, capsules, ovules, elixirs, solutions or suspensions that may contain flavorings or coloring agents for immediate, delayed or controlled release applications. The medicaments, drugs and pharmaceutical compositions of the present invention can also be administered via intracavernous injection.

[0278] Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate and glycine, disintegrants such as starch (preferably corn starch, potato starch or tapioca starch), sodium starch glycolate, cross-linked sodium carboxymethylcellulose and certain complex silicates, and granulating binders such as polyvinyl pyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and acacia. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0279] Solid compositions of similar types can also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar, or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the medicaments, drugs, and pharmaceutical compositions of the present invention can be combined with various sweeteners or flavorings, coloring agents or dyes, with emulsifiers and / or suspending agents, and with diluents such as water, ethanol, propylene glycol, and glycerol, and combinations thereof.

[0280] Combination therapy of the present invention can be administered parenterally, for example intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they can be administered by infusion techniques. They are preferably used in the form of sterile aqueous solution, and this sterile aqueous solution can contain other substances, for example, and enough salts or glucose are made so that solution and blood are isotonic. If necessary, then the aqueous solution should be appropriately buffered (preferably, being buffered to pH is 3 to 9). By well-known standard pharmaceutical techniques among those skilled in the art, it is easily realized to prepare suitable parenteral formulations under aseptic conditions.

[0281] Drugs and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Drugs and pharmaceutical compositions may be present in unit doses or multi-dose containers (e.g., sealed ampoules and vials), and may be stored under freeze drying (lyophilization) conditions that only require the addition of a sterile liquid carrier (e.g., water for injection) immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0282] The combination therapy of the present invention can also be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or aerosol spray presentation by using a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes such as 1,1,1,2-tetrafluoroethane (HFA134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. The pressurized container, pump, sprayer or nebulizer can contain a solution or suspension of the active agent, for example using a mixture of ethanol and a propellant as a solvent, which may additionally contain a lubricant, for example sorbitan trioleate. Capsules and cartridges for inhalers or insufflators (e.g. made of gelatin) can be formulated as a powder mix containing the agent of the present invention and a suitable powder base (such as lactose or starch).

[0283] Alternatively, the combination therapy of the present invention can be administered in the form of a suppository or vaginal suppository, or they can be topically applied in the form of a lotion, solution, cream, gel, ointment or dusting powder. The medicaments, drugs and pharmaceutical compositions of the present invention can also be administered transdermally, for example, by using a skin patch. They can also be administered by an ocular route, particularly for the treatment of ocular diseases.

[0284] For ophthalmic use, the combination therapies of the invention may be formulated as micronized suspensions in isotonic, pH-adjusted sterile saline, or preferably, as solutions in isotonic, pH-adjusted sterile saline, optionally in combination with a preservative such as benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

[0285] For topical application to the skin, the combination therapy of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in a mixture of, for example, 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 formulated as a suitable lotion or cream suspended or dissolved in a mixture of, for example, one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0286] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0287] In general, in humans, local administration of the combination therapy of the invention at or near the tumor site is the preferred route, particularly intratumoral or peritumoral administration.

[0288] For veterinary use, the compositions of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice, and the veterinarian will determine the administration regimen and route of administration most suitable for the particular animal.

[0289] The eighth aspect of the present invention relates to a method for prognosing or predicting the responsiveness of a combination therapy described herein. In some embodiments, the method includes determining a biomarker signature of a test sample, which enables prognosis or prediction of an individual from which a sample is obtained. The biomarker signature can be based on one or more of the biomarkers selected from the group consisting of Ccl2, Ccl3, Ccl4, Cxcl10, and Ifng, and the biomarkers listed in Table 36, Table 37, and / or Table 38.

[0290] For example, this aspect of the invention provides a method of prognosing responsiveness to a combination therapy as described herein, comprising or consisting of the following steps:

[0291] (a) providing a sample (such as an ex vivo sample) from an individual to be tested; and

[0292] (b) determining a biomarker signal for the test sample by measuring the presence and / or amount of one or more biomarkers in the test sample, the one or more biomarkers selected from one or more biomarkers selected from Ccl2, Ccl3, Ccl4, Cxcl10, and Ifng, and the biomarkers listed in Table 36, Table 37, and / or Table 38;

[0293] Wherein the presence and / or amount of one or more biomarkers in the test sample is indicative of the responsiveness of the individual to the combination therapy.

[0294] The method is intended to cover situations where no surgical steps are required on the human or animal body. Therefore, what we mean by "providing a sample" includes the meaning that the sample has been provided and no active steps (such as surgical steps) are required to obtain such a sample. In this case, the sample can be referred to as an ex vivo sample.

[0295] In some embodiments, the sample is a blood sample. In some preferred embodiments, the sample is a serum or plasma sample derived from an unfractionated blood sample. In some preferred embodiments, the sample is a serum sample.

[0296] By "biomarker" we mean any naturally occurring biomolecule or component or fragment thereof, the measurement of which can provide information useful for diagnosing cancer (e.g., pancreatic cancer), optionally low chemotherapy-resistant cancer. Thus, in the context of Ccl2, Ccl3, Ccl4, Cxcl10, and Ifng and Tables 36 to 38, a biomarker can be a nucleic acid molecule encoding a protein or a portion thereof, such as mRNA or cDNA. Alternatively, a biomarker can be a protein or a polypeptide fragment thereof or a carbohydrate portion thereof.

[0297] By "prognosing" or "prognostic" we include predicting the likely course (i.e., responsiveness) of a medical condition. Responsiveness can be determined based on a positive outcome for a medical condition. For example, in cancer (e.g., pancreatic cancer), optionally a chemotherapy-resistant cancer, responsiveness can be determined based on increased survival compared to relevant controls and / or decreased tumor size / volume compared to relevant controls.

[0298] The methods of the invention are also suitable for testing samples from any individual suspected of having cancer (e.g., pancreatic cancer), optionally a chemotherapy-resistant cancer, or at risk of developing it. The methods of the invention are suitable for testing samples from any individual undergoing treatment for cancer (e.g., pancreatic cancer), optionally a chemotherapy-resistant cancer. The treatment may be chemotherapy as described herein (e.g., mFOLFIRINOX), an anti-CD40 antibody (e.g., mitazalimab), or a combination thereof alone.

[0299] In some preferred embodiments, the treatment is a combination of mFOLFIRINOX and mitazalimab.

[0300] It will be understood by those skilled in the art that in addition to measuring the biomarkers in a sample from the individual to be tested, the methods of the invention may also include measuring those same biomarkers in one or more control samples. By "relevant control" of the methods of the invention, we include the meaning of an individual who (i) is not undergoing treatment with a combination therapy as described herein; (ii) is undergoing treatment with a chemotherapy as described herein (e.g., mFOLFIRINOX), but is not being administered an anti-CD40 antibody (e.g., mitazalimumab), preferably wherein the chemotherapy is the same as used in the combination therapy; and / or (iii) is undergoing treatment with an anti-CD40 antibody (e.g., mitazalimumab), but is not undergoing treatment with a chemotherapy as described herein (e.g., mFOLFIRINOX), preferably wherein the anti-CD40 antibody is the same as used in the combination therapy.

[0301] Therefore, in one embodiment, the method further comprises or consists of the following steps:

[0302] (c) providing one or more (negative) relevant control samples as described herein; and

[0303] (d) determining a biomarker signature for one or more control samples by measuring the presence and / or amount of one or more biomarkers measured in step (b) in the control samples;

[0304] Responsiveness to the combination therapy is identified where the presence and / or amount of the one or more biomarkers measured in step (b) in the test sample is different from the presence and / or amount of the one or more biomarkers measured in step (d) in the control sample.

[0305] "Different from the presence and / or amount in the control sample" comprises that the presence and / or amount of the one or more biomarkers in the test sample is different from the presence and / or amount of the one or more control samples (or a predefined reference value representing it). Preferably, the presence and / or amount in the test sample differs from the presence or amount in one or more control samples (or the average value of the control samples) by at least ±5%, for example, at least ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, ±20%, ±21%, ±22%, ±23%, ±24%, ±25%, ±26%, ±27%, ±28%, ±29%, ±30%, ±31%, ±32%, ±33%, ±34%, ±35%, ±36%, ±37%, ±38%, ±39%, ±40%, ±41%, ±42%, ±43%, ±44%, ±45%, ±46%, ±47%, ±48%, ±49%, ±50%, ±51%, ±52%, ±53%, ±54%, ±55%, ±56%, ±57%, ±58%, ±59%, ±60%, ±61%, ±62%, ±63%, ±64%, ±65%, ±66%, ±67%, ±68%, ±69%, ±70%, ±71%, ±72%, ±73%, ±74%, ±75%, ±76%, ±77%, ±78%, ±79%, ±80%, ±81%, ±82%, ±83%, ±84%, ±85%, ±86%, ±87%, ±88%, ±89%, ±90%, ±91%, ±92%, ±93%, ±94%, ±95%, ±96%, ±97%, ±98%, ±99%, ±100%, ±100%, ±100%, ±100%, ±100%, ±100%, ± %, ±23%, ±24%, ±25%, ±26%, ±27%, ±28%, ±29%, ±30%, ±31%, ±32%, ±33%, ±34%, ±35%, ±36%, ±37%, ±38%, ±39%, ±40%, ±41%, ±42%, ±43%, ±44%, ±45%, ±46%, ±47%, ±48%, ±49%, ±50%, ±51%, ±52%, ±53% %, ±54%, ±55%, ±56%, ±57%, ±58%, ±59%, ±60%, ±61%, ±62%, ±63%, ±64%, ±65%, ±66%, ±67%, ±68%, ±69%, ±70%, ±71%, ±72%, ±73%, ±74%, ±75%, ±76%, ±77%, ±78%, ±79%, ±80%, ±81%, ±82%, ±83%, ±84%, ±85%, ±86%, ±87%, ±88%, ±89%, ±90%, ±91%, ±92%, ±93%, ±94%, ±95%, ±96%, ±97%, ±98%, ±99%, ±100%, ±125%, ±150%, ±175%, ±200%, ±225%, ±250%, ±275%, ±300%, ±350%, ±400%, ±500% or at least ±1000%.

[0306] Alternatively or in addition, the presence or amount in the test sample differs from the average presence or amount in the control sample by at least>1 standard deviation relative to the average presence or amount in the control sample, e.g., by ≥1.5, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥11, ≥12, ≥13, ≥14, or ≥15 standard deviations relative to the average presence or amount in the control sample. Any suitable means can be used to determine the standard deviation (e.g., direct method, sum of squares, Welford method), however, in one embodiment, a direct method (i.e., square root of [sum of squares of samples minus mean, divided by the number of samples]) is used to determine the standard deviation.

[0307] Alternatively or in addition, by "different from the presence and / or amount in the control sample", we mean that the presence or amount in the test sample is not correlated with the amount in the control sample in a statistically significant manner. For example, the presence or amount in the test sample can differ from the presence or amount in the control sample in a statistically significant manner by an adjusted p-value of ≤0.05, for example, ≤0.04, ≤0.03, ≤0.02, ≤0.01, ≤0.005, ≤0.004, ≤0.003, ≤0.002, ≤0.001, ≤0.0005, or ≤0.0001. In some other embodiments, the presence or amount in the test sample can differ from the presence or amount in the control sample in a statistically significant manner by an adjusted p-value of ≤0.05, e.g., ≤0.04, ≤0.03, ≤0.02, ≤0.01, ≤0.005, ≤0.004, ≤0.003, ≤0.002, ≤0.001, ≤0.0005, or ≤0.0001. In some embodiments, -Log 10 p-value >1.0, e.g., >1.1, >1.2, >1.3, >1.4, >1.5, >1.6, >1.7, >1.8, >1.9, >2.0, >2.1, >2.2, >2.3, >2.4, >2.5, >2.6, >2.7, >2.8, >2.9, or >3.0. For example, in some embodiments, -Log 10 p-value>1.87.

[0308] Any suitable means for determining p-values ​​known to those skilled in the art may be used, including z-test, t-test, Student's t-test, f-test, Mann-Whitney U test, Wilcoxon signed-rank test, and Pearson chi-square test.

[0309] Alternatively or additionally, by "different from the presence and / or amount in the control sample" we include that the presence or amount in the test sample does not correlate with the amount in the control sample when determined as described in Example 12. For example, we mean or include that the presence or amount in the test sample may be different from the presence or amount in the control sample in a statistically significant manner, where -Log 10 p value>1.0, such as>1.1,>1.2,>1.3,>1.4,>1.5,>1.6,>1.7,>1.8,>1.9,>2.0,>2.1,>2.2,>2.3,>2.4,>2.5,>2.6,>2.7,>2.8,>2.9 or>3.0; preferably, wherein -Log 10p value>1.87;And / or the existence or amount in the test sample is different from the existence or amount of the control sample, wherein the Log2 fold change>1 (that is, more than 1), such as>1.1,>1.2,>1.3,>1.4,>1.5,>1.6,>1.7,>1.8,>1.9 or>2, or the Log2 fold change<-1 (that is, less than-1, which means that the fold change value further deviates from 0), such as<-1.1,<-1.2,<-1.3,<-1.4,<-1.5,<-1.6,<-1.7,<-1.8,<-1.9 or<-2. In some preferred embodiments, the log2 fold change is greater than 1.

[0310] In some embodiments, the biomarker signature is based on biomarkers (as described herein) that are upregulated by the combination therapy compared to a relevant control, wherein the biomarkers are selected from the group consisting of Ccl2, Ccl3, Ccl4, Cxcl10, and Ifng.

[0311] In some embodiments, the biomarker signature is based on biomarkers that are upregulated by the combination therapy compared to chemotherapy alone (e.g., mFOLFIRINOX), preferably compared to an equivalent chemotherapy for the combination therapy. For example, the biomarker signature can be based on upregulation of biomarkers selected from the group consisting of Adar, Parp9, Dhx58, Dhx60, Slfn4, Ifit3, Ifit3b, Herc6, Usp18, Ifi44, Hmgcs2, Orm1, and combinations thereof.

[0312] In some embodiments, the biomarker signature is based on biomarkers that are upregulated by the combination therapy compared to the administration of an anti-CD40 antibody (e.g., mitrazalimab) alone, preferably compared to an equivalent anti-CD40 antibody for combination therapy. For example, the biomarker signature can be based on the upregulation of a biomarker selected from the group consisting of Acer2, Mapre3, Lancl3, Eda2r, Hmgcs2, Car3, and combinations thereof. In some embodiments, the biomarker signature is based on biomarkers that are downregulated by the combination therapy compared to the administration of an anti-CD40 antibody (e.g., mitrazalimab) alone, preferably compared to an equivalent anti-CD40 antibody for combination therapy. For example, the biomarker signature can be based on downregulation of a biomarker selected from the group consisting of Bud31, Gm33111, Ing2, Asf1a, Atf4, Gpbp1, Cd200r3, Zbtb44, Mtss2, Mospd1, Supt4a, Eif1, Gm15931, Ms4a2, Mcpt8, Rag1, and combinations thereof.

[0313] In some embodiments, the biomarker signature is based on biomarkers that are upregulated by the combination therapy compared to an untreated control (i.e., an individual who has not yet been subjected to a combination therapy or chemotherapy or anti-CD40 antibody monotherapy as disclosed herein). For example, the biomarker signature can be based on the upregulation of biomarkers selected from the group consisting of Zfp750, Eda2r, Psrc1, Cdkn1a, Ifit3, Ifit3b, Usp18, Mapre3, Upp1, Psme1, Parp9, Clca3a1, Gbp7, Ddx60, Ligp1, Herc6, Slfn4, Ifi44, and combinations thereof. In some embodiments, the biomarker signature is based on biomarkers that are downregulated by the combination therapy compared to an untreated control (i.e., an individual who has not yet been subjected to a combination therapy or chemotherapy or anti-CD40 antibody monotherapy as disclosed herein). For example, the biomarker signature can be based on the downregulation of biomarkers selected from the group consisting of Cd209a, Abca9, Hpgd, Fcgrt, and combinations thereof.

[0314] In some embodiments, the biomarker signature is based on biomarkers that are upregulated by the combination therapy compared to the corresponding monotherapy, preferably an equivalent chemotherapy (e.g., mFOLFIRINOX) or anti-CD40 antibody (e.g., mitazalimab) as used in the combination therapy. For example, the biomarker signature can be based on the upregulation of Hmgcs2.

[0315] In some embodiments, the biomarker signature is based on biomarkers that are upregulated by the combination therapy compared to anti-CD40 antibody (e.g., mitazalimumab) monotherapy, preferably compared to an equivalent anti-CD40 antibody as used in the combination therapy, or an untreated control as described herein. For example, the biomarker signature can be based on upregulation of Mapre3 and / or Eda2r.

[0316] In one embodiment, the method of the present invention may further comprise or consist of the following steps:

[0317] (e) providing one or more (positive) control samples from individuals who are undergoing or have undergone treatment with the combination therapy; and

[0318] (f) determining a biomarker signature of the control sample by measuring the presence and / or amount of the one or more biomarkers measured in step (b) in the control sample;

[0319] Responsiveness to the combination therapy is identified where the presence and / or amount of the one or more biomarkers measured in step (b) in the test sample corresponds to the presence and / or amount of the one or more biomarkers measured in step (f) in the control sample.

[0320] Therefore, the method of the present invention may comprise steps (c)+(d) and / or steps (e)+(f).

