Multispecific antibodies for use in the treatment of disease

Abstract

A multispecific antibody is provided, comprising a first portion that binds and activates CD40, a second portion that specifically binds to dendritic cells (DCs), and a third portion that comprises an altered Fc region of the multispecific antibody to increase the specificity and affinity of binding to FcyRIIb, and uses thereof.

Description

[Technical Field]

[0001] Related Applications This application claims priority from Israel Patent Application No. 272194, filed January 22, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement A 57,172 byte ASCII file entitled "85611 Sequence Listing.txt", created on January 21, 2021, which was filed concurrently with the filing of this application, is incorporated herein by reference.

[0003] Technical Field The present invention, in some embodiments thereof, relates to multispecific antibodies for use in the treatment of disease. [Background technology]

[0004] Activation of the immune system to eliminate tumor cells can be achieved by either blocking inhibitory checkpoint molecules such as PD-1 or activating stimulatory molecules such as CD40 [1][2]. CD40, a member of the tumor necrosis factor receptor (TNFR) family, stimulates immune responses, including the production of cytotoxic T cells, when crosslinked by its natural ligand, CD40L [3][4]. Anti-CD40 agonist Abs, which mimic CD40L, have been proposed as an efficient approach to crosslink CD40, promoting DC maturation and the subsequent production of tumor antigen-specific cytotoxic T cells. Mechanistically, CD40 activation is a proximal event in T cell priming; therefore, anti-CD40 Abs may be important in converting non-inflammatory tumors to inflammatory tumors and generating effective T cell immunity [3]. Indeed, the anti-tumor activity of anti-CD40 agonist Abs has been demonstrated in numerous animal models of various tumors [5].

[0005] Several previous publications have demonstrated that binding of inhibitory FcγRIIB is an absolute requirement for the in vivo antitumor activity of agonistic Abs targeting mouse CD40, as well as other members of the TNFR family. This requirement is due to extensive cross-linking of CD40 antibodies by FcγRIIB expressed on neighboring cells. Such cross-linking enhances CD40 clustering on the cell surface, resulting in enhanced CD40 signaling [6][7]. However, similar requirements for human Abs have been questioned in several recent studies. These studies have been limited by the use of in vitro experimental systems. Alternatively, these human Abs have been evaluated in wild-type mice, limiting their ability to mimic the complex and unique cellular distribution, binding affinity, and functionality of human FcγRs.

[0006] CP-870,893 is a human anti-CD40 Ab currently under clinical evaluation. It is composed of a human IgG2 isotype and the anti-CD40 clone 2141. Surprisingly, this antibody is not only a potent anti-human CD40 agonist, but also exhibits only modest antitumor activity in patients with pancreatic ductal adenocarcinoma (PDA) or other solid tumors. [8][9] It has been found that the modest in vivo activity of CP-870,893 may be due to its Fc domain being of the human IgG2 subtype, resulting in very weak interaction with human FcγRIIB.

[10] Importantly, it has been observed that the antitumor activity of anti-CD40 agonist Abs can be enhanced through Fc engineering targeting FcγRIIB. Previous studies have tested numerous Fc-engineered 2141 variants in isogenic mice that are fully humanized for CD40 and FcγR and lack the mouse homologs of these receptors. The "V11" Fc variant, which selectively enhances FcγRIIB binding, was selected as the optimal clinical candidate. The Fc-engineered 2141 variant V11 exhibited significantly enhanced immunostimulatory activity in vivo, manifested as superior antitumor potency compared to the original IgG2 subclass variant, in several mouse tumor models.

[10] Although the Fc-engineered 2141-V11 variant can enhance therapeutic antitumor immunity compared to the parent IgG when administered at their respective maximum tolerated doses (MTDs) in patients, its use at the respective MTDs for both subclasses may be suboptimal in humans. To prepare the Fc-modified 2141 antibody for clinical studies, dosing and delivery regimens were optimized to maximize antitumor activity and minimize toxicity. Intratumoral administration of Fc-modified 2141 resulted in the widest therapeutic window compared with parental 2141 Ab administration and systemic Ab administration.

[11] Based on these studies, a second-generation Fc-modified 2141 (Fc-modified "F11") was developed and administered via intratumoral injection to patients with solid tumors (ClinicalTrials.gov identifier NCT04059588).Although intratumoral administration shows promise in some patients, it is not suitable for all patients and may be limited to patients with localized solid tumors or solid tumors metastatic to the skin, as well as tumors amenable to radiographically directed therapy.

[0007] Additional background technology: U.S. Patent No. 20160376371, U.S. Patent No. 20170253659, International Publication No. 2017004016, International Publication No. 2018213747, Mazor, Yariv, et al. "Improving target cell specificity using a novel monovalent bispecific IgG design." MAbs. Vol. 7. No. 2. Taylor & Francis, 2015. Summary of the Invention

[0008] According to an aspect of some embodiments of the present invention there is provided a multispecific antibody comprising a first portion that binds to and activates CD40 and a second portion that specifically binds to dendritic cells (DCs).

[0009] According to some embodiments of the invention, the multispecific antibody is a bispecific antibody.

[0010] According to some embodiments of the invention, the second moiety binds to a DC marker selected from the group consisting of CD11c, CD11b, DEC-205, BDCA-1, CD8, CD8α, CD103, and MHC-Class II (e.g., HLA-DR), CD141, FLT3, CD13, CD1c, Clec9a, and XCR1.

[0011] According to some embodiments of the invention, the second moiety binds to CD11c.

[0012] According to some embodiments of the invention, the second moiety binds to DEC-205.

[0013] According to some embodiments of the invention, the second moiety binds to Clec9a.

[0014] According to some embodiments of the invention, the second moiety binds to XCR1.

[0015] According to some embodiments of the present invention, a multispecific antibody comprises a first portion comprising, in its heavy chain from its N-terminus to its C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 19 to 21, and in its light chain from its N-terminus to its C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 22 to 24.

[0016] According to some embodiments of the invention, the multispecific antibody is a trifunctional antibody.

[0017] According to some embodiments of the invention, the multispecific antibody comprises: FcγRIIb and a third portion comprising an altered Fc region of the multispecific antibody to increase the specificity and affinity of binding to the target protein.

[0018] According to some embodiments of the invention, the modified Fc region comprises mutations similar to SEQ ID NO:2.

[0019] According to some embodiments of the invention, the multispecific antibody comprises knobs-into-holes mutations.

[0020] According to some embodiments of the invention, the mutations comprise the CH3 domain of the first antibody comprising Y349C / T366S / L368A / Y407V for the bispecific antibody and the CH3 domain of the second antibody comprising S354C / T366W for the multispecific antibody.

[0021] According to some embodiments of the invention, the multispecific antibody comprises SEQ ID NO:5 and SEQ ID NO:6, and any of SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:17 and SEQ ID NO:18.

[0022] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the multispecific antibody.

[0023] According to an aspect of some embodiments of the present invention there is provided a nucleic acid sequence encoding the heavy and / or light chain of the multispecific antibody.

[0024] According to an aspect of some embodiments of the present invention there is provided an expression vector comprising the nucleic acid sequence.

[0025] According to an aspect of some embodiments of the present invention there is provided a cell transformed with the expression vector.

[0026] According to an aspect of some embodiments of the present invention there is provided a method for preparing a multispecific antibody, the method comprising the steps of: (a) culturing the cells under conditions that allow expression of the multispecific antibody; (b) isolating the multispecific antibody from the cells; and A method is provided, comprising:

[0027] According to an aspect of some embodiments of the present invention there is provided a method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition to stimulate an immune response in the subject.

[0028] According to some embodiments of the invention, the subject has a tumor and an immune response against the tumor is stimulated.

[0029] According to some embodiments of the invention, the subject has a chronic viral infection and an immune response against the viral infection is stimulated.

[0030] According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition so as to treat cancer in the subject.

[0031] According to some embodiments of the invention, the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancer.

[0032] According to an aspect of some embodiments of the present invention there is provided a method of treating a chronic viral infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition so as to treat the chronic viral infection in the subject.

[0033] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition for use in the treatment of cancer and / or chronic viral infections.

[0034] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0035] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. While particular reference will now be made in detail to the drawings, it is emphasized that the details shown are for purposes of illustration and are illustrative descriptions of embodiments of the invention. In this regard, the description provided with the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Brief explanation of the drawings]