[0321] By "corresponding to the presence and / or amount in the control sample" we include that the presence and / or amount is the same as that in the positive control sample; or is closer to the presence and / or amount in one or more positive control samples than one or more negative control samples (or a predefined reference value representing the same). Preferably, the presence and / or amount is within ±40% of the presence and / or amount of one or more positive control samples (or the average of the positive control samples), for example, within ±39%, ±38%, ±37%, ±36%, ±35%, ±34%, ±33%, ±32%, ±31%, ±30%, ±29%, ±28%, ±27%, ±26%, ±25%, ±24%, ±23%, ±22%, ±21%, ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.05% or 0% of one or more positive control samples.

[0322] Alternatively or additionally, the presence or amount in the test sample differs by ≤5 standard deviations relative to the mean presence or amount in the positive control sample, e.g., ≤4.5, ≤4, ≤3.5, ≤3, ≤2.5, ≤2, ≤1.5, ≤1.4, ≤1.3, ≤1.2, ≤1.1, ≤1, ≤0.9, ≤0.8, ≤0.7, ≤0.6, ≤0.5, ≤0.4, ≤0.3, ≤0.2, ≤0.1, or 0 standard deviations relative to the mean presence or amount in the control sample, provided that the standard deviation ranges for the different and corresponding biomarker expressions do not overlap (e.g., are contiguous, but not overlapping).

[0323] Alternatively or in addition, by "corresponding to the presence and / or amount in the control sample", we mean that the presence or amount in the test sample is related to the amount in the control sample in a statistically significant manner. For example, the presence or amount in the test sample is not statistically different from the presence or amount in the control sample, wherein the p-value is >0.05, such as >0.06, >0.07, >0.08, >0.09 or >0.1.

[0324] Alternatively or additionally, by "corresponding to the presence and / or amount in the control sample" we include that the presence or amount in the test sample is correlated with the amount in the control sample when determined as described in Example 12. For example, we mean or include that the presence or amount in the test sample is correlated with the presence or amount in the control sample, where Log 10The p-value < 3.0, such as < 2.9, < 2.8, < 2.7, < 2.6, < 2.5, < 2.4, < 2.3, < 2.2, < 2.1, < 2.0, preferably < 2.5. And / or the presence or amount in the test instance is related to the presence or amount of the control sample, with a Log2 fold change < 2 (i.e., less than 2), such as < 1.9, < 1.8, < 1.7, < 1.6, < 1.5, < 1.4, < 1.3, < 1.2, < 1.1 or < 1, or a Log2 fold change > -2 (i.e., exceeding -2, which means the fold change value moves towards 0), such as > -1.9, > -1.8, > -1.7, > -1.6, > -1.5, > -1.4, > -1.3, > -1.2, > -1.1 or > -1.

[0325] Differential expression (upregulation or downregulation) of the biomarker, or the lack thereof, can be determined by any suitable means known to those skilled in the art. The differential expression is determined to be at least less than 0.05 (p = < 0.05), such as at least < 0.04, < 0.03, < 0.02, < 0.01, < 0.009, < 0.005, < 0.001, < 0.0001, < 0.00001 or at least < 0.000001 of the p-value.

[0326] Those skilled in the art will understand that the differential expression can relate to a single biomarker or to multiple biomarkers considered in combination (i.e., as a biomarker signature). Thus, the p-value can be related to a single biomarker or to a group of biomarkers. In fact, when considered alone, a biomarker with a differential expression < p-value greater than 0.05 can still be used as a biomarker according to the present invention when considering its expression level in combination with one or more other biomarkers.

[0327] In a preferred embodiment of the eighth aspect of the present invention, the method is repeated on an individual. Thus, steps (a) and (b) (or the previous method) can be repeated using samples collected from the same individual at different times relative to the original sample being tested. Such repeated testing can enable the assessment of responsiveness to combination therapy and, if appropriate, the selection of alternative options to be employed.

[0328] Thus, in one embodiment, the method is repeated using test samples based on the treatment cycles and / or regimens described herein. For example, the method can be repeated using test samples collected from each cycle consisting of: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or longer.

[0329] Alternatively or additionally, the method can be repeated at least once, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times or more.

[0330] Alternatively or additionally, the method is repeated continuously.

[0331] In some embodiments, the method is repeated until responsiveness to the combination therapy is observed in the individual using the methods of the invention and / or conventional clinical methods.

[0332] Examples

[0333] Example 1 – Sequence Information

[0334] Anti-CD40 Antibody Clone G12 (Antibody ADC-1013)

[0335] This clone is described in WO 2016 / 023960. An exemplary antibody comprising the CDR sequences of ADC-1013 is called mitazalimab.

[0336] (a) CDR sequences (defined according to IMGT numbering, with core CDR sequences underlined)

[0337]

[0338] (b) Variable region sequence

[0339] Variable light chain (V L ) Amino acid sequence – SEQ ID NO:7 (CDRs are underlined)

[0340] QSVLTQPPSASGTPGQRVTIS CTGSSSNIGAGYNVY WYQQLPGTAPKLLIY GNINRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYY CAAWDKSISGLV FGGGTKLTVLG

[0341] Variable heavy chain (V H ) Amino acid sequence – SEQ ID NO: 8 (CDRs are underlined)

[0342] EVQLLESGGGLVQPGGSLRLSCAAS GFTFSTYGMH WVRQAP GKGLEWLSYISGGSSYIFYADSVRGR FTISRDNSENALYLQMNSLRAEDTAVYY CARILRGGSGMDL WGQGTLVTVSS

[0343] Variable light chain (V L ) Nucleotide sequence – SEQ ID NO:9

[0344] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGCACTGGGAGCAGCTCCAACATCGGGGCGGGTTACAATGTATACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACATCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATAAGAGCATTTCTGGTCTGGTTTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT

[0345] Variable heavy chain (V H ) Nucleotide sequence - SEQ ID NO: 10

[0346] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTTTCATATATTAGTGGTGGTAGTAGTTACATTTTCTACGCAGACTCAGTGAGGGGCCGATTCACCATCTCCAGAGACAACTCCGAGAACGCGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAATATTAAGAGGCGGGAGCGGTATGGACCTCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA

[0347] (c) Exemplary constant region amino acid sequences

[0348] Human Ig lambda light chain C2 region (NCBIAAA59107.1) - SEQ ID NO: 11

[0349] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0350] Human Igγ-1 heavy chain constant region (UniProtP01857.1) - SEQ ID NO: 12

[0351] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0352] Human CD40 sequence – SEQ ID NO: 13

[0353] >gi|117606560|gb|ABK41937.1|CD40 molecule, TNF receptor superfamily member 5 [Homo sapiens]

[0354] MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0355] Example 2 – OPTIMIZE-1 Clinical Study

[0356] General information

[0357] ●Study title: A phase 1b / 2, open-label, multicenter study evaluating the clinical efficacy of mitazalimab in combination with chemotherapy in metastatic pancreatic ductal adenocarcinoma.

[0358] ●Region: Europe.

[0359] • Expected number of sites: 6 to 8 sites for Part 1 and up to 15 sites for Part 2.

[0360] ●Expected number of patients: Up to 80 patients.

[0361] ● Patient population: Patients with previously untreated, measurable and histologically confirmed metastatic pancreatic ductal adenocarcinoma. Patients must be 18 years of age or older and have an Eastern Cooperative Oncology Group performance status (ECOG) of 0 or 1 and good organ function by laboratory assessment.

[0362] Study Medicinal Product, Dosage, and Administration

[0363] Mitazalimab was evaluated in combination with chemotherapy-modified FOLFIRINOX (mFOLFIRINOX). Mitazalimab and mFOLFIRINOX were administered by intravenous infusion on 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.

[0364] Mitazalimab: An agonistic human monoclonal (IgG1) antibody targeting CD40 that is being developed for cancer immunotherapy. In Part 1 (Phase 1b) of the study, the dose of mitazalimab was escalated from a starting dose of 450 μg / kg to 900 μg / kg (2 dose levels were evaluated) to achieve the recommended Phase 2 dose (RP2D) of mitazalimab in combination with mFOLFIRINOX. In Part 2 of the study, the RP2D of mitazalimab in combination with mFOLFIRINOX was administered to all patients.

[0365] Chemotherapy was administered as background treatment as 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 plus nab-paclitaxel were classified as non-investigational medicinal products.

[0366] mFOLFIRINOX: mFOLFIRINOX treatment consists of oxaliplatin (85 mg / m 2 ), folinic acid (400 mg / m 2 ), irinotecan (150 mg / m 2 ) and 5-fluorouracil (5-FU, 2400 mg / m 2). If leucovorin supply issues occur or to comply with local standards of nursing practice, other approved comparable folinates may be used under the supervision of an experienced clinician. In the event that mFOLFIRINOX is found to be unsafe and not tolerated with the lowest dose of mirtazalimab, mFOLFIRINOX will be changed to gemcitabine plus nab-paclitaxel.

[0367] Gemcitabine plus nab-paclitaxel (if applicable): Gemcitabine plus nab-paclitaxel is approved in Europe for first-line treatment of adult patients with metastatic adenocarcinoma of the pancreas. Gemcitabine plus nab-paclitaxel was administered in this study only if mFOLFIRINOX was found to be unsafe and intolerant in combination with the lowest dose level of mitazalimab.

[0368] Study Design

[0369] This is a Phase 1b / 2, open-label, multicenter study designed to evaluate the safety, tolerability, and efficacy of mitazalimab in combination with chemotherapy in patients with metastatic pancreatic ductal adenocarcinoma.

[0370] This study consists of two parts (such as Figure 1 shown):

[0371] Part 1 (Phase 1b): Two dose levels of mitazalimab in combination with mFOLFIRINOX are evaluated to determine the tolerable mitazalimab dose (RP2D) for Part 2 of the study. Part 1 follows a Bayesian optimal interval (BOIN) design with at least 3 patients enrolled at each dose level. A minimum of 6 patients are evaluated at the RP2D in Part 1. If the lowest dose of mitazalimab (450 μg / kg) is found to be unsafe and not tolerated in combination with mFOLFIRINOX, mFOLFIRINOX treatment may be changed to gemcitabine plus nab-paclitaxel.

[0372] Part 2 (Phase 2): In Part 2 of the study, up to 60 enrolled patients were administered mitazalimab and mFOLFIRINOX (or gemcitabine plus nab-paclitaxel) to explore the clinical efficacy of mitazalimab in combination with chemotherapy. When the objective response rate (ORR) of a total of 23 patients (including patients from both Part 1 and Part 2) was evaluable, a Simon two-stage design was performed, and an interim analysis was performed for discontinuation due to futility or efficacy.

[0373] In Part 1, at each dose level, there was a staggered dosing of at least 11 days between the first dose of mitazalimumab administered to the first patient and the first dose of mitazalimumab administered to the second patient.

[0374] All patients were monitored for at least 4 hours after the end of the first infusion of mitazalimumab and for at least 2 hours after the second infusion of mitazalimumab. If no infusion-related reactions were observed with the most recent infusion (2nd or later infusion), monitoring of patients for subsequent infusions could be shortened to 1 hour. The monitoring period could be extended for all patients by the Data Review Committee (DRC) based on emerging safety data.

[0375] See also Figure 2 In the event that the lowest planned dose level of mirtazalimab (450 μg / kg) was found to be unsafe or intolerant in combination with mFOLFIRINOX in Part 1, mFOLFIRINOX could be switched to gemcitabine plus nab-paclitaxel, following the same mirtazalimab dose-escalation schedule. If the lowest planned dose level of mirtazalimab was found to be unsafe or intolerant in combination with gemcitabine plus nab-paclitaxel in Part 1, the study was terminated.

[0376] When the last patient in Part 1 completes the DLT assessment period and the data have been collected and reviewed by the DRC, the decision to proceed to Part 2 is made. Up to 60 patients are planned to be enrolled in Part 2.

[0377] An interim analysis was performed to allow stopping for futility or efficacy when efficacy data were 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 in all patients dosed at the RP2D of mirtazalimab. The interim analysis included efficacy data for up to 17 weeks of study treatment (8 cycles for mFOLFIRINOX and 4 cycles for gemcitabine plus nab-paclitaxel). Depending on the results of the interim analysis, a decision was made as to whether the study should stop further enrollment or continue to enroll an additional 37 patients.

[0378] For each corresponding combination of chemotherapy, the dosing schedule and visit assessment schedule were the same for Part 1 and Part 2 in the study.

[0379] Duration of treatment

[0380] Patients can receive treatment with mitazalimab and mFOLFIRINOX for up to 12 treatment cycles (approximately 6 months; one 21-day treatment period / dose-limiting toxicity (DLT) assessment period (Cycle 1) followed by eleven 14-day treatment cycles). However, treatment can be extended beyond 12 cycles until progression to unacceptable levels of toxicity is observed in the patient.

[0381] Patients can receive treatment with mirtazalimab and gemcitabine plus nab-paclitaxel for up to 6 treatment cycles (approximately 6 months; one 35-day treatment cycle (Cycle 1), of which the first 28 days will be the DLT assessment period, followed by five 28-day treatment cycles) if applicable.

[0382] Patients may continue study treatment within the above limits until disease progression (PD) or significant clinical deterioration, as long as they tolerate the treatment and agree to continue, based on the investigator's judgment. After completion of study treatment, patients will have an end-of-treatment visit and continue the treatment follow-up period to assess disease and survival status.

[0383] Dosage, dose schedule, and dose escalation

[0384] Both mFOLFIRINOX and gemcitabine plus nab-paclitaxel are well-established and effective chemotherapy regimens for the treatment of advanced pancreatic cancer. A central principle of the dose-finding portion of this study is to never compromise the intensity of the standard chemotherapy. If the adverse event is considered to be related to chemotherapy based on the clinical judgment of the investigator, the treatment can be adjusted to the regimen in a standard manner as summarized in the dose adjustment section in Example 4 (for mFOLFIRINOX) and Example 5 (for gemcitabine plus nab-paclitaxel). However, when unexpected toxicity or unexpected intensity toxicity associated with these chemotherapy is encountered during the incremental portion of this study and the investigator believes that this toxicity may impair the effectiveness of chemotherapy, the investigator can stop mitazalimab. If this occurs during the DLT period of the study, and the adverse event is not considered as a DLT, the patient can be replaced.

[0385] In Part 1, two dose levels of 450 μg / kg and 900 μg / kg of mitazalimab were planned to be administered in combination with mFOLFIRINOX. During the first treatment cycle (first 21 days) for each patient, mitazalimab was administered on days 1 and 10, and the mFOLFIRINOX infusion started on day 8. During the following 14-day treatment cycles, mFOLFIRINOX was administered on day 1 of each cycle, and mitazalimab was administered on day 3, see Figure 3In case the number of DLTs at a certain dose level meets the stopping criteria, see Table 3 for escalation rules based on the number of patients with DLTs, chemotherapy mFOLFIRINOX can be switched to chemotherapy gemcitabine plus nab-paclitaxel at the lowest dose level of mitazalimab, as Figure 2 If chemotherapy is changed from mFOLFIRINOX to gemcitabine plus nab-paclitaxel, see the dosing schedule in Example 5.

[0386] Dose escalation in Part 1 followed a BOIN design with at least 3 evaluable patients per dose level. According to the BOIN design, each time a patient completes a cohort, a decision is made as to 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, based on the decision rule presented in Table 3 below. A range of 3 to 9 patients was included at each dose level. In this study, the de-escalation from 450 μg / kg mitazalimab in combination with mFOLFIRINOX was to change chemotherapy to gemcitabine plus nab-paclitaxel, as previously described.

[0387] Table 3: Escalation rules based on the number of patients with DLTs

[0388]

[0389] NA = not applicable.

[0390] a If a higher dose level has been evaluated and the number of DLTs at that higher dose level leads to tapering, then discontinue.

[0391] Dose escalation is determined by the DRC after reviewing the safety data obtained during the DLT evaluation period and the available data from all patients in the study after the DLT evaluation period. Dose escalation is permitted when the DRC defines the dose and dose schedule for the next dose level. Based on the available data, the DRC may decide to introduce intermediate dose levels between pre-planned dose levels as well as to change the dosing frequency.

[0392] Mitazalimab in combination with mFOLFIRINOX: The DLT assessment period was defined as the time from the first dose of mitazalimab (Day 1) until Day 21 of the first treatment cycle (Cycle 1). Mitazalimab in combination with gemcitabine plus nab-paclitaxel: The DLT assessment period was defined as the time from the first dose of mitazalimab (Day 1) until Day 28 of the first 35-day treatment cycle (Cycle 1). This period allowed the DRC to evaluate the early safety of mitazalimab in combination with chemotherapy.

[0393] DLT was defined as one of the following toxicities graded according to CTCAE version 5.0 (ie, drug-related AE):

[0394] Grade 4 neutropenia lasting more than 7 days

[0395] Grade 4 thrombocytopenia lasting more than 7 days

[0396] Grade 4 infusion-related reactions

[0397] Grade 3 infusion-related reactions that do not resolve to a lower grade within 24 hours of onset

[0398] Grade 4 AST, ALT, and / or bilirubin

[0399] Grade 3 AST, ALT, and / or bilirubin that does not resolve to a lower grade within seven days

[0400] Any non-hematologic toxicity of grade 3 or higher, except for the following:

[0401] ○ Laboratory abnormalities without clinical consequences that resolve to ≤ Grade 2 within 14 days (including electrolyte abnormalities that respond to medical intervention)

[0402] ○ Fatigue

[0403] ○Nausea and / or vomiting lasting less than 48 hours

[0404] ○ Diarrhea that resolves to less than grade 3 by the end of the DLT period

[0405] The same DLT criteria apply to the combination of mitazalimab with mFOLFIRINOX and gemcitabine plus nab-paclitaxel. DLTs are considered related to study treatment unless there is a clear, well-documented alternative explanation for the adverse event (AE). AEs that meet the above criteria but occur after the DLT assessment period are not defined as DLTs unless the event occurs during the DLT assessment period. If applicable, the event may be reported as an AE / serious adverse event (SAE).

[0406] The RP2D for mitazalimab in combination with mFOLFIRINOX or gemcitabine plus nab-paclitaxel as obtained in Part 1 was administered to all patients in Part 2. Adjustments in the administration of mFOLFIRINOX or gemcitabine plus nab-paclitaxel were permitted as described in Examples 4 and 5, respectively. The same dosing schedule of mitazalimab in combination with each corresponding chemotherapy was applicable to both Parts 1 and 2 of the study.

[0407] Goals and End Points

[0408] Part 1

[0409]

[0410]

[0411] Part 2

[0412]

[0413] Evaluate

[0414] Evaluations include demographics, medical history (including previous anticancer treatment), height and weight, vital signs (blood pressure, pulse, oxygen saturation, and temperature), physical examination, ECG, ECOG performance status, and clinical laboratory tests (clinical chemistry, hematology, urinalysis), concomitant medications, and AE collection. Blood samples will be collected for pharmacokinetic, pharmacodynamic, and immunogenicity analyses. Antitumor activity will be assessed by evaluating CT scans according to RECIST v.1.1 guidelines. The post-treatment follow-up period includes disease / survival status assessments, subsequent cancer-related therapies, and antitumor activity (CT scans). The post-treatment follow-up period will continue for up to 2 years after the last patient is enrolled (LPI).

[0415] statistics

[0416] Statistical methods

[0417] No formal statistical assumptions were defined for Part 1 (Phase 1b). For Part 2, the primary endpoint of ORR was compared between mitazalimab and historical controls using a one-sided exact binomial test at the 10% significance level. All secondary and exploratory endpoints were summarized using appropriate descriptive statistics. Patients in Part 1 who received the same dose regimen as patients in Part 2 were combined with patients enrolled in Part 2 for statistical analysis and data summary.

[0418] Sample volume

[0419] In Part 1, it is estimated that at least 9 patients will be enrolled for the planned 2 dose levels, 3 patients at the lower dose level and 3+3 patients at the higher dose level. The actual number of patients in Part 1 depends on the data obtained during the study.

[0420] For Part 2, the sample size was based on an extended Simon two-stage design, including interim analyses of futility and efficacy, and assuming a 15% withdrawal rate. A total of 23 patients at RP2D (Part 1 + Part 2) with data evaluable for ORR were included in the futility and efficacy analyses. To reach this number, an estimated 27 patients need to be enrolled. If the study continues, depending on the results of the interim analysis, an additional 37 patients may be enrolled to include a total of 54 evaluable patients.

[0421] List of abbreviations and terms

[0422]

[0423]

[0424] Visit Assessment Schedule

[0425] The following visit assessment table applies to when mitazalimab is administered in combination with mFOLFIRINOX and includes all assessments to be performed during the study. Table 4 lists the study assessments to be performed during screening and treatment cycle 1. Table 5 lists the assessments to be performed during treatment cycle 2 and subsequent cycles and at the end of treatment. The assessments to be performed during the post-treatment follow-up period and at the end of the study are listed in Table 6. The timing of the different assessments is indicated by an "X".

[0426] Many pre- and post-operative medications can be administered at specific times relative to the administration of mitazalimumab and mFOLFIRINOX, respectively (up to 3 days prior to infusion).

[0427] The assessment timetable applies to both Part 1 and Part 2 of your studies.

[0428] The visit assessment schedule to be used when mitazalimab is administered in combination with gemcitabine plus nab-paclitaxel is found in Example 5.

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435] Inclusion criteria

[0436] Patients were eligible for inclusion in the study if all of the following criteria applied:

[0437] 1. Written informed consent has been provided

[0438] 2. ≥18 years old when signing the informed consent form (ICF)

[0439] 3. Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1

[0440] 4. Have been diagnosed with previously untreated metastatic pancreatic ductal adenocarcinoma (histologically documented)

[0441] 5. Have measurable disease according to RECIST v.1.1

[0442] 6. No previous chemotherapy for pancreatic ductal adenocarcinoma

[0443] 7. No prior abdominal radiotherapy (except palliative radiotherapy for non-target lesions)

[0444] 8. Life expectancy ≥ 3 months

[0445] 9. Have acceptable hematology laboratory values ​​defined as:

[0446] a. Neutrophil count ≥ 1.5x without growth factor stimulation within 3 weeks prior to blood test

[0447] 10 9 / L

[0448] b. Platelets ≥ 100 x 10 9 / L

[0449] c. Hemoglobin ≥ 6.2mmol / L (about 100g / L) (possibly after blood transfusion)

[0450] 10. Have acceptable clinical chemistry laboratory values ​​defined as:

[0451] a. Bilirubin ≤1.5x ULN (bile drainage allowed)

[0452] b.AST≤3x ULN (regardless of liver metastasis)

[0453] c.ALT ≤ 3x ULN (regardless of liver metastasis)

[0454] d. Creatinine ≤ 1.5 x ULN or glomerular filtration rate (GFR) ≥ 45 mL / min (for calculation of GFR, see Example 6)

[0455] e.INR ≤ 1.5 x ULN

[0456] f. Albumin ≥ 28 g / L

[0457] 11. For women of childbearing potential 1 :

[0458] a. Negative highly sensitive serum (β-human chorionic gonadotropin [β-hCG]) pregnancy test at screening

[0459] b. Willing to use highly effective contraceptive methods during the study treatment period and for at least six months thereafter

[0460] 12. Males of childbearing potential must use effective contraceptive methods (i.e. surgical sterilization or condoms containing spermicide) during the study treatment period and for at least six months thereafter.

[0461] 13. Willing to comply with all research procedures;

[0462] 1 Women are considered of childbearing potential (WOCBP) after menarche and until they become postmenopausal, unless permanently infertile. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. The postmenopausal state is defined as the absence of menstruation for 12 consecutive months in the absence of an alternative medical reason. A high follicle stimulating hormone (FSH) level in the postmenopausal range can be used to confirm the postmenopausal state of women who are not using hormonal contraception or hormone replacement therapy. However, a single FSH measurement is not sufficient in the absence of 12 months of amenorrhea.

[0463] Exclusion criteria

[0464] Patients were excluded if they met any of the following criteria:

[0465] 1. Other types of pancreatic non-ductal tumors, including endocrine tumors or acinar cell adenocarcinoma, cystic adenocarcinoma, and ampullary carcinoma

[0466] 2. Other current cancer or history of cancer within the previous 3 years before signing the ICF, except for cervical cancer in situ or basal cell carcinoma or squamous cell carcinoma treated with local excision only

[0467] 3. Patients with known CNS metastasis or carcinomatous meningitis

[0468] 4. Contraindications to any component of study treatment (mitazalimumab and chemotherapy if applicable)

[0469] 5. History of chronic diarrhea, inflammatory disease of the colon or rectum, or unresolved partial or complete intestinal obstruction

[0470] 6. History of myocardial infarction, uncontrolled angina, unstable arrhythmia, or New York Heart Association class II or higher congestive heart failure within 12 months of first administration of mitazalimumab

[0471] 7. With a QTc > 450 milliseconds

[0472] 8. Have uncontrolled concurrent illnesses, including active infections

[0473] 9. Have a known history of HIV, hepatitis B or active hepatitis C infection

[0474] 10. Female patients who are pregnant or breastfeeding

[0475] 11. Received attenuated vaccine within 28 days after the first dose of study treatment

[0476] 12. Any situation that the investigator believes will put the patient at greater risk or prevent the patient from complying with the study.

[0477] 13. Participation in another investigational drug or device study and any intervention within the previous 4 weeks before the first administration of mitazalimumab

[0478] Additional exclusion criteria applicable only to mFOLFIRINOX treatment:

[0479] 14. Have received previous treatment with irinotecan or platinum-containing chemotherapy

[0480] 15. Have pre-existing peripheral neuropathy greater than grade 1

[0481] 16. Known Gilbert's disease

[0482] 17. With known genotype UGT1A1*28 / *28

[0483] 18. Known fructose intolerance (malabsorption)

[0484] 19. Complete dihydropyrimidine dehydrogenase (DPD) deficiency

[0485] Additional exclusion criteria applicable only to gemcitabine plus nab-paclitaxel treatment:

[0486] 14. A history of slowly progressive dyspnea and dry cough, or a history of conditions such as sarcoidosis, silicosis, idiopathic pulmonary fibrosis, pulmonary allergies, pneumonia, or multiple allergies

[0487] 15. History of peripheral arterial disease (e.g., claudication, Leo Buerger's disease)

[0488] 16. History of connective tissue disorders (e.g., lupus, scleroderma, arteritis nodosa)

[0489] deal with

[0490] Investigational Medicinal Product (IMP) - Mitazalimab

[0491] The IMP mitazalimab is an agonistic human monoclonal (IgG1) antibody targeting CD40 that is being developed for cancer immunotherapy.

[0492] manufacture

[0493] Mitazalimab is manufactured by Biogen (USA) in accordance with GMP. The product is manufactured in a fed-batch bioreactor using a stable Chinese hamster ovary (CHO) cell line. The product does not contain any components of animal origin and is safe from the perspective of adventitious factors.

[0494] Formulation, packaging and labeling

[0495] Mitazalimab can be provided in a disposable glass vial in the form of a lyophilized cake, in which case it can be reconstituted with sterile water for injection (WFI) before use. Each vial requires the addition of 2 mL of sterile WFI to fully dissolve the cake. Each vial contains 40 mg of mitazalimab, and the resulting concentration after reconstitution will be 20 mg / mL. The exact concentration will be indicated on the master vial. Packaging, labeling and distribution to clinical sites will be performed by Fisher Clinical Services (FCS). Packaging and labeling will be completed at the FCS site in Allentown, Pennsylvania, USA, and distribution to the site will be completed by the sponsor in accordance with good manufacturing practices (GMP) by FCS in Germany. For additional details on preparation, packaging and labeling, please refer to the Mitazalimab Investigational Medicinal Product File (IMPD).

[0496] Handling, preparation and storage

[0497] According to local regulations, mitazalimab vials can be stored refrigerated at a temperature of 2°C-8°C at a local pharmacy and protected from light in a safe area. The vials must not be used after the expiration date. Preparation of mitazalimab solution for infusion will be carried out at the local pharmacy according to the instructions in the pharmacy manual. After reconstitution, mitazalimab will be diluted in normal saline or 5% dextrose in water. According to local regulations, the prepared mitazalimab solution for infusion is preferably stored at room temperature in a safe area protected from light. The mitazalimab solution for infusion can be infused within 16 hours from reconstitution of the lyophilized cake. Please refer to the pharmacy manual for detailed instructions on the preparation, handling and storage of study drugs.

[0498] Administration of mitazalimab

[0499] Mitazalimab will be administered intravenously during a 2-hour rate-controlled infusion. Baseline body weight will be used to calculate the mitazalimab dose throughout the study. If the pre-dose body weight assessment assessed at the beginning of each treatment cycle changes by >10% relative to baseline, the mitazalimab dose can be recalculated and adjusted accordingly. All patients will be monitored for at least 4 hours after the end of the first infusion of mitazalimab and for at least 2 hours after the second infusion of mitazalimab. If no infusion-related reactions were observed at the time of the most recent infusion (2nd or later infusion), monitoring of patients may be shortened to 1 hour for subsequent infusions.

[0500] Mitazalimab dose adjustments

[0501] The dose and frequency of mitazalimab administration can be adjusted for individual patients in the following ways:

[0502] ●Dose reductions may be necessary due to AEs:

[0503] ■ If the adverse event is possibly, probably, or definitely related to mirtazalimumab and results in a treatment delay of more than two weeks, the mirtazalimumab dose will be reduced by 50%. If the dose has been reduced once, mirtazalimumab treatment will be discontinued; and / or

[0504] ■If the adverse event is severe or greater than Grade 2 and is possibly, probably, or definitely related to mirtazalimumab, mirtazalimumab will be withheld until the AE resolves to the higher of baseline grade or Grade 1 or lower; at this time, mirtazalimumab may be resumed at the investigator's discretion at the previous dose or, if the dose has not been reduced, at a 50% dose reduction; the investigator may also choose to forgo rechallenge.

[0505] ●Pre-dosing may be introduced due to AEs (eg, infusion-related AEs).

[0506] Management of infusion-related reactions

[0507] If clinically significant symptoms of an infusion-related reaction occur, the infusion may be temporarily stopped (interrupted). After recovery, the infusion may be resumed for at least 30 minutes at 50% of the rate at which the reaction occurred. If clinically significant symptoms do not reappear after 30 minutes, the infusion rate may be increased according to the original infusion plan. A lower infusion rate may be selected, for example, if clinically indicated.

[0508] Depending on the timing and severity of the reaction, the investigator may consider administering supportive medications such as antihistamines, acetaminophen, or corticosteroids in addition to the mandatory premedication and postmedication described herein.

[0509] The DRC may introduce changes to the mitazalimumab infusion if severe infusion-related reactions are observed.

[0510] If it is considered that the AEs associated with the infusion (e.g., infusion-related reactions) are not well controlled by the above schedule, the DRC may decide to introduce a pre-dose of mitazalimab one day before the first administration. The pre-dose will be up to 10% of the full dose. After the decision is made by the DRC, a pre-dose may be prescribed for all enrolled patients.

[0511] If symptoms are related to the infusion, cytokine release syndrome may be indistinguishable from an infusion-related reaction. Cytokine release syndrome may also have many symptoms similar to infection or even sepsis. Cytokine release syndrome, as reported with CD3-targeted antibodies or CAR T-cell infusions, has not been observed with mitazalimab.

[0512] When reporting a reaction thought to be related to the infusion, the preferred term will be infusion-related reaction rather than cytokine release syndrome. Grade 2 or higher infusion-related reactions should be reported as adverse events of special interest (AESI).

[0513] Management of damaged liver parameters

[0514] Liver injury is defined by the following changes in liver parameters:

[0515] ●In 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 an increase in bilirubin >2 times ULN measured in the same blood draw; and / or

[0516] ●For patients with impaired liver function at baseline, i.e., AST or ALT above ULN, an AST and / or ALT elevation >5 times ULN and a combined total bilirubin elevation >2 times ULN measured in the same blood draw.

[0517] If laboratory values ​​are observed consistent with liver injury as defined above, the following laboratory tests may be repeated within 48 to 72 hours: ALT, AST, and bilirubin (total and direct). If hepatobiliary toxicity is observed, treatment may be suspended to allow evaluation of other causes, such as biliary obstruction / stent failure. Other causes may require follow-up according to Example 7 Drug-Induced Liver Injury (DILI). Grade 3 or higher increases in AST or ALT and Grade 2 or higher increases in bilirubin should be reported as AESI.

[0518] Non-investigational medicinal products

[0519] Mitazalimab pre- and post-operative medication

[0520] Premedication and postoperative medication can be given in conjunction with each mitazalimumab administration. Premedication can be started 3 days prior to the mitazalimumab infusion. Advantageous premedication and postoperative medication are listed in Table 7 below and include the type of medication and intake time associated with the mitazalimumab infusion.

[0521] Antihistamine H1 and leukotriene inhibitors can be given as premedication, for example starting 3 days before mitazarimab administration.

[0522] Table 7 Preoperative and postoperative medications to be administered with mitazalimab

[0523]

[0524]

[0525] mFOLFIRINOX

[0526] The mFOLFIRINOX used in this study consists of the components oxaliplatin, leucovorin, irinotecan, and 5-fluorouracil (5-FU) and is considered standard of care by the European Society for Medical Oncology (ESMO) and the National Comprehensive Cancer Network (NCCN) for first-line treatment of advanced pancreatic cancer based on evidence from large phase 3 trials.

[0527] The original “complete” FOLFIRINOX regimen defined in ACCORD 11 / 0402 (a phase 3 study of 342 patients) consisted of a 400-mg bolus of 5-FU followed by 2400 mg / m2 of 5-FU over 46 hours. 2 5-FU, 400 mg / m 2 Folic acid, 85 mg / m 2 Oxaliplatin and 180 mg / m 2irinotecan. Over the past decade, it has become common practice to administer a less-intense version of this regimen (collectively referred to as mFOLFIRINOX) to reduce side effects. Current practice guidelines consider the mFOLFIRINOX regimen to be equivalent to full FOLFIRINOX as the 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. Accessible via the following webpage: https: / / www.nccn.org / professionals / physician_gls / pdf / pancreatic.pdf).

[0528] Based on phase 2 trials, other variants of FOLFIRINOX have been suggested as acceptable alternatives that retain comparable clinical efficacy with an improved side effect profile. For example, these have included reducing all components by 20%

[30] or reducing the 5-FU bolus to 300 mg and irinotecan to 135 mg / m 2

[31] ’s solution.