[0036] [Figure 1A-1] Figure 1A shows the sequences of the antibody (Ab) variable domains used in constructing multispecific antibodies according to some embodiments of the present invention. The sequences of the heavy chain (VH) and light chain (VL) variable domains of N418Ab and HD-109Ab were sequenced from the RNA of the respective hybridomas by amplification of cDNA ends ("anchored" PCR). The sequence of 2141 was previously identified in a patent application for this Ab. The sequences of the CDRs are underlined. In the Sequence Listing, 2141 is identified by SEQ ID NO:78 and SEQ ID NO:38, N418 by SEQ ID NO:7 and SEQ ID NO:8, HD109 by SEQ ID NO:39 and SEQ ID NO:40, and 10B4 by SEQ ID NO:15 and SEQ ID NO:16. [Figure 1A-2] Same as above [Figure 1B-1] FIG. 1B shows the sequence of an antibody or fragment thereof that can be used according to some embodiments of the present invention. [Figure 1B-2] Same as above [Figure 1B-3] Same as above [Figure 1B-4] Same as above [Figure 1B-5] Same as above [Figure 2-1] Figure 2 shows the constructs generated for the production of monospecific and bispecific Abs. Variable domains were amplified from hybridoma cDNA (N418 and HD-109) or synthesized de novo (2141) and cloned in frame with the IgG1 constant domain in expression vectors as shown. VH: heavy chain variable domain; VL: light chain variable domain; CH1-3: heavy chain constant domains 1-3; CL: light chain constant domain; KiH: knob-into-hole mutation. [Figure 2-2] Same as above [Figure 3A] Figures 3A-3B show SDS-PAGE and size-exclusion chromatogram analyses of monospecific and bispecific Abs. (Figure 3A) SDS-PAGE analysis of five Abs. The homogeneous bands in the non-reduced sample confirm that N418 and HD-109 form a single heterodimer with the 2141 Ab. Reduction of the sample confirms that the bispecific heterodimer is composed of four Ab chains, two of which originate from each partner Ab. [Figure 3B] (FIG. 3B) Size exclusion chromatogram analysis of monospecific and bispecific Abs, where the numbers indicate the molecular weight of the Abs. [Figure 4-1] Figures 4(A) to 4(D) show the dual antigen-binding properties of the bispecific Abs. (Figure 4(A)) ELISA for binding to hCD40. Quantitative ELISA assay of standard binding for anti-CD40 (red) monospecific Ab and anti-CD40 / CD11c (black)-CD40 / DEC-205 (blue) bispecific Ab against recombinant huCD40 protein. The anti-CD40 / CD11c-CD40 / DEC-205 bispecific Ab recognizes huCD40 similarly to the monospecific anti-CD40 parent Ab. [Figure 4-2](Figure 4(B)) ELISA binding to DEC-205 or CD11c. Quantitative standard binding ELISA assays of anti-CD40 (red)-DEC-205 or CD11c (black) monospecific Abs and anti-CD40 / DEC-205 or CD40 / CD11c (blue) bispecific Abs against recombinant DEC-205 or CD11c proteins. Both anti-CD40 / DEC-205 or CD40 / CD11c bispecific Abs and the monospecific anti-DEC-205 or CD11c parent Abs recognize DEC-205 or CD11c, respectively, but not the monospecific anti-CD40 Abs. (Figure 4(C)) Simultaneous ELISA binding to DEC-205 or CD11c and huCD40. Sandwich ELISA quantification assay for simultaneous binding of anti-CD40 (red)-DEC-205 or CD11c (black) monospecific Abs and anti-CD40 / DEC-205 or CD40 / CD11c (blue) bispecific Abs to recombinant DEC-205 or CD11c and huCD40 proteins. Only anti-CD40 / DEC-205 and CD40 / CD11c bispecific Abs, respectively, simultaneously bind to both proteins. [Figure 4-3] (Figure 4D) The CD40 / CD11c (2141 / N418) bsAb and 2141 were used to stain splenocytes from humanized CD40 / FcgR mice. As measured by CD19 gating, the bsAb preferentially binds to DCs and has reduced binding to B cells compared to the CD40 parental mAb. [Figure 5] Figure 5 shows in vitro stimulation of DCs with anti-human CD40 bispecific Ab. Human DCs were activated with CD40 / DEC-205 bsAb. Activation was detected by upregulation of activation of immature human DCs cultured in the presence of the indicated bsAb. Activation was determined by upregulation of different surface activation markers (CD86 and CD54 are shown). Data are representative of four donors. [Figure 6A]Figures 6A-6B show that FcγR-mediated cross-linking is required for CD40 / DC bsAb activity. Increasing doses of anti-human CD40 / DEC-205 or the Fc variant of the CD40 / CD11c bsAb described in (Figure 6A) were incubated with immature human DCs. Upregulation of CD86 and CD54 activation markers was analyzed by flow cytometry. Representative data from one of four donors are shown. [Figure 6B] T cell activation was measured by flow cytometry analysis of OVA-specific CD8+ T cells in the blood of hCD40 / FcγR mice immunized with OVA in the presence of the Fc variants of the CD40 / DC bsAb indicated (Figure 6B). Each point represents an individual mouse. Data are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. [Figure 7-1] Figures 7A-7D show the improved therapeutic window achieved by using bsAbs to reduce hepatotoxicity and increase activity. (Figure 7A) Dose-dependent T cell activation assays were performed by flow cytometry analysis of OVA-specific CD8+ T cells in the blood of humanized CD40 / FcgR mice immunized with OVA in the presence of the indicated anti-CD40 mAbs or bsAbs. Each point represents an individual mouse. [Figure 7-2] (Figure 7(B)) Dose-dependent toxicity of liver transaminases in response to increasing amounts of anti-CD40 antibody is shown. Mice were treated with increasing doses of anti-CD40 mAb or bsAb, and liver transaminases (AST and ALT) were measured. Each point represents an individual mouse. (Figure 7(C)) The CD40 / DC bsAb has an improved liver toxicity profile compared to that of the monospecific 2141 CD40 Ab. The horizontal axis (efficacy) represents the mean OVA-specific CD8+ T cells in the blood of the humanized CD40 / FcgR mice shown in panel A. The vertical axis (liver toxicity) represents the mean AST and ALT liver transaminases shown in panel B. [Figure 7-3](Figure 7(D)) Determining the MTD for liver toxicity in humanized mice demonstrates that CD40 / CD11c bsAb significantly improves T cell activity without exceeding the toxicity of monospecific CD40 mAbs. T cell activation assays were performed by flow cytometry analysis of OVA-specific CD8+ T cells in the blood of humanized CD40 / FcgR mice immunized with OVA in the presence of the indicated anti-CD40 mAbs or bsAbs (upper panel). Liver transaminases in response to anti-CD40 antibodies. Mice were treated with anti-CD40 mAbs or bsAbs, and liver transaminases (AST and ALT) were measured. Each point represents an individual mouse (lower panel). [Figure 8-1] Figures 8A-8H show the cell populations mediating the efficacy and toxicity of CD40 mAbs. (Figures 8A and 8B) T cell activation after CD40 mAb treatment was measured by flow cytometry of OVA-specific CD8+ blood T cells from C57BL / 6 (green) and Batf3- / - (blue) mice immunized with OVA (Figure 8A) or inoculated with B16-OVA tumors (Figure 8B). Left: Representative flow plots gated on CD8+ cells, showing the mean ± SEM. Right: Percentage of gated cells; each point represents an individual mouse. (Figure 8C) C57BL / 6 and Batf3- / - mice inoculated with MC38 or MCA-205 tumor cells and treated with CD40 mAbs are shown. Results are presented as the mean ± SEM (n = 8-13 per group). [Figure 8-2](Figure 8D) Blood liver transaminase levels after CD40 mAb injection in the indicated strains. (Figure 8E+Figure 8F) 24 h before CD40 mAb injection, hCD40 / FcγR mice were injected with clodronate liposomes (Figure 8E) or anti-CD42b (Figure 8F). Blood AST and ALT levels were measured (left), and livers were removed and analyzed (right, representative liver H&E sections; scale bar = 200 μm). (Figure 8G) 24 h before CD40 mAb injection, hCD40 / FcγR mice were injected with clodronate liposomes. Platelet counts 24 h later. (Figure 8(H)) hCD40 / FcγR mice were injected with CD40 mAb, and 2.5 hours later, LNs and spleens were harvested and single-cell suspensions were analyzed for intracellular IL-6 expression by flow cytometry. Top: Representative cell sorter plots. Bottom: Grouped IL-6 staining intensity. Data represent mean ± SEM. *P ≤ 0.05, **p ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns: not significant. [Figure 8-3] Same as above [Figure 8-4] Figure 8(I) shows CD40 expression on the indicated cells in MC38 tumor-bearing mice. Tumors, draining lymph nodes (LNs), spleens, and livers were excised for flow cytometry analysis. Kupffer cells (KCs), macrophages (MFs), dendritic cells (DCs), classical type 1 DCs (cDC1s), and classical type 2 DCs (cDC2s) are shown. Delta geometric mean fluorescence intensity (ΔMFI) is shown. Figures 8(J)-8(K) show the target selection of bsAbs. Expression of CD11c and DEC-205 on the indicated cell types in the MC38 tumors, draining lymph nodes (LNs), spleens, and livers of tumor-bearing mice, and on platelets (K) of naive mice, is shown. CD41 served as a positive control marker for platelets. The geometric mean fluorescence intensity (MFI) of DEC-205 and the geometric mean fluorescence intensity (ΔMFI) of CD11c delta are shown. Each dot represents an individual mouse (Fig. 8(J)). FACS analysis of a representative mouse is shown (Fig. 8(K)). [Figure 8-5]Figures 8(L)-8(R) show the cell populations mediating the efficacy and toxicity of CD40 mAb. (Figure 8(L)) The amount of Kupffer cells in the livers of C57BL / 6 (green) or Batf3- / - (blue) mice was analyzed by flow cytometry. (Figure 8(M)) C57BL / 6 mice were injected with clodronate liposomes. 24 hours later, the livers were removed, and single-cell suspensions were analyzed by flow cytometry for the frequency of the indicated cell populations. (Figure 8(N)) C57BL / 6 mice were injected with clodronate liposomes 24 hours before CD40 mAb injection. 24 hours later, blood AST and ALT levels were measured. (Figure 8(O)-8(P)) hCD40 / FcγR mice were injected with anti-CD42b 24 hours before CD40 mAb injection. Platelets were measured 24 hours later (Figure 8(O)), and livers were excised and analyzed (Figure 8(P)). Representative liver H&E sections; scale bar = 100 μm. (Figure 8(Q)) Serum IL-6 and TNF-α levels after CD40 mAb injection. hCD40 / FcγR mice were injected with 2141 CD40 mAb, and serum was collected 3 hours later. Cytokine levels were measured by ELISA. (Figure 8(R)) Intracellular IL-6 expression after CD40 mAb injection. hCD40 / FcγR mice were injected with 2141 CD40 mAb. 2.5 hours later, blood was analyzed by flow cytometry. Kupffer cells (KCs), macrophages (MFs), dendritic cells (DCs), classical type 1 DCs (cDC1s), and classical type 2 DCs (cDC2s) were detected. Each point represents an individual mouse and data are presented as mean ± SEM. *P<0.05, **p≦0.01, ***P≦0.001, ****P≦0.0001, ns: not significant. [Figure 9]Figures 9(A)-9(B) show that FcγR-mediated cross-linking is required for CD40 / DC bsAb activity. (Figure 9(A)) T cell activation was measured by flow cytometry analysis of OVA-specific CD8+ T cells in the blood of hCD40 / FcγR mice immunized with OVA in the presence of the Fc variants of the CD40 / DC bsAb. Each point represents an individual mouse. Data are presented as mean ± SEM. *p≦0.05, **p≦0.01. (Figure 9(B)) Binding of human bsAb Fc variants to human FcγRIIB is shown. Binding of the Fc variants of the anti-CD40 / DC bsAb to recombinant hFcγRIIB was assessed by ELISA. [Figure 10-1] Figures 10(A) to 10(E) show that CD40 / CD11c bsAb induced superior antitumor responses compared with CD40 mAb when administered at safe doses. (Figure 10(A)) Representative H&E staining of livers from hCD40 / FcγR mice treated with CD40 mAb or CD40 / CD11c bsAb at the indicated doses (n = 4 per group) is shown. (Figure 10(B)) IL-6 and TNF-α secretion after CD40 mAb or CD40 / CD11c bsAb treatment. (Figure 10(C)) T cell activation was measured by flow cytometry analysis of OVA-specific CD8+ T cells in the blood of humanized CD40 / FcγR mice immunized with OVA in the presence of the indicated mAb or bsAb. [Figure 10-2](Figure 10(D)) hCD40 / FcγR mice were inoculated with MC38 or B16-F10 tumor cells. Once tumors were established and reached an average volume of 50 mm, mice were treated with CD40 mAb or CD40 / CD11c bsAb at their designated MTDs. Tumor volumes were measured with a caliper every 3 to 4 days (n = 9 to 11 per group). (Figure 10(E)) hCD40 / FcγR mice were inoculated with B16-F10 tumor cells. Mice with established tumors were treated with the indicated mAb / bsAb. Tumor volumes were measured with a caliper every 3 to 4 days (n = 9 to 10 per group). Each point represents an individual mouse, and data are presented as the mean ± SEM. *P < 0.05, **p ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention, in some embodiments thereof, relates to multispecific antibodies for use in the treatment of disease.