[0529] Other clinical trials seeking to combine novel agents with FOLFIRINOX have chosen similar modifications of FOLFIRINOX, for example, no 5-FU bolus was administered in a study combining FOLFIRINOX with the hedgehog inhibitor IPI-926.

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

[33] .

[0530] The mFOLFIRINOX regimen used in this study was identical to that used in the phase 3 PRODIGE 24 trial of adjuvant pancreas and differed from the original FOLFIRINOX regimen in that the 5-FU bolus was omitted and the irinotecan dose was 150 mg / m 2

[34] .

[0531] Administration of mFOLFIRINOX

[0532] Mitazalimab can be administered in combination with the chemotherapy regimen mFOLFIRINOX as a first choice. mFOLFIRINOX can consist of the components oxaliplatin, leucovorin, irinotecan and / or 5-FU. The recommended parameters for the timing and sequence of infusions of the components of mFOLFIRINOX are described in Table 8 below.

[0533] Leucovorin is one of several folinates that can be used in combination with 5-FU for cytotoxic therapy. Leucovorin strengthens the inhibitory effect of 5-FU on thymidylate synthase, thereby enhancing the antimetabolite activity of 5-FU. Alternative folinates of leucovorin can be utilized based on local availability, including but not limited to calcium folinate, calcium leucovorin, disodium folinate and disodium leucovorin. Administration based on folinic acid as defined in the relevant SmPC can be followed. When leucovorin is mentioned in this application, it is acknowledged that similar approved folinates can be used.

[0534] Variations in administration are permitted and can be accomplished based on drug dosing and adjustment guidelines such as those described in Example 4.

[0535] Weight and height at baseline can be used to calculate the dose of the mFOLFIRNOX component. If the patient's body surface area (BSA) changes >10% from baseline, the dose can be readjusted. If the patient's BSA changes ≤10%, no adjustment is required unless the center has a standard procedure for adjusting the dose based on the current BSA according to institutional guidelines.

[0536] Table 8 mFOLFIRINOX Administration

[0537]

[0538] Recommended infusion schedule and sequence:

[0539] ●IV oxaliplatin within 2 hours, immediately thereafter;

[0540] ●Leucovorin or a similar approved folinate within 2 hours;

[0541] ●Administer irinotecan over 90 minutes (starting 30 minutes after the start of the leucovorin infusion), followed by;

[0542] ●5-FU infusion over 46-48 hours.

[0543] If one of the mFOLFIRINOX components is stopped, one or more of the other components may be given. In the event that the combination of mFOLFIRINOX with the lowest planned dose level of mitazalimab (450 μg / kg) is considered unsafe and not tolerated, mFOLFIRINOX may be switched to gemcitabine plus nab-paclitaxel, and / or a lower dose of mitazalimab may be used. The dose schedule and dosing adjustments allowed for gemcitabine plus nab-paclitaxel are described in Example 5.

[0544] Pre- and post-operative medications for mFOLFIRINOX.

[0545] Premedication and postmedication can be given in conjunction with each mFOLFIRINOX administration. The recommended premedication should be started one hour before the administration of oxaliplatin (the first component of mFOLFIRINOX). The recommended premedication and postmedication are listed below.

[0546] Recommended premedication may include one or more of the following given 30-60 minutes prior to the infusion:

[0547] NK1 receptor antagonists, such as Aprepritant, 125 mg PO, 60 minutes before infusion, and may be continued during chemotherapy days. Aprepritant is a cytochrome 2C9 inducer and may inactivate some oral contraceptives. Therefore, women of childbearing potential may use alternative forms of contraception if Aprepritant is administered.

[0548] ●5-HT3 receptor antagonists, such as ondansetron, 8 mg PO, 30 minutes before infusion and can be continued during the days of chemotherapy.

[0549] Corticosteroids, such as dexamethasone, 8 mg IV or PO 30 minutes before infusion.

[0550] Postoperative medications may include the following:

[0551] G-CSF, such as Neulasta, 6 mg SC, on day 4 after initiation of the mFOLFIRINOX regimen (at least 24 hours after completion of the continuous 5-FUiv infusion).

[0552] Alternative premedication and postmedication with mFOLFIRINOX were permitted.

[0553] Gemcitabine and nab-paclitaxel (if applicable)

[0554] Gemcitabine and nab-paclitaxel are considered standard of care for first-line treatment of advanced 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 3 trials.

[0555] Because of the possibility of irreversible infertility resulting from treatment with gemcitabine or nab-paclitaxel, participants should be informed to seek advice regarding germline donation and cryopreservation prior to study treatment.

[0556] Gemcitabine plus nab-paclitaxel may be provided / obtained according to the clinical study protocol and in accordance with local guidelines. Gemcitabine and nab-paclitaxel are to be stored and handled according to the package insert and stored in a secure place under appropriate storage conditions.

[0557] Administration of gemcitabine and nab-paclitaxel

[0558] In the event that mFOLFIRINOX is found not feasible for a particular subject, gemcitabine plus nab-paclitaxel may be given in combination with mitazalimab. The recommended (exemplary) parameters for the timing and sequence of infusions of gemcitabine and nab-paclitaxel are listed in Table 9 below, respectively. Variations in administration are permitted if the drug dosing and adjustment guidelines are followed, see Example 5.

[0559] Table 9 Administration of gemcitabine and nab-paclitaxel

[0560]

[0561] Recommended infusion schedule and sequence:

[0562] ●Infuse nab-paclitaxel over 30 to 40 minutes;

[0563] ●Infuse gemcitabine over 30 minutes immediately following the completion of the nab-paclitaxel infusion.

[0564] Research Assessment

[0565] Demographics and medical history

[0566] The age, sex, race, and ethnic origin of the study participants were recorded. Clinically significant abnormal findings observed during the physical examination and non-serious AEs occurring pre-treatment (ie, after signing the ICF but before the first dose) may be recorded as medical history.

[0567] Cancer disease states, including cancers other than the disease under study

[0568] The date of initial histopathological diagnosis of pancreatic adenocarcinoma is recorded, as well as the disease stage at diagnosis and the current disease stage. Surgery for cancer may be recorded, including information on whether the surgery was with curative intent. Radiation therapy may also be recorded when it was given for palliative purposes.

[0569] Any prior anticancer treatment for cancer diseases other than pancreatic adenocarcinoma may be recorded (if applicable), including:

[0570] ● Treatment characteristics, including surgery, radiation therapy, and chemotherapy (if applicable);

[0571] The start and end dates of treatment;

[0572] ●Best response (CR, PR, SD, PD, and NE);

[0573] ●Reason for discontinuation (for treatment discontinued before initially planned).

[0574] Weight and height

[0575] Body weight (excluding coat and shoes) was measured at Screening and during treatment and rounded to the nearest kilogram. The dose calculation of mitazalimumab was based on body weight. The dose of mitazalimumab could be recalculated in the event of a change in body weight of more than 10% from baseline measurement. The dose calculation of the components of mFOLFIRINOX, gemcitabine, and nab-paclitaxel was based on BSA. The dose of the components of mFOLFIRINOX, gemcitabine, and nab-paclitaxel could be recalculated in the event of a change in BSA of more than 10% based on baseline measurement calculated using body weight. Additional weight assessments could be performed based on the discretion of the investigator at scheduled visits or at additional (unscheduled) visits. Height was measured at Screening (without shoes) and rounded to the nearest centimeter.

[0576] Vital signs

[0577] Vital sign measurements include systolic and diastolic blood pressure, pulse rate, oxygen saturation and temperature. Preferably, the same equipment is used in each visit, and blood pressure is measured after the patient has rested for 10 minutes. Any new or aggravated clinically significant abnormal findings compared to the pre-treatment assessment will be reported as AEs. Additional vital sign assessments may be performed based on the investigator's judgment during regular visits or during additional (unplanned) visits.

[0578] Physical examination

[0579] Physical examination may include general appearance and examination of the following body systems: eyes, mouth and throat, lymph node areas, respiratory system, cardiovascular system, abdomen, extremities, and skin. Neurological examination may be performed based on the investigator's judgment. Any new or aggravated clinically significant abnormal medical findings compared to the pre-treatment assessment may be reported as AEs. Additional assessments of physical examination may be performed based on the investigator's judgment at regular visits or at additional (unplanned) visits.

[0580] Electrocardiogram (ECG)

[0581] A standard 12-lead ECG was recorded and the ECG was interpreted in its entirety by the Investigator or, if applicable, the Investigator delegated this task to a cardiologist. The ECG printout was signed and dated after review and interpretation. For the ECG recording, the patient had to be at rest and in a horizontal or semi-recumbent position for at least 10 minutes. The same assessment method could be used throughout the study. Any abnormality observed or occurring during the ECG (e.g., vomiting, coughing) could result in a repeat of the ECG. Any new or worsening clinically significant abnormal ECG finding compared to the pre-treatment assessment could be reported as an AE. Additional ECG recordings could be performed based on the Investigator's discretion at scheduled visits or at additional (unplanned) visits.

[0582] ECOG performance status

[0583] The researchers scored the patients' performance status in activities of daily living using the Eastern Cooperative Oncology Group (ECOG) performance status scale

[35] provided in Table 10 below.

[0584] Table 10 ECOG physical status

[0585]

[0586]

[0587] Clinical laboratory tests

[0588] Timings for clinical laboratory assessments are specified in Section 0 Visit Assessment Schedule. However, more frequent testing may be performed if indicated by the patient's clinical condition or by abnormalities that the investigator deems warrant more frequent monitoring. Screening laboratory results must be available and reviewed by the investigator prior to the first dose of mirtazalimumab. Results from clinical laboratory tests are summarized in the clinical study report. Therefore, worsening of these parameters compared to pretreatment values ​​will be reported as AEs only if they meet any of the criteria for an SAE or are a reason for modification of study treatment. Worsening of laboratory values ​​clearly due to disease progression will not be reported as an AE.

[0589] Procedures for the collection, preparation, and processing of blood and urine samples are performed in accordance with local procedures. The DPD test obtained at screening can be analyzed at a central laboratory or in accordance with local procedures. The clinical laboratory tests to be performed are listed in Table 11. Although urinalysis tests can be performed and evaluated by field personnel, all tests can be performed by local laboratories. If an abnormal urinalysis result (urine test strip) is considered clinically significant, a microscope can be used to measure the sediment, i.e., red blood cells, white blood cells, epithelial cells, crystals, casts, and bacterial cultures.

[0590] Table 11 Clinical laboratory tests

[0591]

[0592]

[0593] Immunogenicity (anti-drug antibodies)

[0594] Blood samples (serum) for immunogenicity testing were obtained according to the Visit Assessment Schedule in Section 0 for the combination of mitazalimumab and mFOLFIRINOX, and according to the Visit Assessment Schedule in Example 5 for the combination of mitazalimumab and gemcitabine and nab-paclitaxel. Samples may also be collected at additional time points, at regularly scheduled visits, or at additional (unscheduled) visits, based on the investigator's discretion.

[0595] If the infusion of mitazalizumab is interrupted due to an AE, samples for immunogenicity (except during the infusion) and PK samples can be collected at the time of the interruption or as soon as possible considering patient safety. Samples used for immunogenicity testing can be used for anti-drug antibody (ADA) analysis (i.e., antibodies against mitazalizumab). Samples analyzed for immunogenicity and confirmed to be positive can be tested for neutralizing antibodies. Other analyses may be performed to further characterize the immunogenicity of mitazalizumab. If deemed necessary, PK samples collected at other time points noted in the visit assessment schedule may be analyzed for immune response. Details on sample collection and processing will be provided in the laboratory manual.

[0596] Pharmacokinetics (PK)

[0597] For the combination of mitazalimab and mFOLFIRINOX, blood samples (serum) are obtained for analysis of mitazalimab concentration and PK analysis according to the Section 0 Visit Assessment Schedule and for the combination of mitazalimab and gemcitabine and nab-paclitaxel according to Example 5. PK samples may be collected at additional time points, at regular visits, or at additional (unplanned) visits at the discretion of the investigator. Samples for PK analysis are preferably obtained from a peripheral vein on the opposite side of the arm infused with mitazalimab. If the infusion of mitazalimab is interrupted due to an AE, PK samples and samples for immunogenicity may be collected at the time of interruption or as soon as possible for patient safety (except during the first infusion, when samples for immunogenicity do not need to be obtained).

[0598] The following PK parameters will be derived for mitazalimab:

[0599] ●C max

[0600] ●T max

[0601] AUC (0-T)

[0602] If the data allow, other PK parameters can be derived, such as:

[0603] AUC 0-∞

[0604] AUC τ

[0605] ● Elimination half-life (T 1 / 2 )

[0606] Total serum clearance (CL)

[0607] ● Volume of distribution (V d )

[0608] Evaluation of antitumor activity

[0609] Computed tomography (CT) scan

[0610] CT scans of the chest / abdomen / pelvis may be obtained according to local practice. CT scans of other body regions may also be obtained if needed to evaluate the tumor (e.g., a CT scan of the neck is required for patients with cervical lymph nodes or head and neck tumors). Additional CT scans may be obtained based on the investigator's discretion at scheduled visits or at additional (unscheduled) visits. The use of intravenous contrast was at the discretion of the radiologist performing the scan, but imaging must be consistent for each patient throughout the study.

[0611] If a CT scan is not feasible at the discretion of the investigator, magnetic resonance imaging (MRI) may be performed. The same scanning modality is preferably used throughout the study. The investigator and / or radiologist identifies the tumors to be followed throughout the study.

[0612] CT scans were performed at screening (within 28 days of first administration), 9 weeks after the start of study treatment (at the end of cycle 4 for mFOLFIRINOX combination and at the end of cycle 2 for gemcitabine plus nab-paclitaxel treatment), and every 8th week thereafter (assuming no treatment delays). Additional CT scans may be performed at unplanned visits at the discretion of the investigator. CT scans were evaluated according to RECIST v.1.1. Patients with a response (PR or CR) may have a confirmatory CT scan at least 4 weeks later to confirm the response. If a patient has progressive disease, the patient should stop treatment.

[0613] Assessment of tumor response

[0614] Tumor response was assessed by the study site according to RECIST v.1.1 guidelines using CT scans according to Example 3 RECIST v.1.1. Patients with PD could discontinue study treatment. However, if the investigator considered patients with suspected progression to be clinically stable, they were allowed to continue treatment until clinical or radiographic progression was documented.

[0615] Pharmacodynamics

[0616] The purpose of pharmacodynamic biomarkers is to characterize changes in intratumoral and systemic immune activation associated with the mode of action of mitazalimab and to correlate pharmacodynamic effects with clinical responses. Disease progression and treatment response will also be followed by liquid biopsies, analyzing biomarkers such as carbohydrate antigen 19-9 (CA19-9) and circulating tumor DNA (ctDNA). All biomarker analyses in blood and tumor biopsies are exploratory in nature. All exploratory analyses are performed in fit-for-purpose laboratories as data from the analyses are intended for scientific use and decision making only. These data may be included in scientific publications but will not be included in clinical study reports.

[0617] Blood

[0618] For the combination of mitazalimumab and mFOLFIRINOX, blood samples will be obtained according to the Section 1 Visit Assessment Schedule, and for the combination of mitazalimumab and gemcitabine plus nab-paclitaxel, blood samples will be obtained according to Example 5.

[0619] Different types of blood samples will be obtained and evaluated for the following biomarkers of drug efficacy:

[0620] Cytokines and Chemokines: Serum samples are analyzed for levels of cytokines and chemokines involved in immune activation by mitazalimumab, including but not limited to IFN-γ, TNF-α, IL12p70, IL-6, MCP-1, IP-10, MIP-1α, MIP-1β, and IL-8; for example, using a 30-plex kit with Luminex, MSD, or the like.

[0621] Immunophenotyping: Whole blood samples will be used for immunophenotyping of whole blood to quantify immune cell populations and immune cell activation that are directly or indirectly involved in immune activation with mitazarimab using flow cytometry.

[0622] The following biomarker panels were analyzed:

[0623] T / NK / NKT cells (eg, CD45, CD3, CD8, CD4, CD16, CD56, CCR7, CD45RA + activation markers CD25, Ki67);

[0624] B cells (eg, CD45, CD19, CD27, IgD+ activation markers CD86, CD83, CD54, HL-DR); and / or

[0625] ●Monocytes and DCs (CD45, CD14, CD16, CD11c, CD123 + activation markers CD54, CD86, HLA-DR, CD83).

[0626] Whole blood RNA: Whole blood RNA samples will be collected for analysis of gene signatures involved in immune activation with mitazalimumab (including but not limited to: immune score, IFNγ gene profile, APC gene profile, and T cell gene profile) and prognostic tumor gene profiles (e.g., TMB and MSI-high), and can be analyzed using, for example, Nanostring IO 360, RNAseq, or the like.

[0627] Whole blood DNA: Cell-free whole blood DNA samples will be collected for evaluation of circulating tumor DNA (ctDNA). Changes in tumor-specific genes (e.g., KRAS) will be tracked as biomarkers for monitoring treatment response. Whole blood DNA can be collected for evaluation of TCRb clonality.

[0628] CA19-9: Changes in levels of the pancreatic serum protein CA19-9 will be followed as a biomarker for monitoring disease progression and treatment response.

[0629] Tumor biopsy

[0630] For the combination of mitazali monoclonal antibody and mFOLFIRINOX, tumor biopsies are collected according to the visit assessment schedule of Chapter 1, and for the combination of mitazali monoclonal antibody and gemcitabine plus albumin-bound paclitaxel, tumor biopsies are collected according to Example 5. In the case where the tumor is inaccessible or the biopsy procedure exposes the patient to a higher risk as judged by the investigator, the collection of biopsies can be omitted. If a biopsy cannot be obtained at the time of screening, archived biopsy materials (i.e., tissue sections or tissue blocks, preferably from the tumor biopsy collected most recently before enrollment) can be collected if possible. If a biopsy (fresh or archived) is not obtained at the time of screening, no additional biopsies will be collected during the study. Biopsy (core biopsy, 18-gauge needle) will be obtained under local anesthesia. Throughout the study, tumor biopsies are preferably obtained from the same tumor. Imaging can be used to guide biopsies. Tumor tissue collected by puncture biopsy or excisional biopsy is an acceptable alternative to skin tumors. Before performing a tumor biopsy, anticoagulants can be suspended according to medical instructions. Biopsies can be formalin fixed and paraffin embedded.

[0631] Analysis of a tumor biopsy may include:

[0632] Immunohistochemistry: Archived or freshly collected tumor biopsies can be analyzed for CD40 target expression and immune cell infiltration at baseline and correlated with clinical response. On-treatment biopsies will be compared with baseline biopsies for analysis of immune cell infiltration and immune activation induced by mitazarimab for mechanistic evidence.

[0633] The following immunohistochemistry (IHC) panels:

[0634] CD40 target expression

[0635] T cell infiltration and activation status

[0636] ●Infiltration of macrophages with M1 / M2 differentiation and activation status;

[0637] ●Cell surface markers associated with immune regulation, such as PD-L1; and / or

[0638] • Other markers describing mechanistic evidence, such as, for example, markers of fibrosis and matrix (eg, fibronectin and type I collagen) macrophage function associated with macrophage function.

[0639] Genetic analysis: Baseline and on-treatment tumor biopsies will be collected for analysis of gene signatures involved in immune activation with mitazalimab (including but not limited to: immune score, IFNγ gene profile, APC gene profile, and T cell gene profile) and prognostic tumor gene profiles (e.g., TMB and MSI-high) and can be analyzed using, for example, Nanostring IO

[0640] 360, RNAseq or similar analysis.

[0641] Any tumor biopsy material remaining after the described analyses was stored in a biobank for possible future analysis.

[0642] Disease / survival status (follow-up after treatment)

[0643] During post-treatment follow-up, disease and survival status were collected, see Table 4 in Section 0 Visit Assessment Schedule for the combination of mirtazalimumab and mFOLFIRINOX and Table 22 in Example 5 for the combination of mirtazalimumab and gemcitabine plus nab-paclitaxel.

[0644] The following will be assessed:

[0645] ● Survival; and / or

[0646] ●Current disease stage.

[0647] If the patient dies, the date and cause of death will be collected and recorded. Disease / vitality status can be followed up via the patient's medical record (as permitted by local regulations) or telephone contact. Where permitted by local law, public records can be used to record death for the purpose of obtaining vital status.

[0648] Subsequent cancer-related therapy (post-treatment follow-up)

[0649] During the post-treatment follow-up period, all subsequent cancer-related therapies were collected. Subsequent cancer-related therapies could be followed up via the patient's medical dose (as permitted by local regulations) or telephone contact.

[0650] statistics

[0651] Research Hypothesis

[0652] For Part 1 (Phase 1b), no formal statistical assumptions were defined.

[0653] For Part 2 (Phase 2), the null hypothesis for the primary endpoint of ORR was:

[0654] H0: ORR = 30%

[0655] Test the null hypothesis against the alternatives:

[0656] H1: ORR>30%

[0657] Sample size considerations

[0658] In part 1, it was estimated that at least 9 patients would be enrolled at both dose levels, with at least 3 patients at each dose and at least 6 patients at RP2D. For part 2, the sample size calculation was based on the assumption of a 30% ORR for mFOLFIRINOX [4]. It was assumed that mitraz was added to increase the ORR to approximately 45%. It was assumed that gemcitabine plus nab-paclitaxel had a similar ORR (29%) to mFOLFIRINOX [5], and all other assumptions were the same, so the sample size estimate was considered valid for both treatment options. The sample size estimate was based on an extended Simon two-stage design divided into futility and efficacy

[36] . The optimal design was selected because it maximized the expected sample size. At the interim analysis, there were two limits for the number of responders: one was stopping due to futility and the other was stopping due to efficacy. If the number of responders was between these limits, the study would continue. Table 12 presents the sample sizes.

[0659] Table 12 Extended Simon two-stage design

[0660]

[0661]

[0662] Assuming a 15% withdrawal rate (not evaluable for response), the interim analysis requires a total of 27 enrolled patients at RP2D to reach 23 evaluable patients. Patient recruitment continues during the interim analysis. If the study continues after the interim analysis, assuming the same withdrawal rate as above, a total of 64 patients will need to be enrolled at RP2D to reach 54 evaluable patients.

[0663] Statistical methods

[0664] Main Benefits

[0665] The primary endpoint is the objective response rate (ORR), which is defined as the proportion of patients who reach a confirmed complete response (CR) or partial response (PR) according to RECIST v.1.1 at any time during the study. For interim analysis, ORR is based on the response assessment for mitazali monoclonal antibody combined with mFOLFIRINOX after 8 cycles or for mitazali monoclonal antibody combined with gemcitabine plus albumin-bound paclitaxel after 4 cycles (i.e., about 4 months after the first administration). For interim analysis, it is not necessary to confirm the response according to RECIST v.1.1. The primary endpoint ORR between mitazali monoclonal antibody and historical controls is compared using a one-sided exact binomial test performed at a 10% significance level. A bilateral exact binomial 90% confidence interval is also presented. In addition, ORR is presented for each study visit. Patients who cannot evaluate ORR and complete at least 2 cycles are considered non-responders.

[0666] Secondary effects

[0667] All secondary efficacy endpoints are presented using descriptive statistics. The best overall response (BOR) of each patient will be defined as the best response from RECIST v.1.1 categories CR, PR, SD and PD at any time during the study. Disease control rate will be defined as CR, PR or SD at each visit, as determined by RECIST v.1.1. Duration of response (DoR) is defined as the number of days from the initial response of CR or PR to disease progression or death (whichever comes first) caused by underlying disease. The duration of stable disease (SD) is defined as the number of days from the first administration of mitazali monoclonal antibody to disease progression or death (whichever comes first). The time to the next anticancer therapy is defined as the number of days from the first administration of mitazali monoclonal antibody to the start of the next treatment. Kaplan-Meier curves are presented for the duration of DoR, SD and the time to the next anticancer therapy. Progression-free survival (PFS) was defined as the number of days from the first dose of mitazalimumab to disease progression or death from any cause, whichever occurred first. Overall survival (OS) was defined as the number of days from the first dose of mitazalimumab to the day 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.

[0668] Security Analysis

[0669] The number of patients experiencing AEs and the number of events are summarized by SOC and preferred term. In addition, the following AEs are presented:

[0670] The incidence of DLTs (Part 1);

[0671] Grade 3 or higher AEs;

[0672] Serious AEs

[0673] AEs leading to discontinuation of study treatment; and

[0674] ●AEs leading to death.

[0675] Analyze the population

[0676] Patients in Part 1 who were on the same dosing regimen as patients in Part 2 were combined with patients in Part 2 for statistical analysis and data summary.

[0677] Full Analysis Set (FAS)

[0678] The FAS included all patients with at least 1 post-baseline efficacy measurement. The FAS population was used for all efficacy and exploratory endpoints.

[0679] Security Set

[0680] The safety set included all patients who received any study treatment (mitazalimab) and was used for all endpoints related to the safety objective.

[0681] Each Protocol Set (PP)

[0682] The PP set included all patients who completed at least one treatment cycle and had at least 1 post-baseline efficacy measurement without major protocol deviations. In addition to the FAS, the PP was used for all endpoints related to efficacy objectives.

[0683] Dose escalation in part 1

[0684] Part 1 follows a Bayesian optimal interval (BOIN) design with at least 3 evaluable patients per dose level. BOIN is described in statistical detail in Ref.

[37] and in clinical application in Ref.

[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 cohort of patients is completed, an assessment is made as to 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, based on the decision rules presented in Table 3. The BOIN design has the simplicity of the 3+3 design by Dose escalation / decrease is determined by comparing 0 / 3, 1 / 3, 2 / 3, 0 / 6, 1 / 6, and 2 / 6. In the BOIN design, this decision is based on With two predetermined fixed bounds, λ e and λ d Among them is the DLT rate observed at the current dose level (number of patients with DLT / number of patients treated). In this study, the target toxicity level, i.e., the target DLT rate, is 30%, and the boundary λ e =0.236 and λ d =0.359.

[0685] Interim analysis in Part 2

[0686] According to the extended Simon two-stage design, an interim analysis was performed when 23 evaluable patients at RP2D completed 17 weeks of treatment (8 treatment cycles for the combination of mirtazalimab and mFOLFIRINOX or 4 cycles for the combination of mirtazalimab and gemcitabine plus nab-paclitaxel). The cutoffs for stopping due to futility or efficacy were ≤7 or ≥12 responders (ORR), i.e., the study continued if there were 8-11 responders.

[0687] Example 3: Appendix 1 Response Evaluation Criteria in Solid Tumors - RECIST v.1.1 Guidelines

[0688] The revised RECIST v.1.1 guidelines are available at:

[0689] https: / / ctep.cancer.gov / protocoldevelopment / docs / recist_guideline.pdf

[0690] definition

[0691] In this trial, the international standards (version 1.1) [Eur J Cancer.45 (2009) 228-247] proposed by the solid tumor response evaluation criteria (RECIST) committee were used to evaluate response and progression. Only the change of the maximum diameter (one-dimensional measurement) of the tumor lesion was used in the RECIST v.1.1 standard. Note: Using the standards provided below, lesions are measurable or unmeasurable. The term "evaluable" about measurability will not be used because it does not provide additional meaning or accuracy.

[0692] Measurable disease

[0693] Measurable disease is defined by the presence of at least one measurable lesion. A measurable lesion is defined as a lesion that can be accurately measured in at least one dimension [the longest diameter (LD) in the measurement plane to be recorded], where the smallest dimension is:

[0694] ●10mm through CT scan (CT scan slice thickness is no more than 5mm);

[0695] ●Measurement by 10-mm caliper during clinical examination (lesions that cannot be accurately measured with caliper should be recorded as non-measurable); and / or

[0696] ●20mm through chest x-ray.

[0697] Malignant lymph nodes: When evaluated by CT scan (CT scan slice thickness not exceeding 5 mm), lymph nodes must be ≥15 mm in the short axis to be considered pathologically enlarged and measurable.

[0698] Unmeasurable disease

[0699] All other lesions (or disease sites), including small lesions (pathologic lymph nodes <10 mm in longest diameter or ≥10 to <15 mm in short axis), were considered non-measurable disease. Lesions considered truly non-measurable included: leptomeningeal disease, ascites, pleural / pericardial effusions, cutaneous / pulmonary lymphangitis, inflammatory breast disease, and abdominal masses / abdominal organomegaly that were detected by physical examination but not examined by CT or MRI.

[0700] Bone lesions, cystic lesions, and lesions previously treated with local therapy may be considered as follows:

[0701] Bone lesions:

[0702] Bone scans, positron emission tomography (PET) scans, or plain radiographs are not considered adequate imaging techniques to measure bone lesions. However, these techniques can be used to confirm the presence or absence of bone lesions.

[0703] A lytic bone lesion with an identifiable soft tissue component that can be assessed by cross-sectional imaging techniques (ie, CT or MRI) if the soft tissue component meets the definition of measurability above

[0704] Or mixed lytic-blastic lesions can be considered measurable lesions.

[0705] ●Blastogenic bone lesions are not measurable.

[0706] Cystic lesions:

[0707] ●Lesions that meet the criteria for radiologically defined simple cysts should not be considered malignant lesions (neither measurable nor non-measurable) because, by definition, they are simple cysts.

[0708] • "Cystic lesions" thought to represent cystic metastases may 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 preferably selected as target lesions.

[0709] Lesions treated with previous topical therapy:

[0710] • Tumor lesions located in previously irradiated areas or areas subjected to other local therapies are generally not considered measurable unless there is demonstrated progression of the lesion.

[0711] Target lesions

[0712] All measurable lesions (up to two lesions per organ and a total of five lesions representing all affected organs) can be identified as target lesions and recorded and measured at baseline. Target lesions should be selected based on the size of the lesion (lesion with the largest diameter) and its suitability for accurate repeated measurements (either by imaging technology or clinically). The sum of the diameters of all target lesions (longest axis for non-nodal lesions and short axis for nodal lesions) is calculated and reported as the baseline total diameter. The baseline total diameter is used as a reference for characterizing objective tumor response.

[0713] Lymph node evaluation

[0714] For lymph nodes, measurements can be made of the short axis, which is defined as perpendicular to the LD of the lymph node being evaluated in the measurement plane:

[0715] ●If the short axis is ≥15 mm, it is a target lesion;

[0716] ●If the short axis is ≥10 but <15 mm, it is a non-target lesion;

[0717] ●If the short axis is <10mm, it is normal.

[0718] For baseline, the actual short-axis measurement was added to the sum of the LDs of non-nodal lesions.

[0719] Non-target lesions

[0720] 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 tracked as "present," "absent," or, in rare cases, "definitely progressed." In addition, multiple non-target lesions involving the same organ can be recorded as a single item on the case report form (e.g., "multiple enlarged pelvic lymph nodes" or "multiple liver metastases").

[0721] Guidelines for assessing measurable disease

[0722] All measurements can be made and recorded in metric notation using a ruler or caliper. All baseline assessments can be performed as close to the start of treatment as possible, but no more than 4 weeks before the start of treatment. It is preferred to use the same assessment method and the same technology to characterize each identified and reported lesion at baseline and during follow-up. When both methods are used to assess the anti-tumor effect of treatment, imaging-based assessment is superior to assessment by clinical examination.

[0723] Clinical lesions. Clinical lesions are considered measurable only if they are superficial and ≥10 mm in diameter as assessed using calipers (e.g., skin nodules). In the case of skin lesions, documentation by color photography is recommended, including a ruler to estimate the size of the lesion. When a lesion can be assessed by both clinical examination and imaging, imaging assessment should be performed as it is more objective and can be reviewed at the end of the trial.

[0724] Chest x-ray. Chest CT is preferred over chest x-ray, especially when progression is the important endpoint. Lesions on chest x-ray can be considered measurable if they are well-circumscribed and surrounded by aerated lung.

[0725] Conventional CT and MRI. This guideline defines the measurability of lesions on CT scans based on the assumption that CT slice thickness is 5 mm or less. When a CT scan has a slice thickness >5 mm, the minimum size of a measurable lesion should be twice the slice thickness. In some cases (e.g., for body scans), MRI is acceptable. Ultrasound (US). US should not be used to measure neoplastic lesions. US examinations cannot be reproduced in their entirety for later independent review because they are operator-dependent. If a new lesion is identified by US, confirmation by CT or MRI is recommended. MRI may be used instead of CT if radiation exposure is a concern at the time of CT.

[0726] Endoscopy, laparoscopy. These techniques are not recommended for objective tumor assessment. However, such techniques may be used to confirm a complete pathological response when a biopsy is obtained or to determine recurrence in trials where a complete response or recurrence after surgical resection is an endpoint.

[0727] Tumor markers. Tumor markers alone cannot be used to assess objective tumor response. If markers are initially above the upper limit of normal, they must be normalized for subjects to be considered complete clinical responses.

[0728] Cytology, histology. These techniques can be used to differentiate between a partial response (PR) and a complete response (CR) in rare circumstances (e.g., residual disease in tumor types such as germ cell tumors, where known residual benign tumor may remain).

[0729] Response Standard

[0730] Evaluation of target lesions

[0731]

[0732] Target lymph node assessment

[0733] Lymph nodes identified as target lesions may have their actual short-axis measurements recorded (measured in the same anatomical plane as the baseline examination), even if the node regresses to less than 10 mm during the trial. To qualify for a CR, each lymph node may achieve a short axis of <10 mm. For PR, SD, and PD, the actual short-axis measurements of the lymph nodes should be included in the sum of the target lesions.

[0734] Target lesions becoming "too small to measure"

[0735] All lesions (nodal and non-nodal) recorded at baseline can have their actual measurements recorded at each follow-up assessment, even if very small (e.g., 2 mm). If the radiologist believes that the lesion has disappeared, the measurement can be recorded as 0 mm. If the lesion is considered to be present and faintly visible but too small to be measured, a default value of 5 mm can be specified.

[0736] Lesions that split or merged during treatment

[0737] When non-nodular lesions are fragmented, the longest diameters of the fragmented parts can be added to calculate the target lesion sum. Similarly, when lesions are merged, the plane between them can be maintained, which will help obtain diameter measurements for each individual lesion. If the lesions have truly merged so that they are no longer separable, the vector of longest diameters can be the maximum longest diameter of the "merged lesion".

[0738] Evaluation of non-target lesions

[0739]

[0740]

[0741] New lesions

[0742] The finding of a new lesion can be unequivocal (i.e., not attributable to differences in scanning technology, 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 anatomic locations not scanned at baseline in a follow-up trial are considered new lesions and indicate disease progression. If a new lesion is equivocal, continued therapy and follow-up assessments can clarify whether it represents true new disease. If a repeat scan confirms that this is definitely a new lesion, the date of the initial scan can be used to declare progression.