[0038] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following detailed description or illustrated by the examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0039] The therapeutic use of anti-CD40 monoclonal agonist antibodies (mAbs) is an approach aimed at exploiting the ability of the immune response to eliminate tumors. This approach has been demonstrated to be effective in numerous animal models of various tumors. However, human anti-CD40 mAbs have shown only modest anti-tumor activity in patients with solid tumors.

[0040] In considering embodiments of the present invention, the inventors have devised multispecific molecules that target CD40 in a dendritic cell-specific manner. Antibodies that preferentially bind FcγRIIb have also been devised.

[0041] As shown below and in the Examples section that follows, we have established the cellular pathways that mediate the efficacy and toxicity of anti-CD40 antibodies. We designed a CD40 agonist mAb with maximal antitumor activity by reducing dose-limiting toxicity. We engineered a new class of molecule in the form of an Fc-engineered multispecific (bispecific) antibody that targets both CD40 and dendritic cell markers. Specifically, the 2141-V11 variant (with enhanced specificity for FcγRIIb) is specific for DCs, the cell population that mediates its antitumor activity, while simultaneously reducing binding to CD40 in other cell populations. To achieve this specific delivery, we designed a novel strategy that utilizes a bispecific Ab format on an IgG scaffold as the entirety. Such a design has the advantage of increased specificity for defined cell populations while maintaining the ability to engage the appropriate FcγR pathways. This antibody possesses many beneficial properties, including (i) defined cell specificity mediated by dual human Fab recognition and (ii) enhanced FcγRIIB binding mediated by Fc engineering. To generate the desired bispecific Ab combination, the two heavy and two light chains of two pre-existing Abs must be properly assembled. Therefore, we synthesized the four chains that make up each bsAb. We utilized knob-into-hole technology (specified point mutations in the CH3 domains of the heavy chains) to enable heterodimerization of the desired heavy chains

[12] . We applied CrossMab technology (swapping the heavy chain CH1 and light chain CL1 domains of one of the two Abs that make up the bsAb) to ensure proper association of the light chains with their cognate heavy chains. The variable domains of 2141 were cloned from the previously generated monospecific 2141 Ab into the bispecific Ab construct. The variable domains of anti-DEC-205 (HD-109), CD11c (N418), Clec9a, and XRC1 (MARX10) Abs were sequenced and cloned from the HD-109

[13] and N418

[14] hybridomas, respectively.The variable domains of Clec9a (10B4) and XRC1 (MARX10) were synthesized based on their sequences described in U.S. Patent Application No. 20130273150(A) and European Patent No. 2641915(A1), respectively. Bispecific Abs were constructed with wild-type (WT) IgG and the "V11" Fc scaffold (point mutations were introduced using PCR-mediated site-directed mutagenesis) to preserve the optimal high degree of cross-linking by FcγRIIB required for CD40 activation in vivo.

[0042] The inventors have established a synergistic effect between the CD40-binding moiety, DC targeting, and FcγRIIB binding in vivo, which exhibits optimal antitumor activity and minimal toxicity, suggesting improved therapy through checkpoint modulation (e.g., anti-PD-L1).

[0043] It is believed that the antibodies of some embodiments of the present invention have improved specificity and therefore therapeutic efficacy, and therefore may be successfully used in the clinic.

[0044] Thus, according to one aspect of the present invention there is provided a multispecific antibody comprising a first portion that binds to and activates CD40 and a second portion that specifically binds to dendritic cells (DCs).

[0045] According to one aspect of the invention, a first portion that binds to and activates CD40, a second portion that specifically binds to dendritic cells (DCs), FcγRIIb and a third portion comprising an altered Fc region of the multispecific antibody to increase the specificity and affinity of binding to the target protein.

[0046] As used herein, "CD40" refers to "TNF receptor superfamily member 5" (TNFRSF5).

[0047] The sequence of human CD40 (NP_001241.1), including the 20 amino acid signal sequence, is provided in SEQ ID NO:41.

[0048] CD40 interacts with CD40 ligand (CD40L), which is also known as TNFSF5, gp39, and CD154. Unless otherwise stated or clear from the context, references herein to CD40L refer to human CD40L ("huCD40L"). Human CD40L is further described in MIM:300386. The sequence of human CD40L (NP_000065.1) is provided in SEQ ID NO:42.

[0049] It will be understood that antibodies to CD40 bind to human CD40 and / or mouse CD40. Antibodies that bind to both human and mouse CD40 are typically referred to as "pan-specific antibodies."

[0050] As mentioned, the first portion binds to and activates CD40 (mimicking CD40L) and is therefore termed "agonistic." Agonistic activity can be assayed by testing for upregulation of CD54 or CD86 on human dendritic cells and / or by testing in vivo T cell activation assays (e.g., binding to CD40 confirmed by ELISA).

[0051] Complementarity determining sequences (CDRs) of the first portion that can be used in multispecific antibodies according to some embodiments of the invention can be found in the antibodies listed below. Anti-CD40 2141 (also known as CP870,893) is represented by SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:12.

[0052] Thus, according to embodiments of the present invention, there is provided a multispecific antibody comprising a first portion comprising, from N- to C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 19 to 21 in the heavy chain and, from N- to C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 22 to 24 in the light chain. CD40 V11 and bispecific assemblies containing mutations to optimize FcγRIIB binding are represented by SEQ ID NOs: 5 and 6 (V11 and V21, respectively). H and V L ) is available. Anti-CD40 antibodies: 12D6 and 5F11 are described in WO 2017 / 0253659, which is incorporated herein by reference in its entirety. APX005M (Apexigen): Johnson M, Fakih M, Bendell J, et al. 2017. First-in-human study of the CD40 monoclonal agonist antibody APX005M in subjects with solid tumors. J. ImmunoTher. Cancer 5(Suppl. 3):89 (Abstr.) ·SGN-40:C.-L. Law et al., “Preclinical antilymphoma activity of a humanized anti-CD40 monoclonal antibody, SGN-40.,” Cancer Res., vol. 65, no. 18, pp. 8331-8338, Sep. 2005. · SEA-CD40-DOI:10.1200 / JCO.2018.36.15_suppl.3093 Journal of Clinical Oncology 36, no. 15_suppl (May 20, 2018) 3093-3093.

[0053] Other CD40 agonist antibodies are available in the art.

[0054] As mentioned above, the multispecific antibody comprises a second portion that specifically binds to a dendritic cell (DC).

[0055] As used herein, "specific" refers to a binding preference for DCs compared to other cells, such as peripheral blood cells, or platelets. According to some embodiments, "specific" refers to the absence of binding to macrophages (Kupffer or non-Kupffer) and platelets because the target is not expressed or is expressed at a lower density compared to DCs, as measured by flow cytometry analysis.

[0056] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to an antibody that binds to an epitope on a given antigen but not to other antigens. Typically, an antibody (i) binds to an epitope of about 10 mAb, as measured by surface plasmon resonance (SPR) technology on a BIACORE® 2000 surface plasmon resonance instrument, for example, using a given antigen (e.g., a recombinant DC marker) as the analyte and the antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than m, e.g., about 10 -8 Under M, 10 -9 Less than M or even 10 -10 and (ii) binds to a predetermined antigen with an affinity that is at least two-fold greater than the affinity with which it binds to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to human CD40 or a DC marker" has an equilibrium dissociation constant (KD) that is 10 M or less. -6 M or less, e.g., about 10 -7 Less than M, about 10 -8 Under M, 10 -9 Less than M or even 10 -10 K below M D This refers to an antibody that binds to soluble or cell-bound human CD40 or a DC marker.