[0743] Assessment of best overall response

[0744] The best overall response is the best response recorded from the start of treatment until disease progression / recurrence (with the minimum measurement recorded since the start of treatment as a reference for disease progression). The best overall response allocation of the subject depends on the discovery of both target disease and non-target disease, and takes into account the appearance of new lesions. In addition, depending on the nature of the trial, confirmatory measurements may also be required. Specifically, in non-randomized trials with response as the primary endpoint, PR or CR is preferentially confirmed to regard either of them as the "best overall response". Table 13 provides a summary of the calculation of the overall response status of subjects with measurable disease at baseline at each time point.

[0745] Table 13 Time Point Responses: Subjects with Target (+ / - Non-Target) Disease

[0746]

[0747] Determination of best response for studies requiring confirmation of CR or PR: A complete or partial response may be claimed only if each criterion is confirmed by repeat assessment at least 4 weeks later. In this case, the best overall response can be interpreted as in Table 14.

[0748] Table 14 Best overall response when confirmation of CR and PR is required

[0749]

[0750]

[0751] Confirmatory measurements / duration of response

[0752] confirm

[0753] To designate a status of PR or CR, changes in tumor measurements can be confirmed by repeat assessments, preferably 4 weeks after the response criteria are first met. In the case of SD, preferably follow-up measurements meet SD criteria at least once after the start of the trial, with a minimum interval of 7 weeks.

[0754] Duration of overall response

[0755] The duration of overall response was measured from the time the measurement criteria for CR or PR were met (whichever was recorded first) until the first day of objectively documented relapse or disease progression (the smallest measurement recorded since the start of treatment was used as a reference for disease progression). The duration of overall CR was measured from the time the measurement criteria for CR were first met until the first day of objectively documented relapse.

[0756] Duration of disease stability

[0757] Stable disease was measured from the start of treatment until progression criteria were met, with the smallest measurement recorded since the start of treatment as reference.

[0758] Example 4: Appendix 2mFOLFIRINOX and Permitted Dose Modifications

[0759] Guidelines for dose modifications of mFOLFIRINOX that were permitted during the study are described in this appendix. All other information related to mFOLFIRINOX treatment is described throughout the protocol.

[0760] Rules for missed doses and schedule adjustments

[0761] Dose modifications for mFOLFIRINOX treatment are outlined below. Toxicity is graded based on CTCAE v 5.0. Dose modifications should be made based on the system showing the greatest degree of toxicity. For both hematologic and non-hematologic toxicities, the dose will be reduced one level at a time.

[0762] • Three levels of dose adjustment were allowed for each component of mFOLFIRINOX according to the following criteria (Table 15).

[0763] If toxicity requiring dose adjustment occurs after the third dose reduction of any component, additional dose reductions are not permitted. However, alternative treatments may be discussed with the medical supervisor.

[0764] Table 15 Dosage Adjustment of mFOLFIRINOX

[0765]

[0766] Patients may discontinue mFOLFIRINOX and mitazalimab treatment if treatment is maintained for more than 4 consecutive weeks for treatment-related toxicity. However, if the patient is clinically benefiting at the end of the 4-week maintenance, the investigator may contact the medical monitor for possible continuation of protocol-based therapy. Patients who discontinue study treatment may have an end-of-treatment visit and enter the post-treatment follow-up period. Following the end-of-treatment visit, patients will receive recommendations from the investigator regarding standard of care treatment.

[0767] Determinations regarding the need for dose adjustments of 5-FU, irinotecan, oxaliplatin, and / or leucovorin may be made based on guidelines according to the system outlined below. Management of toxicities and supportive care, except as noted below, may be performed at the discretion of the investigator.

[0768] Dose adjustments for neutropenia and / or thrombocytopenia at the start of a cycle or during a cycle

[0769]

[0770]

[0771] Prophylactic G-CSF is recommended when there is a delay in treatment due to hematologic toxicity involving neutrophils. G-CSF may be started first 24 hours after the end of cytotoxic chemotherapy. No dose adjustment is required for other hematologic toxicities. However, red blood cell transfusions may be considered for hemoglobin <9.5 g / dL or with significant symptoms of anemia or as per institutional guidelines.

[0772] Measurement of hematologic toxicity during the cycle (nadir)

[0773]

[0774] Dosage Adjustment for Diarrhea

[0775]

[0776] For symptoms of diarrhea (and / or abdominal cramps) that occur at any time during the treatment cycle, it is recommended that patients should be instructed to take antidiarrheal drugs, such as loperamide (2 mg every 2 hours, until diarrhea subsides for 12 hours; 4m4 hours are allowed at night) or diphenoxylate / atropine (Lomotil) as treatment for diarrhea. For persistent diarrhea (i.e., lasting more than 48 hours), treat with broad-spectrum antibiotics, oral fluoroquinolones for 7 days. If diarrhea is severe, hospitalization should be considered for parenteral rehydration and change to iv antibiotics. Acute diarrhea and abdominal cramps that develop during or within 24 hours after irinotecan administration may occur as part of the cholinergic syndrome. For irinotecan-related cholinergic reactions, the infusion time can be increased to alleviate these symptoms, and prophylactic atropine according to institutional guidelines is allowed.

[0777] Dose Modification for Drug-Related Hepatotoxicity

[0778] For all hepatobiliary toxicities, treatment was maintained and non-drug causes, such as biliary obstruction / stent failure, were evaluated (see also Example 7). Once the underlying cause was corrected and improved, therapy was resumed at the previous dose level (5-FU and oxaliplatin only, without irinotecan), and irinotecan was added once toxicity improved to ≤ Grade 1.

[0779] The following dosage adjustment guidelines can be used for hyperbilirubinemia:

[0780] 1) Grade 2 and 3 hyperbilirubinemia: Omit irinotecan until ≤ Grade 1 and resume at the same dose level;

[0781] 2) Grade 4 Hyperbilirubinemia: Maintain therapy until ≤ Grade 1 and resume at the next dose level once underlying etiology is corrected*.

[0782] * Notice : If the etiology of hyperbilirubinemia is due to biliary obstruction (i.e., reversible and unrelated to therapy), discussion with the medical monitor may allow the option to continue irinotecan at the original level once liver function tests resolve to ≤ Grade 1.

[0783] Dosage Adjustments for Mucositis

[0784] Mucositis as toxicity is caused by 5-FU. If grade 3-4 toxicity occurs, the continuous 5-FU IV infusion can be reduced by 25% for the remainder of the course.

[0785] Dose Adjustments for Peripheral Neuropathy

[0786] In case of peripheral neuropathy, only oxaliplatin was adjusted:

[0787] 1) Grade peripheral neuropathy: Continue monitoring at the same dose level;

[0788] 2) Grade 2 peripheral neuropathy lasting more than 14 days: reduce oxaliplatin dose by one level;

[0789] 3) Grade 3 peripheral neuropathy: If G3 peripheral neuropathy persists for more than 14 days, reduce the oxaliplatin dose to 65 mg / m 2 , discontinue oxaliplatin;

[0790] 4) Grade 4 peripheral neuropathy: Discontinue oxaliplatin and continue 5-FU, irinotecan, and leucovorin at the same dose level. If it resolves to < Grade 1, then the patient may be treated with oxaliplatin based on follow-up considerations after discussion with the medical supervisor.

[0791] Restorative therapy for specific situations.

[0792] Hand-foot syndrome

[0793] Hand-foot syndrome as toxicity is caused by 5-FU. If grade 3-4 toxicity occurs, the continuous 5-FU IV infusion can be reduced by 25% for the remaining course of treatment.

[0794] Dose modifications for other clinically significant non-hematologic* toxicities (except alopecia and grade 3 nausea and vomiting that is responsive to medical therapy within 72 hours)

[0795]

[0796] Gastrointestinal ulcer, whether bleeding or not: Withhold 5-FU until symptoms resolve. Acute laryngopharyngeal dysesthesia is caused by oxaliplatin and can be managed by institutional practice, which may include extending the duration of the oxaliplatin infusion to 6 hours and infusing 1 g of calcium gluconate and 1 g of magnesium sulfate within 15 minutes before all subsequent oxaliplatin infusions.

[0797] Dose Modifications for Infusion-Related Reactions

[0798] In the event of an infusion-related reaction to any chemotherapy component given per protocol, institutional guidelines or those described below may be followed. Infusion reactions will be defined according to the National Cancer Institute CTCAE (version 5.0) definition of allergic reaction or anaphylaxis, as described below.

[0799]

[0800]

[0801] Cardiotoxicity

[0802] In cases of cardiac pain (eg, angina or myocardial infarction), 5-FU therapy may be discontinued.

[0803] Extravasation

[0804] Severe reactions due to extravasation of irinotecan or oxaliplatin have been reported

[39] .

[0805] General recommendations in case of extravasation are as follows:

[0806] - Stop the infusion immediately,

[0807] -Do not remove the needle or catheter,

[0808] - Aspirate / suction the maximum amount of infiltrated product through the needle,

[0809] - Apply ice to the wetted area for 15 to 20 minutes every 4 to 6 hours over a 72 hour period,

[0810] - Apply topical cortical therapy.

[0811] Examine the infiltrate periodically over the next few days to verify whether further treatment is needed. If in doubt, do not hesitate to request a surgical consultation.

[0812] Example 5: Appendix 3 Gemcitabine plus nab-paclitaxel administration, including dosing schedule, visit assessment form, and potential dose adjustments (if applicable)

[0813] Please note that treatment with gemcitabine plus nab-paclitaxel was only available for this study in case the Data Review Committee (DRC) formally decided to change chemotherapy mFOLFIRINOX to gemcitabine plus nab-paclitaxel during part 1 of this study.

[0814] Dosage Considerations

[0815] Dose adjustments should be made based on the system showing the greatest degree of toxicity. For both hematologic and non-hematologic toxicities, the dose may be reduced by one level at a time.

[0816] At least two levels of dose reduction were allowed for each drug according to the following criteria, see Table 16. If toxicity requiring dose modification occurred after the second or further dose reduction of either gemcitabine or nab-paclitaxel, no additional dose reductions were allowed.

[0817] Table 16 Dose adjustment of gemcitabine and nab-paclitaxel

[0818]

[0819] If treatment maintenance continues for >3 consecutive weeks for treatment-related toxicity, the patient may discontinue all study treatment. However, if the patient benefits clinically after the end of the 3-week maintenance, the treating physician may contact the medical supervisor to possibly extend therapy. Patients who discontinue study treatment may have an end-of-treatment visit and enter a post-treatment follow-up period according to Tables 21 and 22, respectively. In the event that toxicity justifies discontinuation of an agent, only the individual offending agent need be removed from the regimen, and treatment may otherwise be continued as per the regimen. Decisions regarding the need for dose adjustments of nab-paclitaxel and / or gemcitabine may be made based on the following guidelines.

[0820] Table 17: Doses for neutropenia and / or thrombocytopenia at the start of a cycle or during a cycle Volume Adjustment

[0821]

[0822] Prophylactic G-CSF is recommended when there is a delay in treatment due to hematologic toxicity involving neutrophils. G-CSF can be started first 24 hours after the end of cytotoxic chemotherapy. If hematologic toxicity is limited to platelet counts, a dose adjustment of gemcitabine alone can be considered. Other hematologic toxicities do not necessarily require dose adjustments. However, red blood cell transfusions can be considered for hemoglobin <9.5 g / dL or with significant symptoms of anemia or as per institutional guidelines.

[0823] Table 18 Dose Modifications for Other Clinically Significant Non-Hematologic* Toxicity

[0824]

[0825] For all other grade ≥3 non-hematologic toxicities (*except nausea, vomiting, alopecia, and pulmonary embolism and adverse events of special interest (AESI) described below).

[0826] ●Withhold dose of either or both agents until improvement to ≤ Grade 1;

[0827] ●Resume at the next lower dose level.

[0828] Hepatotoxicity and dose reduction of gemcitabine plus nab-paclitaxel

[0829] If AST or ALT or both increase to less than 5x ULN, gemcitabine can be continued without dose reduction;

[0830] If AST or ALT or both increase by more than 5x ULN, but less than 20x ULN, the gemcitabine dose may be reduced by 25%;

[0831] ● Gemcitabine may be discontinued if AST or ALT or both increase more than 20x ULN.

[0832] Dosage Schedule

[0833] The first treatment cycle with mitazalimumab and gemcitabine plus nab-paclitaxel will last 35 days, mitazalimumab will be administered on days 1, 10, and 24, and gemcitabine plus nab-paclitaxel will be administered on days 8, 15, and 22. During the following 28-day treatment cycles, mitazalimumab will be administered on days 3 and 17, and gemcitabine plus nab-paclitaxel will be administered on days 1, 8, and 15. A maximum of 6 treatment cycles will be allowed for the combination of mitazalimumab and gemcitabine plus nab-paclitaxel, see Figure 4 .

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841] Example 6: Appendix 4 Calculation of Glomerular Filtration Rate

[0842] The glomerular filtration rate (GFR) can be estimated based on a commonly used and accepted formula (ie, one of the following formulas).

[0843] Cockcroft Gault formula:

[0844]

[0845] Unit: GFR [ml / min], age [years], weight [kg], serum creatinine [mg / dl], F S is the sex correction factor: in males, F S =1, in women, F S =0.85

[0846] Modification of Diet in Renal Disease (MDRD) formula:

[0847]

[0848] Unit: GFR [ml / min], age [years], serum creatinine [mg / dl], F S is the sex correction factor: in males, F S =1, in women, F S =0.762

[0849] Variants of the MDRD formula:

[0850]

[0851] Unit: GFR [ml / min], age [years], serum creatinine [mg / dl], F S is the sex correction factor: in males, F S =1, in women, F S =0.742

[0852] Example 7: Appendix 5 Drug-induced liver injury (DILI)

[0853] Changes in liver laboratory parameters can be further evaluated using the following procedures:

[0854] program

[0855] Repeat the following laboratory tests:

[0856] ALT, AST, and Bilirubin (Total and Direct) - Within 48 to 72 hours. If ALT and / or AST > 3x ULN and total bilirubin elevation > 2x ULN (if normal values ​​at baseline / screening) is confirmed, or ALT and / or AST > 5x ULN and total bilirubin elevation > 2x ULN (if elevated values ​​at baseline / screening), the results of the following laboratory parameters must be available to the Investigator and Sponsor as soon as possible.

[0857] In addition, the following items were obtained:

[0858] ●A detailed history of current symptoms and concurrent diagnoses, as well as medical history;

[0859] A history of concomitant medication use (including over-the-counter medications, herbal remedies, and dietary supplements), alcohol use, recreational drug use, and special diets;

[0860] ●History of exposure to environmental chemicals (consider household and workplace exposures).

[0861] Provide abdominal ultrasound or other appropriate imaging to exclude biliary, pancreatic, or intrahepatic pathology, such as bile duct stones or neoplasms.

[0862] Clinical Chemistry

[0863] Alkaline phosphatase, albumin, PT or INR, CK, CK-MB, ceruloplasmin, alpha-1 antitrypsin, transferrin, amylase, lipase, fasting blood glucose, cholesterol, triglycerides, glutamate dehydrogenase, D-dimer, C-reactive protein, gamma-glutamyl transpeptidase.

[0864] Serology

[0865] Hepatitis A (RNA), Hepatitis B (HbsAg, anti-HBs, DNA), Hepatitis C (anti-HCV, RNA), Hepatitis D (anti-IgM, anti-IgG), Hepatitis E (anti-HEV, anti-HEV IgM, RNA if anti-HEV IgM positive), Cytomegalovirus (repeated CMV DNA), anti-smooth muscle antibodies (titer), anti-nuclear antibodies (titer), anti-LKM (liver and kidney microsome) antibodies, anti-mitochondrial antibodies.

[0866] hormone

[0867] Thyroid stimulating hormone;

[0868] hematology

[0869] WBC count + differential, hemoglobin, platelets.

[0870] In cases where AST / ALT remains elevated and previous testing does not provide a possible cause for the elevation, the following tests may be performed: Epstein-Barr virus (VCA IgG, VCA IgM), herpes simplex virus (IgG, IgM), varicella (IgG, IgM), parvovirus (IgG, IgM), toxoplasmosis (IgG, IgM).

[0871] Patients will be closely monitored initially by repeat testing of ALT, AST, and total bilirubin (fractionated for total and direct bilirubin) at least weekly until laboratory ALT and / or AST abnormalities stabilize or return to normal, and then as per protocol. Follow-up may be performed based on medical judgment and good clinical practice (GCP), depending on further laboratory changes or other parameters identified.

[0872] Example 8: Antitumor efficacy of mitazalimab and chemotherapy (FOLFIRINOX) in the chemotherapy-resistant preclinical tumor model MB-49

[0873] Summarize

[0874] The aim of this study was to investigate the antitumor efficacy of the CD40 antibody mitazalimab as a supplement to chemotherapy (FOLFIRINOX) in chemotherapy-resistant preclinical tumor models. The combination of mitazalimab and FOLFIRINOX demonstrated a strong antitumor response in mice with established FOLFIRINOX-resistant MB-49 tumors. These data support the basic 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.

[0875] Materials and methods

[0876] Drugs and Drug Candidates Evaluated

[0877] Note: All mice were dosed with a fixed dose and the mg / kg doses reported herein are based on an average mouse body weight of 20 g (0.02 kg).