[0057] As used herein, "dendritic cells" (DCs) or the plural "dendritic cells" (DCs) refer to cells belonging to a cell population called professional antigen-presenting cells (APCs). DCs have a characteristic morphology, with thin, membrane-like structures (lamellipodia) extending in multiple directions from the dendritic cell body. Several phenotypic criteria are typical but may vary depending on the source of the dendritic cells. These include high levels of MHC molecules (e.g., class I and class II MHC) and costimulatory molecules (e.g., B7-1 and B7-2), and the absence of markers specific for granulocytes, NK cells, B cells, and T cells. Many dendritic cells express specific markers, as listed below. Dendritic cells can initiate primary T cell responses in vitro and in vivo. These responses are antigen-specific. Dendritic cells directly induce a stronger mixed leukocyte reaction (MLR) compared to peripheral blood leukocytes, splenocytes, B cells, and monocytes. Dendritic cells are sometimes assessed by the pattern of cytokine expression by the cells (Zhou and Tedder (1995) Blood 3295-3301). According to certain embodiments, multispecific antibodies may bind to immature DCs and mediate their maturation and activation.

[0058] According to a particular embodiment, the dendritic cells are cDC1 or cDC2.

[0059] According to certain embodiments, the dendritic cells are cDC1 and cDC2.

[0060] According to certain embodiments, the dendritic cell is characterized by marker expression, wherein the second portion binds to a DC marker selected from the group consisting of CD11c, CD11b, Clec9a, XCR1, DEC-205, BDCA-1, CD8, CD8α, CD103, and MHC-class II (e.g., HLA-DR), CD141, FLT3, CD13, and CD1c.

[0061] According to a particular embodiment, the DCs are human DCs.

[0062] According to certain embodiments, the second moiety binds to a DC marker selected from the group consisting of CD141, FLT3, CD13, CD1c, and HLA-DR (MHCII).

[0063] According to certain embodiments, the second portion binds to a DC marker selected from the group consisting of CD11c, CD11b, Clec9a, XCR1, DEC-205, BDCA-1, CD8, CD8α, CD103, MHC-class II (e.g., HLA-DR), CD141, CD13, and CD1c, LILRA4, LAMP5, CLEC4C, IL3RA, and SIGLEC6.

[0064] According to certain embodiments, the DC marker is not LILRA4, LAMP5, CLEC4C, IL3RA, CLEC9A, XCR1, FLT3, or SIGLEC6.

[0065] According to certain embodiments, the second moiety binds to CD11c or DEC-205.

[0066] According to certain embodiments, the second moiety binds to CD11c.

[0067] Thus, according to an embodiment of the present invention, there is provided a multispecific antibody comprising a second portion comprising, in its heavy chain from its N-terminus to its C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 25 to 27, and in its light chain from its N-terminus to its C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 28 to 30.

[0068] According to certain embodiments, the second moiety binds to DEC-205.

[0069] Thus, according to an embodiment of the present invention, there is provided a multispecific antibody comprising a second portion comprising, in its heavy chain from its N-terminus to its C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 31 to 33, and in its light chain from its N-terminus to its C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 34 to 36.

[0070] According to certain embodiments, the second moiety binds to Clec9a.

[0071] Thus, according to an embodiment of the present invention, there is provided a multispecific antibody comprising a second portion comprising, in its heavy chain from N-terminus to C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 52 to 54, and in its light chain from N-terminus to C-terminus, the complementarity determining regions set forth in SEQ ID NOs: 55 to 57 (CDRs of 10B4).

[0072] According to certain embodiments, the second moiety binds to XCR1.

[0073] Thus, according to an embodiment of the present invention, there is provided a multispecific antibody comprising a second portion comprising, in the heavy chain from N-terminus to C-terminus, the complementarity determining regions set forth in SEQ ID NOS: 58 to 60, and in the light chain from N-terminus to C-terminus, the complementarity determining regions set forth in SEQ ID NOS: 61 to 63 (CDRs of MARX10).

[0074] Antibodies capable of binding to CD11c are well known in the art. N418 is available from the ATCC

[14] .

[0075] Antibodies capable of binding to DEC-205 are known in the art, including HD-109, available from Rockefeller University

[13] , HD-20, HD-24, HD-71, HD-73, HD-77, and HD-83.

[0076] Antibodies capable of binding to Clec9a are known in the art: 10B4 and others are described in US Patent Application No. 20130273150(A)

[15] (e.g., 1F6, 397, and 7H11 are described in

[16] ).

[0077] Antibodies capable of binding to XCR1 are known in the art. MARX10 is described in EP 2641915(A1)

[17] .

[0078] It will be understood that any of the portions may include Fc modifications that increase binding to FcγRIIB, such as, but not limited to, V11 mutations (SEQ ID NO: 2), S267E ("SE"), S267E / L382F ("SELF"), G237D / P238D / P271G / A330R ("V9"), and / or E233D / G237D / P238D / H268D / P271G / A330R ("V12") corresponding to the human IgG sequence (positions corresponding to SEQ ID NO: 1).

[0079] According to particular embodiments, the multispecific antibody comprises SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:37 and SEQ ID NO:38.

[0080] According to particular embodiments, the multispecific antibody comprises SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:39 and SEQ ID NO:40.

[0081] According to particular embodiments, the multispecific antibody comprises SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:15 and SEQ ID NO:16.

[0082] According to particular embodiments, the multispecific antibody comprises SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18.

[0083] As used herein, the term "antibody" includes intact molecules as well as functional fragments thereof (which are capable of binding to an epitope of an antigen).

[0084] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0085] According to certain embodiments, antibody fragments include, but are not limited to, single chains, Fab, Fab' and F(ab')2 fragments, Fd, Fcab, Fv, dsFv, scFv, diabodies, minibodies, nanobodies, Fab expression libraries, or single domain molecules such as VH and VL capable of binding to an epitope of an antigen in an HLA-restricted manner.

[0086] Suitable antibody fragments for practicing some embodiments of the present invention include antibody fragments containing essentially the entire variable regions of both the light and heavy chains, such as Fv, single-chain Fv (scFv), disulfide-stabilized Fv (dsFv), Fab, Fab', and F(ab')2, or antibody fragments containing the Fc region of an antibody.

[0087] As used herein, the terms "complementarity determining region" or "CDR" are used interchangeably to refer to the antigen-binding regions found in the variable regions of heavy and light chain polypeptides. Generally, antibodies contain three CDRs in each VH (CDR HI or HI, CDR H2 or H2, and CDR H3 or H3) and three CDRs in each VL (CDR LI or LI, CDR L2 or L2, and CDR L3 or L3).

[0088] The identities of the amino acid residues in a particular antibody that make up the variable regions or CDRs can be determined using methods well known in the art, including the sequence diversity defined by Kabat et al. (see, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), the location of the structural loop regions defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989), the compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (see herein Accelrys®, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268, and the worldwide website www(dot)bioinf-org(dot)uk / abs), available complex crystal structures as defined by contact definition (MacCallum et al. al., J. Mol. Biol. 262:732-745, 1996) and "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).

[0089] As used herein, "variable region" and "CDR" may refer to variable regions and CDRs defined by any approach known in the art, including a combination of approaches.

[0090] Functional antibody fragments that contain all or essentially all of the variable regions of both the light and heavy chains are defined as follows. (i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains; (ii) Single-chain molecule Fv ("scFv"), a genetically engineered single-chain molecule comprising the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker, which is a genetically fused single-chain molecule. (iii) Disulfide-stabilized Fv ("dsFv"), a genetically engineered antibody comprising a light chain variable region and a heavy chain variable region linked by a genetically engineered disulfide bond. (iv) Fab, the fragment of an antibody molecule containing a monovalent antigen-binding portion of the antibody molecule, which can be obtained by treating whole antibody with the enzyme papain to produce an intact light chain and an Fd fragment of the heavy chain consisting of its variable and CH1 domains; (v) Fab', the fragment of an antibody molecule containing a monovalent antigen-binding portion of the antibody molecule can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (to yield two Fab' fragments per antibody molecule); (vi) F(ab')2, the fragment of an antibody molecule containing a monovalent antigen-binding portion of the antibody molecule (i.e., a dimer of Fab' fragments held together by two disulfide bonds) which can be obtained by treating whole antibody with the enzyme pepsin; (vii) single domain antibodies or nanobodies, which are composed of a single VH or VL domain that exhibits sufficient affinity for the antigen; (viii) Fcab, a fragment of an antibody molecule containing the Fc portion of an antibody that has been developed as an antigen-binding domain by introducing antigen-binding capacity into the Fc region of the antibody.

[0091] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0092] Exemplary methods for generating antibodies utilize inducing in vivo production of antibody molecules, screening immunoglobulin libraries (Orlandi DR et al., 1989. Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter G. et al., 1991. Nature 349:293-299), or production of monoclonal antibody molecules by continuous cell lines in culture. These methods include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and Epstein-Barr virus (EBV)-hybridoma technology (Kohler G. et al., 1975. Nature 256:495-497; Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42; Cote RJ. et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole SP. et al., 1984. Mol. Cell. Biol. 62:109-120).

[0093] If the target antigen is too small to elicit an adequate immunogenic response when generating antibodies in vivo, such antigens (haptens) can be conjugated to antigenically neutral carriers, such as keyhole limpet hemocyanin (KLH) or serum albumin [e.g., bovine serum albumin (BSA)] carriers (see, e.g., U.S. Pat. Nos. 5,189,178 and 5,239,078). Conjugation of haptens to carriers can be accomplished using methods well known in the art. For example, direct conjugation to amino groups can be performed, optionally followed by reduction of the imino bond formed. Alternatively, carriers can be attached using condensing agents such as dicyclohexylcarbodiimide or other carbodiimide dehydrating agents. Linker compounds can be used to effect the attachment; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical, Inc., Rockford, IL. The resulting immunogenic complex can then be injected into a suitable mammalian subject, such as a mouse or rabbit. A suitable protocol involves repeated injections of the immunogen in the presence of an adjuvant according to a schedule that promotes the production of antibodies in the serum. The titer of immune serum can be readily measured using immunoassay procedures well known in the art.

[0094] The antisera obtained can be used directly, or monoclonal antibodies can be obtained as described above.

[0095] Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell cultures, or other protein expression systems).

[0096] Antibody fragments can be obtained by conventional pepsin or papain digestion of whole antibodies. For example, antibody fragments can be generated by pepsin cleavage of antibodies to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to generate a 3.5S Fab' monovalent fragment. Alternatively, cleavage using pepsin directly generates two monovalent Fab' fragments and an Fc fragment. These methods are described, for example, in U.S. Patent Nos. 4,036,945 and 4,331,647 by Goldenberg, and the references contained therein, which are incorporated herein by reference in their entireties. See also Porter, RR, Biochem. J. 73: 119-126 (1959). Separation of the heavy chains to form monovalent light-heavy chain fragments, further fragment degradation, or other enzymatic, chemical, or genetic techniques can also be used, provided the fragments bind to the antigen recognized by the intact antibody.

[0097] As noted above, an Fv fragment comprises an association of a VH chain and a VL chain. This association is non-covalent, as described by Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720)]. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises a VH chain and a VL chain connected by a peptide linker. These single-chain antigen-binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is then transformed into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, in Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988), Pack et al., Bio / Technology 11:1271-77 (1993), and U.S. Pat. No. 4,946,778, the entire contents of which are incorporated herein by reference.

[0098] Another form of antibody fragment is a peptide encoding a single complementarity-determining region (CDR). CDR peptides ("minimal units of recognition") can be obtained by constructing genes encoding the CDRs of a desired antibody. Such genes are produced, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0099] As mentioned above, antibody fragments can include the Fc region of an antibody, referred to as "Fcab." Such antibody fragments typically contain the CH2-CH3 domains of an antibody. Fcabs are designed to contain at least one modification in the antibody's loop-forming region, i.e., the CH3 region of the heavy chain. Such antibody fragments can be produced, for example, as follows: a nucleic acid encoding an antibody containing at least one loop-forming region (e.g., an Fc region) is prepared, at least one nucleotide residue in at least one loop-forming region is modified, the modified nucleic acid is transferred to an expression system, the modified antibody is expressed, the expressed modified antibody is contacted with an epitope, and whether the modified antibody binds to the epitope is determined. See, for example, U.S. Pat. Nos. 9,045,528 and 9,133,274, which are incorporated herein by reference in their entireties.

[0100] Humanized antibodies of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab', F(ab'), or other antigen-binding sequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues forming the recipient's complementarity-determining regions (CDRs) are replaced by residues from a CDR (donor antibody) of a non-human species such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0101] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues incorporated from non-human material. These non-human amino acid residues, often referred to as import residues, typically come from an import variable domain. Humanization can essentially be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, following the method of Winter and coworkers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)]. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less of the intact human variable domain has been replaced by the corresponding non-human sequence. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0102] Human antibodies can also be produced using various techniques known in the art, such as phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The technology of Cole et al. and Boerner et al. can also be used to produce human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigen challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in the following scientific publications: Marks et al., Bio / Technology 10,: 779-783 (1992), Lonberg et al., Nature 368: 856-859 (1994), Morrison, Nature 368 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0103] Unless otherwise specified, immunoglobulins can be from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into specific subclasses: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as human IgG1, exist in several allotypes, which often differ from each other by only a few amino acids.

[0104] According to certain embodiments, the antibody is of the IgG1 isotype. Once the antibody is obtained, it can be tested for activity, for example, by ELISA, Western blot, FACS, dot blot, and any other method for evaluating antibodies.

[0105] According to certain embodiments, the antibody is trifunctional in that it binds to Fcγ, CD40, and DC.

[0106] As used herein, a "multispecific antibody" is an antibody that can simultaneously bind to at least two targets, as described above, i.e., different structures, two different antigens, or two different epitopes, one on CD40 and at least one on DCs.

[0107] Specificity refers to the number of antigens or epitopes that the antibody can bind, i.e., bispecific, trispecific, tetraspecific. According to a particular embodiment, the antibody is a bispecific antibody.

[0108] Using these definitions, a natural antibody, eg, an IgG, is bivalent because it has two binding arms but is monospecific because it binds to one type of epitope.

[0109] A "bispecific antibody" is an antibody that can simultaneously bind to two targets of different structures, one on CD40 and at least one other on DCs.

[0110] Valency refers to the number of binding arms or sites an antibody has for a single antigen or epitope, i.e., monovalent, bivalent, trivalent, or multivalent. An antibody being multivalent means that it can utilize multiple interactions in binding to the antigen, thus increasing its binding affinity to the antigen.

[0111] A multispecific, multivalent antibody is a construct that has two or more binding sites with different specificities, such as a diabody, in which one binding site reacts with one antigen and the other binding site reacts with a different antigen.

[0112] As used herein, "moiety" refers to an antibody component of a multispecific (eg, bispecific) antibody capable of binding to an indicated target.

[0113] To produce the multispecific antibodies of some embodiments of the invention, moieties of the invention may be modified in the Fc region, e.g., the CH3 domain (according to Kabat), as is well known in the art. Such modifications ensure correct assembly of the multispecific antibodies via their heavy chains.

[0114] Therefore, the CH3 domain of one heavy chain is altered so that its original interface region matches the original interface region of the CH3 domain of the other heavy chain of the multispecific antibody. In this process, amino acid residues are replaced with amino acid residues with bulkier side chains, creating a protrusion in the interface region of the CH3 domain of the one heavy chain that can be positioned in a cavity in the interface region of the CH3 domain of the other heavy chain. Furthermore, amino acid residues are replaced with amino acid residues with less bulky side chains, creating a cavity in the interface region of the second CH3 domain that matches the original interface region of the first CH3 domain in the trivalent bispecific antibody. This creates a cavity in the interface region of the second CH3 domain that can be positioned in the protrusion in the interface region of the first CH3 domain (this method is also known as the "knob-into-hole" approach by Genentech).

[0115] According to a particular embodiment, the amino acid residue with a bulkier side chain is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

[0116] According to a particular embodiment, the amino acid residue with a less bulky side chain is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

[0117] According to certain embodiments, each CH3 domain is further modified by introducing a cysteine ​​(C) amino acid at the corresponding position in each CH3 domain so that disulfide bridges can be formed between the CH3 domains.

[0118] In certain embodiments, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, or Y407V mutation in the CH3 domain of the "hole chain." For example, an interchain disulfide bridge can be added between the CH3 domains by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C or S354C mutation in the CH3 domain of the "hole chain" (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681). Thus, in another preferred embodiment, the bispecific antibody comprises the Y349C and T366W mutations in one of the two CH3 domains and the E356C, T366S, L368A, and Y407V mutations in the other of the two CH3 domains, or the bispecific antibody comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A, and Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form an interchain disulfide bridge) (numbering always according to the EU index of Kabat). However, other knob-in-hole technologies, such as those described by EP 1 870 459 A1, can also be used alternatively or additionally. A specific example of a bispecific antibody has the R409D; K370E mutation in the CH3 domain of the "knob chain", and the D399K; E357K mutation in the CH3 domain of the "hole chain" (numbering always according to the EU index of Kabat).

[0119] In another embodiment, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, Y407V, and further R409D mutations in the CH3 domain of the "hole chain"; a K370E mutation and a D399K mutation in the CH3 domain of the "knob chain"; and an E357K mutation in the CH3 domain of the "hole chain".

[0120] In another embodiment, the bispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains, or the bispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains, in addition to R409D, and further comprises the mutations K370E and D399K in the CH3 domain of the "knob strand", and the mutation E357K in the CH3 domain of the "hole strand".

[0121] According to certain embodiments, the first mAb (e.g., anti-DC) carries the mutations Y349C / T366S / L368A / Y407V, and the second mAb (e.g., anti-CD40) carries the mutations S354C / T366W (Merchant et al., 1998; Ridgway et al., 1996).

[0122] Alternatively or additionally, at least one of the portions can be expressed in a CrossMab format (CH1-CL swapping) for correct heavy-light chain pairing.

[0123] The basis of CrossMab technology is the crossover of antibody domains in one arm of a bispecific IgG antibody, while the correct heterodimerization of the heavy chains can be achieved by the knob-into-hole technique or charge interactions described above. This format can be achieved by exchanging different domains within Fab fragments. Fab domains within Fab fragments (CrossMab Fab in the form of a cross-linked VH-VL domain (CrossMab VH-VL format), or constant CH1-CL domain (CrossMab CH1-CL format) can be interchanged for this purpose. CH1-CLIn this format, the original light chain and the new VL-CH1 light chain, respectively, do not interact undesirably with the original heavy chain and the VH-CL-containing heavy chain, respectively, and theoretical by-products cannot be formed. Fab In this format, non-functional monovalent antibodies (MoAbs) as well as non-functional Fab fragments may be formed. These by-products can be removed by chromatographic techniques. VH-VL In this format, an undesired by-product with a VL-CH1 / VL-CL domain association (known in Bence Jones proteins) may occur between the heavy chain containing the VL-CH1 and the original unmodified VL-CL light chain. Based on the conserved charge pairs present in the framework of wild-type antibodies, the introduction of repulsive charge pairs into the constant CH1 and CL domains of the wild-type uncrosslinked Fab fragments allows for the formation of CrossMab fragments. VH-VL+ / - This method overcomes the formation of Bence-Jones-like by-products in the ELISA format. Further details regarding the CrossMab technology can be found in Klein et al. Methods 154, 1 February 2019, Pages 21-31c.

[0124] Alternatively, the multispecific antibodies, e.g., bispecific antibodies, described herein can be prepared by conjugating multiple moieties using methods known in the art. For example, each moiety of the multispecific antibody can be generated separately and then conjugated to one another. A variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132, Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. Immunol. 139:2367-2375. Preferred cross-linking agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0125] Alternatively or additionally, cross-linking of the portions of the multispecific antibody can be achieved via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge region is modified to contain an odd number of sulfhydryl groups, preferably one sulfhydryl group, prior to cross-linking.