[0878] Table 23 List of antibodies and controls mentioned in this report

[0879]

[0880] * Together, these four components make up the chemotherapy known as FOLFIRINOX.

[0881] **Vehicle used for mitazalimab and FOLFIRINOX.

[0882] Dosing of FOLFIRINOX

[0883] Since FOLFIRINOX consists of four separate components, it was administered over two days for a total of three cycles. The mice first received oxaliplatin, followed by irinotecan, and the next day received folinic acid, and 2 hours later, the mice were given 5-fluorouracil.

[0884] Administration of mitazalimab

[0885] Mitazalimab was administered at 5 mg / kg in 5% glucose (dextrose buffer) three days after the start of FOLFIRINOX dosing.

[0886] Dosage regimen

[0887] exist Figure 5 The dosing regimens used in the two efficacy studies are outlined in , which details the number of doses and their relationship to each other.

[0888] Cell lines

[0889] Scandion Oncology has produced an MB49 cell line resistant to the three cell inhibitory components (irinotecan, oxaliplatin and 5-fluorouracil) of the combined chemotherapy FOLFIRINOX. Two chemotherapy-resistant MB49 bladder cancer cell lines were produced, namely MB49-FOLFIRINOX-from the beginning and MB49-FOLFIRNINOX-acquired (MB49-FFX-ACQ). Methods for preparing chemotherapy-resistant cell lines (e.g., with 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). Since the de novo cell line grew poorly in culture, its in vivo titration was later not considered, and the experiments described in this report were performed with an MB49 cell line with acquired chemotherapy resistance.

[0890] The MB49 mouse bladder cancer cell line is used as a starting point for the FOLFIRINOX resistance model. Resistance can be established using the following methods:

[0891] 1. Acquired resistance: Cancer cells are exposed to increasing concentrations of drugs until a resistant cell population is generated. Based on previous experience (PMID: 26801902, 25759163, 25596703; Jandu et al., 2016, Jensen et al., 2015 and Hansen et al., 2015), a starting concentration 50-fold lower than the IC50 value of each drug is used, and gradually increased by 2-fold for every three passages of the cells (approximately every three weeks) until the difference in IC50 of each drug increases by at least 5-fold when compared to drug-sensitive cells. This process results in the MB49 cell line having increased resistance to FOLFIRINOX.

[0892] 2. De novo resistance: Cancer cells are exposed to initial high drug concentrations, ranging from 10 times below the IC50 value of each drug to 10 times above the IC50 value of each drug. The goal of this approach is to kill most cancer cells. However, some cancer cells with "de novo" resistance will survive and then be cultured and expanded to establish a drug-resistant cell population.

[0893] Initially, in vivo titrations were performed only on MB49-FFX-ACQ cultured without FOLFIRINOX prior to the cell bank (ELN 148285), and small-scale follow-up titrations were later added to enable comparison with cell lines grown with FOLFIRINOX prior to the master cell bank (ELN 148525).

[0894] Experiments in tumor-bearing hCD40tg mice

[0895] The hCD40tg mouse strain on a C57Bl / 6 background has been 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).

[0896] On day 0, ten to twelve week old female hCD40tg mice were injected subcutaneously in the right hind flank with 0.4 x 10 6MB49-FOLFIRINOX-ACQ. Cells can be cultured with (Study 1) or without FOLFIRINOX (Study 2) prior to inoculation into mice. Controls or antibodies are administered as outlined in Table 24, and mice are monitored shortly after each treatment for potential signs of toxicity. Tumor volume is measured three times per week with calipers and calculated as: ((width / 2×length / 2×height / 2)×4π / 3). Mice are weighed weekly, and weight loss of more than 25% is considered an ethical endpoint, as well as weight loss of more than 2 cm 3 Increased tumor volume, tumor ulceration, or impact on health were also considered ethical endpoints.

[0897] Table 24 Group assignment and experimental settings

[0898]

[0899] Statistical analysis

[0900] The collected data were analyzed for survival using Kaplan Meier and Log-rank (Mantel-Cox), and FACS data were analyzed using Mann-Whitney, nonparametric, 2-tailed, and tumor growth was analyzed using the GraphPad Prism program (*, p<0.05; **, p<0.01).

[0901] result

[0902] Study using MB49-FFX-ACQ co-cultured with FOLFIRINOX

[0903] MB49-FFX-ACQ cells were grown in the presence of 0.3 μM 5-fluorouracil, 4.5 μM oxaliplatin, and 0.12 μM irinotecan (SN38) added to the culture medium until three passages before the master cell bank.

[0904] Figure 7 A shows that FOLFIRINOX reduces tumor growth in chemosensitive tumors. Figure 7 B shows that FOLFIRINOX has no effect in chemoresistant tumors. Figure 7 C shows the synergistic effect of mitazalimab and FOLFIRINOX in chemoresistant tumors.

[0905] The combination of mitazalimab + FOLFIRINOX induced statistically significant tumor growth inhibition compared to vehicle and cured tumor-bearing mice (complete responders: 5 / 10 for mitazalimab + FOLFIRINOX), see Figure 6 and Figure 8 .

[0906] Evaluation and conclusion

[0907] In the efficacy study conducted, the combination of mitazalimab and FOLFIRINOX demonstrated 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 effective anti-tumor effect against chemotherapy-resistant cancer cells in vivo. The data further validate the potential of mitazalimab in combination with standard of care chemotherapy such as FOLFIRINOX.

[0908] Example 9: Safety data from OPTIMIZE-1, a Phase 1b / 2 study of mitazalimab in combination with mFOLFIRINOX in patients with metastatic pancreatic ductal adenocarcinoma (PDAC): mitazalimab 900 μg / kg was determined to be a safe and recommended dose for the Phase 2 (RP2D) portion of the study

[0909] Mitazalimab is a human CD40 agonistic IgG1 antibody being developed as a cancer immunotherapy. Targeting CD40 initiates the cancer immunity cycle by empowering DCs, leading to tumor-specific T cell priming and activation. In addition, in PDAC, CD40 agonists activate myeloid cells and promote degradation of the desmoplastic tumor stroma, thereby improving the influx of T cells and chemotherapeutic agents into the tumor.

[0910] Mitazalimab has been shown to be safe and well tolerated (at doses up to 1200 μg / kg) and has shown signs of clinical activity in solid tumors in a phase I study (NCT02829099). Most drug-related adverse events (AEs) were grade 1 or 2.

[0911] OPTIMIZE-1 (NCT04888312) is a Phase 1b / 2, open-label, multicenter study designed to evaluate the safety, tolerability, and efficacy of mitazalimab in combination with mFOLFIRINOX in adults diagnosed with previously untreated metastatic PDAC.

[0912] The first part of the study (Phase 1b) aims to determine the RP2D of mitazalimumab plus 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 at each dose level.

[0913] In the first 21-day treatment cycle (dose-limiting toxicity assessment period), mitazalimumab was injected intravenously on days 1 and 10, and mFOLFIRINOX was started on day 8. In the second and subsequent cycles, treatment followed a 14-day cycle schedule, with mitazalimumab administered 2 days after mFOLFIRINOX.

[0914] In part 2 (Phase 2) of the study, mitazalimab at RP2D was administered in combination with mFOLFIRINOX. The primary endpoint was the overall response rate defined by RECIST. Progression-free survival and overall survival were evaluated as secondary endpoints.

[0915] In the Phase 1b (dose escalation) portion of this study, 11 patients were treated with mitazalimab: 5 patients at 450 μg / kg mitazalimab dose and 6 patients at 900 μg / kg mitazalimab dose. One patient in the 900 μg / kg dose cohort withdrew from the trial for administration reasons after the first mitazalimab infusion and before receiving mFOLFIRINOX and was not included in the RP2D determination. The main baseline characteristics included: 7 women, 4 men; median age 63 years (range 57-70 years); ECOG 0-1; median time since PDAC diagnosis was 25 days.

[0916] Mitazalimab-related AEs were reported in 9 / 11 patients. Treatment-related AEs occurring in >1 patient were fever (60%), muscle pain (50%), and fatigue (20%). At the 450 μg / kg dose, all mitazalimab-related AEs were grade 1-2. At the 900 μg / kg dose, 4 patients (67%) experienced grade 1-2 mitazalimab-related AEs. One patient at the 900 μg / kg dose experienced mitazalimab-related grade 3 fatigue and grade 3 headache, which led to treatment discontinuation after the first cycle. There were no mitazalimab-related grade 4 or 5 AEs. 1 / 10 patients required mFOLFIRINOX dose reductions, and at the cutoff date, treatment duration ranged from 1-14 weeks.

[0917] Mitazalimab was safe and well tolerated in combination with mFOLFIRINOX. A dose of 900 μg / kg of mitazalimab was selected as the RP2D, and patient enrollment at the RP2D is ongoing.

[0918] OPTIMIZE-1 Safety Summary

[0919] DLT:

[0920] SC-12 (001-009): G3 headache (mitazalimab related). 59-year-old female, h / o NSCLC; progression on C4 scan with new lung lesions. Not investigative.

[0921] SAE:

[0922] SC-01(002-001): SAE G3 new lung cancer (unrelated). 60-year-old female, h / o NSCLC; progression on C4 scan, and new lung lesions. Not research.

[0923] SC-05(103-001): SAE G3 Anorexia (unrelated). 65-year-old male, h / o abdominal pain, ascites. Hospitalized for pain management. Resolved. Study treatment ongoing.

[0924] SC-06(001-001): Revocation of SAE G2 general status change. 66-year-old male. Alternative reason: Progression.

[0925] SC-14(001-011): SAE G3 supraventricular tachycardia (unrelated). 60-year-old female. Resolved. Study treatment ongoing.

[0926] AESI:

[0927] ●None (infusion-related reactions ≥G2; cytokine release syndrome ≥G2; LFTs ≥5x ULN; bilirubin ≥1.5x ULN).

[0928] Mitazalimab-related (11 patients at 2 dose levels):

[0929] ●Grade 3: Fatigue (2); Headache (1); Hypokalemia (1).

[0930] ●Grade 1-2 (most common): Fever or similar symptoms (6); flu / muscle aches (5); fatigue (3).

[0931] Example 10: OPTIMIZE-1 futility analysis

[0932] As mentioned in the study design of Example 2, a total of 23 patients were subjected to an interim invalidity analysis (results are summarized in Table 28). The patient configuration of the safety group is shown in Table 25, which summarizes the number of subjects screened and their inclusion or exclusion (including reasons for exclusion). The patient disposition of the complete analysis set (FAS) at a dose of 900 μg / kg is shown in Table 26, with corresponding patient demographic data shown in Table 27.

[0933] Table 25: Patient Disposition (Safety Set)

[0934]

[0935]

[0936] Table 26: Patient Disposition (FAS)

[0937]

[0938]

[0939] Table 27: Patient Demographic Data (FAS)

[0940]

[0941] Table 28: Failure Analysis (FAS)

[0942]

[0943]

[0944] The futility analysis (Table 28) showed a promising disease control rate of 91.3% for subjects treated with 900 μg / kg dose of mitazalimab and mFOLFIRINOX. Therefore, these preliminary data show an improvement in cancer treatment with combination therapy. In patients with progressive disease, this may indicate that the patient did not respond to the combination therapy, rather than the combination therapy causing the progression of the disease.

[0945] Importantly, these data suggest that the 900 μg / kg RP2D of mitrazalimab shows promising efficacy. This is the first reported data on a (second generation) CD40 antibody in combination with FOLFIRINOX in this patient population. The ORR of 52.2% is significantly better than the historically reported ORR of 31.6% for FOLFIRINOX alone. Modified FOLFIRINOX uses a lower dose of irinotecan, which means reduced toxicity and better compliance with therapy, thereby improving efficacy.

[0946] At the time of this analysis, a considerable number of patients (18 of 23 patients) were still receiving treatment, indicating promising clinical activity and a potentially manageable safety profile. Only one patient (patient ID 002-024) had to stop mitazalimab treatment due to adverse events (pneumonia), and even this patient showed a partial response in the scan at the end of treatment. In this analysis, 8 of the 12 responders were confirmed to have responded via follow-up scans, and 3 of the 4 unconfirmed responders were still receiving treatment on the analysis date. One patient, SC-22, had an almost complete response, with one target lesion disappearing (only due to persistent non-target lesions, the patient must be formally considered as a "partial response" rather than a "complete response").

[0947] In addition, one patient who delayed starting FOLFIRINOX showed marginal disease progression at the first post-baseline assessment. Although this patient was initially considered a nonresponder, follow-up scans showed tumor shrinkage. Stable tumor shrinkage was observed for this patient during treatment.

[0948] Patients used in the clinical studies were assigned a patient ID and all patients were included in the safety set, but some of them were excluded from the FAS set data for each treatment group, as shown in Table 29 (450 μg / kg mitraz and mFOLFIRINOX co-treatment) and Table 30 (900 μg / kg mitraz and mFOLFIRINOX co-treatment).

[0949] Table 29: List 1 Patient Disposition (Safety Set)—Treatment Group = 450 μg / kg Mitazalimab + mFOLFIRINOX

[0950]

[0951]

[0952] Table 30: List 1 Patient Disposition (Safety Set)—Treatment Group = 900 μg / kg Mitazalimab + mFOLFIRINOX

[0953]

[0954]

[0955] Tumor response assessments were also included in the ineffectiveness analysis, as shown in Table 31. The visits performed on the patients (details of the treatment cycles were as previously described) and the target lesion responses and non-target lesion responses were summarized. The best overall response corresponds to the most positive result of the target lesion response observed in this particular patient, where the order of response from best to worst is: (i) complete response (CR); (ii) partial response (PR); (iii) stable disease (SD); and (iv) progressive disease (PD). Target lesion and non-target lesion responses are as previously described in Example 3.

[0956] Table 31: Tumor response assessments included in the futility analysis of List 2

[0957]

[0958]

[0959]

[0960] Unscheduled visits can occur for a variety of reasons, but most often are due to administration reasons or patient preference. At other times, this may be due to a delay in resuming treatment, for example due to an adverse event or patient preference. Regarding patient ID004-015, this patient received treatment as planned, but had rapid disease progression, resulting in premature cessation of treatment.

[0961] Patient ID 106-027 started FOLFIRINOX relatively late due to safety concerns. The first scan showed marginal disease progression, and therefore the decision was made to continue treatment. After the combination therapy, the disease responded, with two subsequent unplanned scans showing stable disease.

[0962] Example 11: Safety Data of Mitazalimab and mFOLFIRINOX from OPTIMIZE-1 (Phase 1b / 2 Study) and Recommended Dosing for Phase 2

[0963] The primary objective of the Phase 1b phase is to determine the recommended Phase 2 dose. Secondary objectives include evaluation of clinical activity (overall response rate (ORR), disease control rate, and time to next anticancer therapy) and survival outcomes.

[0964] The primary objective of phase 2 is to evaluate the clinical activity of mitazalimab in combination with chemotherapy, as determined by ORR. Secondary objectives include survival outcomes, best overall response (BOR), duration of response (DoR), duration of SD, disease control rate, and time to next anticancer therapy.

[0965] Patient demographics for the data generated in Example 11 are shown in Table 32, with analyses of treatment-emergent adverse events (TEAEs) and mitazalimumab-related adverse events shown in Tables 33 and 34, respectively.

[0966] Table 32: Patient Demographics

[0967]

[0968] Table 33: Treatment Emergent Adverse Events (TEAEs)

[0969]

[0970] Table 34: Mitazalimab-related adverse events

[0971]

[0972] The five patients at 450 μg / kg corresponded to SC-01, SC-03, SC-04, SC-05, and SC-06; and the six patients at 900 μg / kg corresponded to SC-09, SC-10, SC-11, SC-12, SC-13, and SC-14. Figure 8 The duration of treatment for these patients is shown in . Patient SC-13 withdrew from the trial for administration reasons after the first infusion of mitazalimab but before receiving mFOLFIRINOX and was not included in the RP2D determination.

[0973] Several peripheral blood assessments were also performed to evaluate IFN-γ, MCP-1, B cell marginalization, and B cell activation (see Fig. 9 AD and Fig.10 AD). Increases in cytokines and chemokines were observed in peripheral blood, confirming activation of T cells and myeloid cells at both doses of mitazalimab tested (see Fig. 9 A and Fig. 9 B and Fig.10 A and Fig.10 B). In addition, B cell marginalization and B cell activation of the remaining B cells were observed in the peripheral blood after treatment with mitazalimab (see Fig. 9 C and Fig. 9 D and Fig.10 C and Fig.10 D).

[0974] in conclusion

[0975] These data support that the combination of mitazalimab and mFOLFIRINOX is safe and well tolerated. Mitazalimab-related adverse events (AEs) occurring in more than 1 patient included fever (60%), myalgia (50%), and fatigue (20%). Cytokine profiles in peripheral blood confirmed activation of T cells and myeloid cells, which is consistent with the mode of action of mitazalimab. Based on these data, a dose of 900 μg / kg of mitazalimab was selected as the RP2D.

[0976] Example 12: Efficacy and efficacy biomarkers of mitazalimab combined with chemotherapy in preclinical mouse models

[0977] This study demonstrated the synergistic potential of mitazalimab and FOLFIRINOX in vivo in a mouse preclinical model and investigated pharmacodynamic biomarkers to assess the effect of the combination in peripheral blood at early time points.