[0126] According to certain embodiments, the third portion enhances specificity for the FcγRIIb receptor.

[0127] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIb, or equivalently, RcγRIIB). Various properties of human FcγRs are summarized in U.S. Patent Application Publication No. 2017 / 0253659 (and Table 1 therein).

[0128] While most innate effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIb, natural killer (NK) cells in mice and humans selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitory FcγRIIb. Human IgG1 binds most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the type of activating Fc receptor it binds.

[0129] Human FcγRIIB has low affinity for human IgG. As mentioned above, several previous publications have shown that binding of inhibitory FcγRIIB is an absolute requirement for the in vivo antitumor activity of agonistic antibodies targeting mouse CD40 as well as other members of the TNFR family. This binding is due to high-order cross-linking of CD40 antibodies by FcγRIIB expressed on neighboring cells. This cross-linking enhances CD40 clustering on the cell surface, resulting in enhanced CD40 signaling [6][7].

[0130] Thus, according to an embodiment of the present invention, the multispecific antibody comprises: FcγRIIb The multispecific antibody comprises a third portion comprising a modified Fc region to increase the specificity and affinity of binding to the target antigen.

[0131] The altered (variant) Fc region has one or more mutations corresponding to one or more mutations in a human IgG heavy chain (SEQ ID NO: 1) selected from the group consisting of N297A, S267E ("SE"), S267E / L382F ("SELF"), G237D / P238D / P271G / A330R ("V9"), or G237D / P238D / H268D / P271G / A330R ("V11") (SEQ ID NO: 2), or ("V12").

[0132] According to a specific embodiment, the altered Fc is of the V11 variant. As shown in the Examples section below, in vitro and in vivo experiments demonstrated that multispecific antibodies benefit from FcγRIIB binding to effectively activate DCs and T cells ( FIG. 6 ).

[0133] Another aspect described herein relates to nucleic acid molecules encoding the antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA bound to the essentially isolated DNA), or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

[0134] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

[0135] V H and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further modified by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L A DNA fragment encoding V H is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0136] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CHI, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification.

[0137] The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, for example, an IgG1 region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain C1 constant region.

[0138] V L The isolated DNA encoding the regions can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region.

[0139] A variety of prokaryotic or eukaryotic cells can be used as host expression systems to express the antibodies of some embodiments of the present invention. Such expression systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the coding sequence or transformed with recombinant plasmid expression vectors, such as Ti plasmids, containing the coding sequence. Mammalian expression systems can also be used to express the antibodies of some embodiments of the present invention. Expression conditions during culture vary depending on the expression system used.

[0140] The antibody is recovered from the culture after culturing for an appropriate period of time. The phrase "recovering the recombinant antibody" refers to collecting the entire culture medium containing the antibody and does not necessarily imply additional steps of separation or purification. Notwithstanding the above, antibodies of some embodiments of the present invention can be purified using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed-phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization.

[0141] The antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo utilities, including, for example, enhancing immune responses by stimulating CD40 signaling. In preferred embodiments, the antibodies described herein are human or humanized. For example, the multispecific antibodies described herein can be administered to cultured cells, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in various diseases.

[0142] Accordingly, provided herein are methods of modifying an immune response in a subject comprising administering to the subject a multispecific antibody described herein, such that the immune response in the subject is enhanced, stimulated or upregulated.

[0143] As used herein, the term "subject" includes a mammal, such as a human, of any age, suffering from a disorder, e.g., cancer, chronic viral infection, etc. According to certain embodiments, the term encompasses individuals at risk of developing the disorder.

[0144] According to certain embodiments, the subject includes a human patient in whom an enhanced immune response is desired. The method is particularly suitable for treating human patients with disorders that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In certain embodiments, the method is particularly suitable for treating cancer. To achieve antigen-specific enhancement of immunity, the multispecific antibodies described herein can be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a cancer-bearing subject or a virus-carrying subject). When the anti-CD40 antibody is administered together with another agent, they can be administered separately or simultaneously.

[0145] Further encompassed is a method of enhancing an immune response (e.g., T cell-mediated anti-tumor immunity) in a subject, comprising administering to the subject a multispecific antibody described herein, such that the immune response in the subject is enhanced. In a preferred embodiment, the subject is a cancer-bearing subject, and the immune response against the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In certain embodiments, the tumor is PD-L1 negative. The subject may also be a subject carrying a virus, and the immune response against the virus is enhanced.

[0146] Further provided are methods of inhibiting tumor cell growth in a subject, comprising administering to the subject a multispecific antibody described herein to inhibit tumor growth in the subject. Also provided are methods of treating a chronic viral infection in a subject, comprising administering to the subject a multispecific antibody described herein to treat the chronic viral infection in the subject.

[0147] In certain embodiments, the multispecific antibodies described herein are administered to a subject as adjuvant therapy. Treating a subject with cancer using the multispecific antibodies described herein may result in a long-term, durable response, long-term survival of at least 1, 2, 3, 4, 5, or 10 years or more, and recurrence-free survival of at least 1, 2, 3, 4, 5, or 10 years or more compared to current standard treatments. In certain embodiments, treating a subject with cancer using the multispecific antibodies described herein prevents or delays cancer recurrence, for example, for 1, 2, 3, 4, 5, or 10 years or more. Anti-CD40 therapy can be used as first-line or second-line therapy.

[0148]

[0013] Provided herein are methods of treating a subject with cancer, comprising administering to the subject a multispecific antibody described herein, such that the subject is treated, e.g., cancerous tumor growth is inhibited or reduced and / or tumor regression occurs. The multispecific antibodies described herein can be used alone to inhibit cancerous tumor growth. Alternatively, the multispecific antibodies described herein can be used in conjunction with another agent, e.g., another immunogenic agent, a standard cancer treatment, or another antibody, as described below.

[0149] Thus, provided herein is a method of treating cancer, e.g., a method of treating cancer by inhibiting the growth of tumor cells in a subject, comprising administering to a subject a therapeutically effective amount of a multispecific antibody described herein.

[0150] Cancers whose growth can be inhibited using the antibodies of the present invention typically include cancers that respond to immunotherapy. Non-limiting examples of cancers to treat include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer.cancer), germ cell tumors, childhood sarcomas, nasal necrotic lesions, melanoma (e.g., metastatic melanoma, such as cutaneous or intracanal melanoma), bone cancer, skin cancer, uterine cancer, anal canal cancer, testicular cancer, fallopian tube cancer, carcinoma of the endometrium, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, cancer of the ureter, renal pelvis cancer, central nervous system (CNS) neoplasms, central nervous system (CNS) primary malignant lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and cancers of the two major blood cell lineages, namely, myeloid (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid (B, T) cell lineages. Hematological malignancies originating from any of the following: acute myeloid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M3V ... Acute, chronic, lymphoid and / or myeloid leukemias, such as (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic lymphoma (e.g., Ki1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoblastic lymphoma lymphoma, lymphoblastic lymphoma (LBL), hematopoietic malignancies of the lymphoid system, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and Waldenstrom's macroglobulinemia lymphocytic myeloma, including lymphoplasmacytic lymphoma with IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and myeloma, including multiple myeloma; chronic lymphocytic leukemia (CLL); hairy cell lymphoma; hematopoietic tumors of myeloid lineage; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of central nervous system and nervous system including seminoma, malignant teratoma, astrocytoma, and schwannoma. tumors of mesenchymal origin, including tumors of the peripheral nerves and osteosarcoma, as well as other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and malignant teratomas; hematopoietic tumors of the lymphoid system, such as T-cell and B-cell tumors (including but not limited to T-cell prolymphocytic leukemia (T-PLL) including small cell and cerebrospinal cell types, large granular lymphocytic leukemia (LGL) preferably of the T-cell type, a / d T-NHL hepatosplenic lymphocytes, peripheral / mature T-cell lymphoma (pleomorphic and immunoblastic subtypes), angiocentric (nasal) T-cell lymphoma); cancer of the head and neck, renal cancer, rectal cancer, thyroid cancer, acute myeloid lymphoma, and any combination of these cancers. The methods described herein can also be used to treat metastatic cancer, refractory cancer (e.g., cancer that is refractory to previous immunotherapy, e.g., immunotherapy involving blockade of CTLA-4 antibodies or PD-1 antibodies), and recurrent cancer.

[0151] According to certain embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancer.

[0152] The multispecific antibodies described herein can be administered as monotherapy or as the sole immunostimulatory therapy, or can be combined with immunogenic preparations in cancer vaccine strategies, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. Many experimental strategies for tumor vaccination have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; and see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF, which has been shown to be a potent activator of antigen presentation for tumor vaccination.Dranoff et al. (1993) Proc. Natl. Acad. Sci. (USA) 90: 3539-43.

[0153] Other tumor vaccines can include proteins derived from viruses involved in human cancer, such as human papillomavirus (HPV), hepatitis virus (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in conjunction with CD40 activation is heat shock proteins (HSPs) isolated and purified from the tumor tissue itself. These heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are highly efficient in delivering them to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269:1585-1588, Tamura et al. (1997) Science 278:117-120).

[0154] Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have also been directly fused to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with CD40 agonism to activate (elicit) stronger antitumor responses. The multispecific antibodies described herein can also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). CD40 agonism can be effectively combined with chemotherapeutic regimens. In these instances, it may be possible to reduce the dose of the chemotherapeutic agent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). One example of such a combination is the combination of decarbazine with an anti-huCD40 antibody for the treatment of melanoma. Another example of such a combination is the combination of interleukin-2 (IL-2) with a multispecific antibody described herein for the treatment of melanoma. The scientific rationale behind the combination of CD40 agonists and chemotherapy is that cell death caused by the cytotoxic action of most chemotherapeutic compounds results in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with CD40 agonism through cell death are radiation, surgery, and hormone ablation. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with CD40 agonists. Inhibition of angiogenesis leads to tumor cell death, which can provide tumor antigens to the host's antigen presentation pathway.

[0155] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by the tumor. These include, among others, TGF-beta (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies against each of these proteins can be used in combination with anti-huCD40 antibodies to neutralize the effects of immunosuppressants and support the host's tumor immune response.

[0156] The multispecific antibodies described herein can effectively replace the helper activity of T cells. Ridge et al. (1998) Nature 393: 474-478. Activating antibodies against T cell costimulatory molecules, such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), CD137 / 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997)), and ICOS (Hutloff et al. (1999) Nature 397: 262-266), can also enhance the level of T cell activation. PD1 or PD-L1 inhibitors may also be used in combination with the multispecific antibodies described herein.

[0157] There are also several experimental therapeutic protocols involving the ex vivo activation and expansion of antigen-specific T cells and the transfer of these adoptive cells into a recipient, with the aim of stimulating T cells specific for tumor antigens (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. In the presence of the multispecific antibodies described herein, ex vivo activation can increase the frequency and activity of adoptively transferred T cells.

[0158] In another aspect, the invention described herein provides a method of treating an infectious disease in a subject comprising administering to the subject a multispecific antibody described herein, thereby treating the infectious disease in the subject.

[0159] Similar to the tumor application described above, antibody-mediated CD40 agonism can be used alone or in combination with vaccines as an adjuvant to enhance immune responses to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which no effective vaccines currently exist or for which conventional vaccines are insufficiently effective. Such pathogens include, but are not limited to, HIV, hepatitis (types A, B, and C), influenza, herpes, giardiasis, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa. CD40 agonism is particularly useful against established infections by pathogens such as HIV that present altered antigens over the course of the infection. These novel epitopes are recognized as foreign upon administration of anti-human CD40 antibodies and therefore elicit strong T cell responses.

[0160] Some examples of pathogenic viruses that cause infections treatable by the methods described herein include HIV, hepatitis (types A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0161] Some examples of pathogenic bacteria that cause infections treatable by the methods described herein include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, yersinia pestis, leptospira, and lyme disease bacteria.

[0162] Some examples of pathogenic fungi that cause infections that can be treated by the methods described herein include Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), Mucorales (e.g., Mucor, Absidia, Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0163] Some examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0164] In all of the above methods, CD40 agonism can be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy to enhance tumor antigen presentation. See, e.g., Holliger (1993) Proc. Natl. Acad. Sci. (USA) 90:6444-6448; Poljak (1994) Structure 2:1121-1123.

[0165] The multispecific antibodies described herein can be used to enhance antigen-specific immune responses by co-administering the multispecific antibodies described herein with an antigen of interest (e.g., a vaccine). Accordingly, the present application provides a method for enhancing an immune response to an antigen in a subject, comprising administering to the subject (i) the antigen and (ii) a multispecific antibody described herein, thereby enhancing the immune response to the antigen in the subject. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those described in the sections above, such as the tumor antigens (or tumor vaccines) described above, or antigens from viruses, bacteria, or other pathogens described above.

[0166] As mentioned above, the multispecific antibodies described herein can be co-administered with one or more therapeutic agents, e.g., cytotoxic agents, radiotoxic agents. The antibody can be linked to the agent (as an immunoconjugate) or administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be administered in combination with other known therapies, e.g., anti-cancer therapies (e.g., radiation). Such therapeutic agents include, among others, antineoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which themselves are only effective at levels that are toxic or subtoxic to patients. Cisplatin is administered intravenously at a dose of 100 mg / mL once every four weeks, and adriamycin is administered intravenously at a dose of 60 mg / mL to 75 mg / mL once every 21 days. The combination of the multispecific antibodies described herein with chemotherapeutic agents allows the two anticancer agents to act by different mechanisms to exert a cytotoxic effect on human tumor cells, thereby overcoming problems resulting from the development of drug resistance or changes in the antigenicity of tumor cells that render them resistant to antibodies.

[0167] The multispecific antibody (also referred to in the plural as "multispecific antibodies") may be provided to a subject as such or as a pharmaceutical composition in which it is mixed with suitable carriers or excipients.

[0168] As used herein, "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein, including physiologically suitable carriers and other chemical components, such as additives. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0169] As used herein, the term "active ingredient" refers to the multispecific antibody responsible for the biological effect.

[0170] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier," which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the compound being administered. These terms include adjuvants.

[0171] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0172] Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0173] Suitable routes of administration can include oral, rectal, transmucosal, especially nasal, intestinal, or parenteral delivery, such as intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal injections, direct intraventricular injections, intracardiac injections, e.g., into the right or left ventricular cavity, common coronary artery injections, intravenous injections, intraperitoneal injections, intranasal injections, or intraocular injections.

[0174] Traditional approaches for drug delivery to the central nervous system (CNS) include neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion), molecular manipulation of drugs in an attempt to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins containing transport peptides with affinity for endothelial cell surface molecules combined with drugs that cannot cross the BBB by themselves), pharmacological strategies designed to increase the lipid solubility of drugs (e.g., conjugation of water-soluble drugs to lipid or cholesterol carriers), and temporary disruption of BBB integrity by hyperosmotic disruption (obtained from injecting mannitol solutions into the carotid artery or from the use of biologically active agents such as angiotensin peptides). However, each of these strategies has limitations that make them suboptimal delivery methods, including inherent risks associated with invasive surgical procedures, size limitations imposed by limitations inherent in intrinsic transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the CNS, and the potential risk of brain injury that may compromise the BBB within brain regions.

[0175] Alternatively, the pharmaceutical composition can be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue region of a patient.

[0176] The pharmaceutical compositions of some embodiments of the present invention may be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, leaching, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0177] Thus, pharmaceutical compositions for use in some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including pharmaceutically acceptable additives and auxiliaries that facilitate processing of the active ingredient into a formulation. The appropriate formulation will vary depending on the route of administration selected.

[0178] For injection, the active ingredient of the pharmaceutical composition can be formulated in aqueous solution, preferably in physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0179] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by patients. Pharmaceutical preparations for oral use can be made using solid additives, and the resulting mixture can be optionally milled, and the granular mixture can be processed to obtain tablets or dragee cores after adding suitable additives as needed. Suitable additives are, in particular, fillers (e.g., sugars such as lactose, sucrose, mannitol, or sorbitol), cellulose preparations (e.g., corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), and / or physiologically acceptable polymers (e.g., polyvinylpyrrolidone (PVP)). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0180] The sugar-coated core is provided with a suitable coating.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and concentrated sugar solution, which may optionally contain suitable organic solvent or solvent mixture, can also be used.For identification or to show different dose combinations of active compound, dyes or pigments can be added to the tablet or sugar-coated coating.

[0181] Orally usable pharmaceutical compositions include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations for oral administration should be in a dosage appropriate for the selected route of administration.

[0182] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0183] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example, made of gelatin, for use in a dispenser can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0184] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers, optionally containing a preservative. The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0185] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredient in water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as appropriate oily or aqueous injection suspensions. Suitable lipophilic solvents or excipients include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredient, allowing for the preparation of highly concentrated solutions.

[0186] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile, pyrogen-free aqueous solution, before use.

[0187] Pharmaceutical compositions of some embodiments of the present invention may also be formulated as rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0188] Pharmaceutical compositions suitable for use in connection with some embodiments of the present invention include compositions containing the active ingredient(s) in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount refers to an amount of the active ingredient (e.g., a multispecific antibody) that is effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0189] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0190] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0191] Toxicity and therapeutic efficacy of the active ingredients described herein can be evaluated in vitro, in cell cultures, or in experimental animals by standard medical procedures. Data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a dosage range for use in humans. Dosages can vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition. (See, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics," Ch. 1, p. 1.)

[0192] Dosage and interval may be individually adjusted to achieve tissue concentrations of the active ingredient sufficient to induce or suppress the biological effect (minimum effective concentration, MEC). The MEC varies for each preparation but can be estimated from in vitro data. The dose required to achieve the MEC varies depending on individual characteristics and the route of administration. Detection assays can be used to measure plasma concentrations.

[0193] Depending on the targeted specificity, higher doses of multispecific antibodies can be used than those used with monospecific CD40 Abs (see Figures 7A-7D).

[0194] According to certain embodiments, the dosage of the multispecific antibody may be between 0.1 mg / kg and 100 mg / kg.

[0195] According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 80 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 60 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 50 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 40 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 30 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 0.1 mg / kg to 10 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 1 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 10 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 20 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 30 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 40 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 50 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 60 mg / kg to 100 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 70 mg / kg to 100 mg / kg.

[0196] According to certain embodiments, the dose of the multispecific antibody may be 1 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 1 mg / kg to 15 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 1 mg / kg to 10 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 1 mg / kg to 5 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 2 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 4 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 6 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 8 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 10 mg / kg to 20 mg / kg. According to certain embodiments, the dose of the multispecific antibody may be 12 mg / kg to 20 mg / kg. According to certain embodiments, the dosage of the multispecific antibody may be 15 mg / kg to 20 mg / kg. According to certain embodiments, the dosage of the multispecific antibody may be 18 mg / kg to 20 mg / kg. According to certain embodiments, the dosage of the multispecific antibody may be 1 mg / kg to 5 mg / kg. According to certain embodiments, the dosage of the multispecific antibody may be 2 mg / kg to 10 mg / kg. According to certain embodiments, the dosage of the multispecific antibody may be 5 mg / kg to 10 mg / kg.

[0197] According to certain embodiments, the multispecific dose is at least 5-fold, 10-fold, 15-fold, 20-fold, or more than that tolerated by the anti-CD40 monospecific antibody.

[0198] Depending on the severity and responsiveness of the condition to be treated, administration may be single or multiple, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved.

[0199] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.

[0200] The compositions of some embodiments of the present invention may, if desired, be provided in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also provide a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, reflecting the agency's approval of the composition or form for human or veterinary administration. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs or a product insert. Compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a designated condition, as further detailed above.