[0978] The ability of mitazalimab to enhance the response to chemotherapy was demonstrated in human CD40 transgenic (hCD40tg) mice bearing MB49 syngeneic tumors (see Fig.11 hCD40tg mice were subjected to a three-week dosing regimen containing vehicle (dextrose), mitazalimumab, FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil, and folinic acid), or a combination of mitazalimumab and FOLFIRINOX. Since FOLFIRINOX consists of four components: oxaliplatin, irinotecan, folinic acid, and 5-fluorouracil, they were administered over the course of two days. All components were diluted in 5% glucose and administered intraperitoneally in 100 μl, except for folinic acid, which was administered in 200 μl. On day 6 post-inoculation, mice received 2.5mg / kg Oxa Riplatin followed by 25 mg / kg irinotecan On the second day, i.e. day 7, mice received 50 mg / kg folinic acid, 2 hours after 25 mg / kg 5-fluorouracil Mitazalimab was administered on day 9. Mitazalimab was diluted in 5% glucose and administered intraperitoneally at a dose of 5 mg / kg (100 μg) in 100 μl. Repeated administration of mitazalimab with FOLFIRINOX induced long-term survival (see Fig.12 B) and tumor volume control (see Fig.12 A).

[0979] Peripheral blood was collected from mice at the end of the first cycle and RNA sequencing was performed to identify efficacy biomarkers. GLOBINclear TM Kit, Mouse / Rat (AM1981, Invitrogen) and Mouse RiboPure TM - Blood RNA Isolation Kit (AM1951, Invitrogen) extracted RNA from whole blood after erythrocyte lysis. The purified RNA was stored in RNase-free microcentrifuge tubes (AM12425, Invitrogen) and frozen at -80C until further use.

[0980] RNA library preparation and sequencing were performed by the Center for Translational Genomics (CTG, Lund University). RNA integrity was analyzed using a TapeStation 4200 (Agilent Technologies) using Agilent RNA ScreenTape and reagents (5067-5576, 5067-5577, Agilent Technologies). RNA concentration was analyzed using Qubit Flex (Q33327 Invitrogen Thermo Fisher Scientific) using the QuantIT RNA HS Assay Kit (Q33140, Life Technologies). Stranded mRNA Library Prep Kit (20020594, Illumina) was used to prepare cDNA sequencing libraries. The libraries were sequenced using the NovaSeq 6000 system (20012850, Illumina) using the NovaSeq 6000 SP reagent kit, 300 cycles v1.5 20028400). Reads were aligned to the mouse reference genome GRCm38 (mm10) using STAR software (v2.5.0a.13).

[0981] Quality control and thresholding, normalization, dimensionality reduction and visualization, and differential gene expression analysis were performed using R (v4.0.3) and R studio (v1.4.1103). Protein-coding genes with a minimum of 5 reads in 3 or more samples were retained for analysis. For data visualization, the filtered raw counts were normalized using the variance stabilizing transformation method in the DESeq2 package (version 1.38.3). Principal component analysis (PCA) was used to visualize the data and assess the effects of treatment at the transcriptome level.

[0982] To identify differentially expressed genes between treatment groups, filtered raw counts were normalized using the ratio median method in the DESeq2 package. Treatment groups were compared using the negative binomial Wald test (adjusted p-value [adj-p.val] < 0.05, fold change > 2).

[0983] Pathway enrichment analysis was performed using EnrichR (v3.0) by querying the gene ontology database (GO, GO_bioprocess_2018) for differentially expressed genes between treatment groups. Gene set variation transformation (GSVA) ​​was used to select and coordinate recurrent and highly ranked pathways (adjusted p-value < 0.05) across samples. The resulting GSVA scores were plotted as spider plots for comparison of pathway activity between groups.

[0984] Exploratory data analysis confirmed that samples clustered according to treatment regimen (see Fig.13 ), with the group containing mitazalimumab showing the highest transcriptome similarity. Differential gene expression profiles were observed for the different treatment groups (see Fig.14 ).

[0985] The analysis revealed 3356 differentially expressed genes between the groups, and 221 differentially expressed genes between the two groups containing mitazalimab (Table 35). The combination of mitazalimab and FOLFIRINOX induced the highest expression of Ccl2 / 3 / 4, Cxcl10, and Ifng (see Fig.15 ). The expression of these cytokines indicates an early response in the myeloid and T cell compartments in the peripheral blood, reinforcing the mode of action of mitazalimab. Similarly, pathway enrichment and gene set variation analysis yielded examples of interactions between immunotherapeutic and chemotherapeutic agents (see Fig.16 Mitazalimab alone or in combination induced high expression of genes involved in responding to type 1 IFN and TLR signaling (see Fig.15 and Figure 17 ). In addition, combination treatment promoted extracellular matrix organization as well as pyrimidine metabolism; processes associated with CD40-mediated bone marrow activation and 5-fluorouracil, respectively (see Fig.16 and Figure 18).

[0986] Table 35: Comparison of differentially expressed genes between treatment groups

[0987]

[0988] In conclusion, mitazalimab synergizes effectively with FOLFIRINOX to induce long-term survival in preclinical tumor models. The pharmacodynamic biomarkers of mitazalimab identified here are consistent with data from a phase 1 study of mitazalimab in patients with advanced tumors (NCT02829099). Together, these data support the ongoing phase 1b / 2 clinical study of mitazalimab in combination with mFOLFIRINOX (NCT04888312).

[0989] These data also support specific biomarkers associated with the efficacy of the combination therapy, depending on the control in question, as summarized in Table 36, Table 37, and Table 38. These biomarkers are determined based on being above a specific Log2 fold change threshold and above or below a specific p-value threshold. Biomarkers (whether upregulated or downregulated) indicate that the biomarker can be used to indicate the efficacy of the combination therapy relative to the corresponding control (chemotherapy alone, mitazalimab alone, or untreated). That is, an upregulated biomarker means that the gene has a higher expression in the combination therapy and a lower expression in the control; and a downregulated biomarker means that the gene has a lower expression in the combination therapy and a lower expression in the control.

[0990] Table 36: Biomarkers for combination therapy relative to FOLFIRINOX alone, assigned MGI symbols (Baseline (Fig. 17E)

[0991]

[0992] Table 37: Biomarkers for combination therapy relative to mitazalimab alone, designated as mouse genes Group Informatics (MGI) notation (based on Figure 17F)

[0993]

[0994]

[0995] Table 38: Biomarkers for combination therapy relative to untreated controls, assigned MGI symbols (based on Figure 17G)

[0996]

[0997] Example 13: Correlation of patient response with cell abundance and activation in the blood of pancreatic cancer patients

[0998] Association test

[0999] The purpose of this analysis was to identify pharmacodynamic (PD) biomarkers whose abundance at baseline (C1D1) or changes (fold change) in C1D1 abundance at subsequent time points showed association with patient response. PD biomarkers were assessed in the blood of patients enrolled in the clinical study Optimize-1 during the first treatment cycle (C1), including counts, percentages of immune cell populations, and plasma cytokines.

[1000] The association of the baseline (C1D1) abundance of each PD biomarker (Table 39 and Table 40) and the change in the abundance of each PD biomarker relative to the baseline (Table 41) with the patient's overall response OR (PR vs. SD / PD) and disease control DC (PR / SD vs. PD) was assessed using a t-test. For each comparison, a positive fold change indicates that the responder is more abundant than the non-responder, and vice versa. The abundance of the PD biomarker, i.e., the cell count and percentage of the baseline (C1D1), or the change in the abundance of C1D1 on day 8, is associated with the patient's response.

[1001] Of note, cell percentages were subjected to arcsine transformation prior to statistical testing (via t-test) at baseline (C1D1) (Table 39b and Table 40b).

[1002] Table 39: Baseline at C1D1: PR vs. SD / PD (OR)

[1003]

[1004] Table 40: Baseline at C1D1: PR / SD and PD (DC)

[1005]

[1006]

[1007] Table 41: Fold changes in C1D8: PR vs. SD / PD (OR)

[1008]

[1009]

[1010] Non-significant association tests and confounding analyses

[1011] Of note, the levels of activation markers or plasma cytokines did not statistically correlate with OR or DC (data not included).

[1012] In addition, confounder analysis was performed to investigate the associations between clinical variables (clinical variables included: change in tumor lesion size from baseline, modified Glasgow prognostic score at baseline, neutrophil-to-lymphocyte ratio (NLR) at baseline, CA19-9 abundance at baseline, and change in CA19-9 abundance from baseline). As expected, significant (adjusted P < 0.05) correlations were observed within response-related variables (RECIST response, OR, DC, and change in tumor size), but no significant correlations were observed between response variables and other clinical variables.

[1013] Data Quality Control (QC)

[1014] The "Cell Count" dataset was filtered to retain cell types with counts greater than zero in at least five samples. Of the 138 features, 9 features were excluded and 129 features were retained for further analysis. A total of 148 samples and 129 cell count features were obtained for further analysis. The random forest method from the R package missForest was used to impute missing data.

[1015] The “Percentage” dataset was filtered to retain cell types with a relative abundance greater than 0% in at least five samples and less than 100% in at least five samples. Of the 153 features, 3 features were excluded and 150 features were retained for further analysis. The dataset was then filtered to exclude 13 samples with no available cell percentage information or missing data for more than 25% of the features. Subsequently, two features with missing data in more than 20% of the samples were excluded. A total of 149 samples and 148 cell percentage features were obtained for further analysis. The random forest method from the R package missForest was used to impute missing data.

[1016] Example 14: Antitumor efficacy of mitazalimab combined with chemotherapy in the KPCY pancreatic mouse tumor model

[1017] To support the combination of mitazalimab and FOLFIRINOX in a pancreatic mouse tumor model, antitumor efficacy was evaluated in human CD40 transgenic mice (hCD40tg mice) using the KPCY pancreatic mouse tumor cell line, which was derived from a spontaneous PDAC mouse model (KPC) that has the main features of human disease, including mutant Kras and p53 (Li et al., 2018, Immunity, 49, 178–193).

[1018] The antitumor efficacy of mitazalimab combined with FOLFIRINOX was evaluated in KPCY tumor-bearing hCD40tg mice. Human CD40 transgenic mice were inoculated subcutaneously on the right flank with KPCY (3x10 5 cells, clone 2838c3) mouse pancreatic tumor cells (day 0). On day 6, one day before the start of treatment, mice were randomly divided into treatment groups according to tumor size. The average tumor size in each group at the start of treatment was 20 mm 3Mice were treated intraperitoneally once a week with mitazalimab (5 mg / kg, days 10, 17, and 24) and / or FOLFIRINOX (oxaliplatin 2.5 mg / kg), irinotecan (25 mg / kg), 5-fluorouracil (25 mg / kg), and calcium folate (50 mg / kg); (days 7-8, 14-15, and 21-22) or vehicle (5% glucose, days 7-8, 10, 14, 15, 17, 21-22, and 24) for 3 weeks (n=10 per treatment group) ( Fig.19 ). Each FOLFIRINOX treatment was administered over 2 days, with oxaliplatin and irinotecan given on the first day, and calcium folate on the second day, followed by 5-fluorouracil 2 hours later.

[1019] The data demonstrated that a significant synergistic effect on the reduction of tumor growth rate was observed for the combination of mitazalimab and FOLFIRINOX compared to each treatment alone ( Fig. 20 ).

[1020] Example 15: Mitazalimab and mFOLFIRINOX Efficacy Data from Phase 2 of OPTIMIZE-1 (Phase 1b / 2 Study)

[1021] The primary objective of Phase 2 is to evaluate the clinical activity of mitazalimab in combination with chemotherapy, as determined by ORR. Secondary objectives include survival outcomes, best overall response (BOR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), duration of SD, disease control rate, and time to next anticancer therapy.

[1022]

[1023]

[1024]

[1025]

[1026]

[1027]

[1028]

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Claims

1. A combination therapy for treating cancer, optionally a chemotherapy-resistant cancer, in a subject, the combination therapy comprising: a. an antibody or antigen-binding portion thereof that specifically binds to CD40, and b. Chemotherapy.

2. A combination therapy comprising an antibody, or antigen binding portion thereof, and chemotherapy for use in a dosage regimen for treating cancer, optionally a chemotherapy-resistant cancer, wherein the dosage regimen comprises the following steps: a. administering an antibody or antigen-binding portion thereof that specifically binds to CD40, and b. Administering chemotherapy.

3. An antibody or antigen binding portion thereof that specifically binds to CD40 for use in treating cancer, optionally a chemotherapy-resistant cancer, in a subject, wherein the antibody or antigen binding portion thereof is for use in combination with chemotherapy.

4. Use of an antibody or an antigen-binding portion thereof that specifically binds to CD40 in the preparation of a medicament, wherein the medicament is: a. for use in treating cancer, optionally a chemotherapy-resistant cancer, wherein the antibody or antigen-binding portion thereof is for use in combination with chemotherapy; or b. In the combination therapy according to claim 1 or 2.

5. A method of treating cancer, optionally a chemotherapy-resistant cancer, in a subject, the method comprising administering to the subject: a. a therapeutically effective amount of an antibody or antigen-binding portion thereof, which specifically binds to CD40, and chemotherapy; b. a therapeutically effective amount of the combination therapy according to claim 1; c. The combination therapy according to claim 2.

6. A pharmaceutical composition comprising: an antibody or an antigen-binding portion thereof that specifically binds to CD40; and chemotherapy.

7. A kit comprising: an antibody or antigen binding portion that specifically binds to CD40; and chemotherapy.

8. The combination therapy according to claim 1 or 2, the use according to claim 3 or 4, the method of treatment according to claim 5, the pharmaceutical composition according to claim 6 or the kit according to claim 7, wherein the cancer, optionally the chemotherapy-resistant cancer, is pancreatic cancer; Optionally, wherein the pancreatic cancer is an exocrine tumor, such as exocrine gland carcinoma or pancreatic ductal adenocarcinoma (PDAC), or an endocrine tumor.

9. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, wherein the chemotherapy is selected from the group consisting of FOLFIRINOX or variants 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 combination therapy, use, method, pharmaceutical composition or kit according to claim 10, wherein the FOLFIRINOX or variant thereof comprises: a. Oxaliplatin (such as or generic drug) infusion, optionally wherein oxaliplatin: i. 85 mg / m 2 The dosage, ii. intravenous, and / or iii. Apply for 2 hours; b. folinate (such as leucovorin, calcium folinate, calcium folinate, disodium folinate and disodium folinate) infusion, optionally wherein the leucovorin: i. 400 mg / m 2 The dosage, ii. intravenous, and / or iii. Apply for 2 hours; c. Irinotecan (such as ) infusion, optionally wherein irinotecan: i. 150mg / m 2 The dosage, ii. intravenous, and / or iii. Administer 30 minutes after the end of the leucovorin infusion; and / or d. 5-fluorouracil infusion, optionally wherein 5-fluorouracil: i. 2400mg / m 2 The dosage, ii. intravenous, and / or iii. Continue to apply for a duration of 46 to 48 hours (e.g., 2.4 g / m 2 / sky); Optionally, wherein the FOLFIRINOX or variant thereof is administered for multiple treatment cycles, preferably for 4 treatment cycles or 8 treatment cycles.

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

13. The combination therapy, use, method, pharmaceutical composition or kit according to claim 11 or 12, further comprising: a. Medication before the start of treatment, optionally, wherein the medication before the start of treatment includes: i. NK1 receptor antagonists, such as aprepitant, 125 mg PO, 60 minutes prior to infusion and / or continuously during days of chemotherapy, ii. 5-HT3 receptor antagonists, such as ondansetron, 8 mg PO, 30 minutes prior to infusion and / or continuously during days of chemotherapy, and / or iii. Corticosteroids, such as dexamethasone, 8 mg IV or PO, 30 minutes before infusion; and / or b. medication after the start of treatment, optionally wherein the medication after the start of treatment comprises: iG-CSF, such as Neulasta, 6 mg SC, on the fourth day after initiation of the FOLFORINOX regimen or its variants, and / or at least 24 hours after completion of the continuous 5-fluorouracil infusion.

14. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, wherein the antibody or antigen-binding portion thereof is administered at a dose of: a. 50 μg / kg to 1200 μg / kg, such as 450 μg / kg to 900 μg / kg; b.50μg / kg, 100μg / kg, 150μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 350μg / kg, 400μg / kg, 450μg / kg, 500μg / kg, 550μg / kg, 600μg / kg, 650 μg / kg, 700μg / kg, 750μg / kg, 800μg / kg, 850μg / kg, 900μg / kg, 950μg / kg, 1000μg / kg, 1050μg / kg, 1100μg / kg, 1150μg / kg, 1200μg / kg or higher; c.450 μg / kg; and / or d.900μg / kg.

15. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, wherein the antibody or antigen-binding portion thereof is administered more than once.

16. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, wherein the antibody or antigen binding portion thereof comprises the following CDRs:

17. 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: 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. SEQ ID NO:7 plus the light chain of SEQ ID NO:11, and / or SEQ ID NO:8 plus the heavy chain of SEQ ID NO:

12.

18. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, wherein the antibody or antigen binding portion thereof that specifically binds to CD40 comprises or consists of an intact antibody such as an IgG1 antibody.

19. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, 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 preceding claim, 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 preceding claim, wherein the antibody or antigen binding portion thereof and the chemotherapy are administered simultaneously, sequentially or successively with each other.

22. The combination therapy, use, method, pharmaceutical composition or kit according to any preceding claim, 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 preceding claim, 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.

Images