[0201] The multispecific antibodies of the present teachings are also contemplated for use in combination with drugs, for example, immune checkpoint modulators such as anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1, or anti-PDL1. According to certain embodiments, the immune checkpoint modulator is anti-PD1 or anti-PDL1.

[0202] It is expected that many related CD40 agonist antibodies will be developed between the time of this application and the expiration of the patent, and the scope of the term anti-CD40 antibody is intended to include all such new technology a priori.

[0203] As used herein, the term "about" refers to ±10%.

[0204] The words "comprises," "comprising," "includes," "including," "having," and conjugations thereof mean "including but not limited to."

[0205] The term "consisting of" means "including and limited to."

[0206] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0207] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, "a compound" or "at least one compound" includes multiple compounds and may include mixtures thereof.

[0208] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all of the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0209] Whenever a range of numerical values ​​is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "ranging between" a first indicated number and a second indicated number and "ranging from" a first indicated number to a second indicated number are used interchangeably herein and are intended to include the first indicated number and the second indicated number, and all fractional and integer numbers between the first indicated number and the second indicated number.

[0210] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine or those that can be readily developed by practitioners from known manners, means, techniques, and procedures.

[0211] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing, or reversing the progression of the condition, substantially ameliorating the clinical or cosmetic symptoms of the condition, or substantially preventing the appearance of clinical or cosmetic symptoms of the condition.

[0212] When referring to a particular sequence listing, it should be understood that such reference also encompasses sequences that substantially correspond to complementary sequences, including minor sequence variations. Sequence variations are the result of, for example, sequencing errors, cloning errors, or other changes that result in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 bases, or less than 1 in 100 bases, or less than 1 in 200 bases, or less than 1 in 500 bases, or less than 1 in 1000 bases, or less than 1 in 5,000 bases, or less than 1 in 10,000 bases.

[0213] It will be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, multiple features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or with respect to any other described embodiment of the invention, as appropriate. A particular feature described in the context of various embodiments should not be considered an essential feature of that embodiment, unless the embodiment is inoperable without that element.

[0214] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. [Example]

[0215] Reference is now made to the following examples, which together with the above descriptions, illustrate some embodiments of the present invention in a non-limiting fashion.

[0216] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, microscopic, and recombinant DNA techniques. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III, Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998), methodologies such as those described in U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057, “Cell Biology: A Laboratory Handbook”, Volumes I-III, Cellis, JE, ed. (1994), “Culture of Animal Cells - A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition, “Current Protocols in Immunology”, Volumes I-III, Coligan JE, ed.(1994), Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980); available immunoassays are fully described in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, and 3,879,262. The specifications, the specifications of US Pat. Nos. 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, "Oligonucleotide Synthesis” Gait, MJ, ed. (1984), “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985), “Transcription and Translation” Hames, BD, and Higgins SJ, Eds. (1984), “Animal Cell Culture” Freshney, RI, ed. (1986), “Immobilized Cells and Enzymes” IRL Press, (1986), “A Practical Guide to Molecular Cloning” Perbal, B., (1984), and “Methods in Enzymology” Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this specification. The procedures in these documents are believed to be well known in the art, but are provided for the convenience of the reader. All information contained in the above documents is incorporated herein by reference.

[0217] Example 1 Materials and Methods mouse Humanized mice containing human Fc receptors (FcγRa, hFcγRI, FcγRIIaR131, FcγRIIb, FcγRIIIaF158, and FcγRIIIb) and human CD40 were generated and evaluated as previously described

[10]

[15] . Mice aged 8–10 weeks were used in all experiments. All mice were maintained at the Weizmann Institute of Science Animal Facility Center.

[0218] Generation of anti-CD40 / DC monospecific or bispecific Ab Fc variants Anti-human CD40 antibody 2141 is clone 21.4.1 (ATCC accession number PTA-3605, native Fc) referenced in U.S. Patent No. 7,338,660. The heavy and light chain variable regions of 2141 were synthesized (Genewiz). The mouse IgG1 anti-human / mouse DEC-205 parent antibody, clone HD-109, and the Armenian hamster IgG anti-mouse CD11c parent antibody, clone N418, producing hybridomas were provided by Rockefeller University and ATCC, respectively. The heavy and light chain variable regions of HD-109 and N418 were sequenced from hybridoma RNA by amplification of cDNA ends ("anchored" PCR) using RLM-RACE (ThermoFisher) according to the manufacturer's instructions. PCR was performed using the following outer and inner oligonucleotides: for clone HD-109 heavy chain, outer mouse IgG1 heavy chain 5'-TCATTTACCAGGAGAGTGG (SEQ ID NO: 43) and inner mouse IgG1 heavy chain 5'-AGAGGCTCTTCTCAGTATGGTGGTTGTGC (SEQ ID NO: 44); for clone N418 heavy chain, outer hamster IgG heavy chain 5'-GCTCACGTCCACCACCACACATGT (SEQ ID NO: 45) and inner hamster IgG heavy chain 5'-GAAATAGCCCTTGACCAGGCAT CC (SEQ ID NO: 46); for clone HD-109 light chain, outer mouse IgG1 kappa light chain 5'-AACACTCATTCCTGTTGAAG (SEQ ID NO: 47) and inner mouse IgG1 kappa light chain 5'-GCTCTTGACAATGGGTGAAGTTGATGTC (SEQ ID NO: 48); for clone N418 light chain, outer hamster IgG light chain 5'-CTAACACTCATTCCTGTTCAGGGTCTTG (SEQ ID NO: 49) and inner hamster IgG light chain 5'-GCTGCTCAGGCTGTAGGTGCTGTC (SEQ ID NO: 50). The variable region sequences of the parent Abs were cloned from hybridomas and inserted into mammalian expression vectors with monohuman IgG1 or human kappa Fc backbones, or into bispecific vectors as previously described (Merchant et al., 1998; Ridgway et al., 1996; Schaefer et al., 2011).To ensure accurate heavy-light chain pairing, one of the parent mAbs was expressed in a CrossMab format (CH1-CL swapping), while the other mAb maintained its wild-type domain structure (Schaefer et al., 2011). For heavy chain heterodimerization, point mutations Y349C / T366S / L368A / Y407V were introduced into the CH3 domain of the first mAb (anti-DC marker ab), and S354C / T366W were introduced into the second mAb (anti-CD40) (Merchant et al., 1998; Ridgway et al., 1996). To generate the human IgG1 Fc domain variant (N297A, G237D / P238D / H268D / P271G / A330R (V11) N297 (which does not recruit receptors)), site-directed mutagenesis was performed using specific primers based on PCR-based site-directed mutagenesis (Agilent Technologies) according to the manufacturer's instructions. The mutated plasmid sequence was confirmed by direct sequencing (Life Science Core Facility, Weizmann Institute of Science). To produce antibodies, antibody heavy and light chain expression vectors were transiently transfected into Expi293 cells (ThermoFisher). Secreted antibodies in the supernatant were separated by protein G Sepharose 4 Fast Flow (GE). The purified antibody was dialyzed in PBS and sterile filtered (0.22 μm). Purity was assessed by SDS-PAGE and Coomassie staining and estimated to be >90%. Size exclusion chromatography (SEC) was performed on an Äkta Pure25 FPLC system using a Superose 6 Increase 10 / 300GL column (GE Healthcare).

[0219] CD40, DEC-205 and CD11c binding ELISA The binding specificity and affinity of monospecific and bispecific Abs were determined by ELISA using recombinant human CD40 (SINO BIOLOGICAL), human DEC-205 (Sino Biological), and mouse CD11c (R&D Systems). ELISA plates (Nunc) were coated overnight at 4°C with recombinant extracellular domains of human CD40 or human DEC-205 (1 μg / mL / well) or mouse CD11c (5 μg / mL / well). All subsequent steps were performed at room temperature. After washing, the plates were blocked with 1x PBS containing 2% bovine serum albumin for 1 hour, followed by incubation with serially diluted IgG (5-fold serial dilutions in 1x PBS containing 2% bovine serum albumin) for 1 hour. For dual-binding ELISA assays, the plates were incubated with biotinylated human CD40 (Acrobiosystems) for 1 hour. After washing, plates were incubated with horseradish peroxidase-conjugated anti-human IgG (Jackson ImmunoResearch) or horseradish peroxidase-conjugated streptavidin (Biolegend) for 1 hour. Detection was performed using a one-component substrate solution (TMB), and the reaction was stopped by adding 0.18 M sulfuric acid. Absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices), and background absorbance from negative control samples was subtracted.

[0220] Flow cytometry Single cell suspensions were prepared as described above. For surface staining, 0.2–1 × 10 cells were cultured in 100 μL of PBS. 6Cells were seeded at a cell concentration of 1000 ng / ml into a U-bottom 96-well plate (ThermoFisher). Cells were first stained with LIVE / DEAD™ Fixable blue dead cell stain (ThermoFisher), then washed twice with PBS and resuspended in 25 μL of FACS buffer supplemented with human or mouse TruStain Fc block (BioLegened) and incubated at room temperature for 15 minutes. Surface antigens were stained for 30 minutes on ice in FACS buffer. Cells were then washed twice with FACS buffer, resuspended in 150 μL of FACS buffer, and analyzed by flow cytometry. For intracellular IL-6 staining, an additional staining step was performed using the True-Nuclear™ transcription factor buffer Set Kit (BioLegened) and anti-IL-6 (MP5-20F3) (BioLegened) according to the manufacturer's instructions. All samples were analyzed on a CytoFLEX LX (Beckman Coulter). Unless otherwise specified, cell populations were distinguished by the following markers (BioLegend): DC: CD45+ (30F11), CD11c+ (N418), MHCII+ (M5 / 11415.2), F4 / 80- (BM8); macrophages: CD45+, CD11b+ (M1 / 70), MHCII+, F4 / 80+, Ly6C- (HK1.4), Ly6G- (1A8); monocytes: CD45+, CD11b+, Ly6C+, F4 / 80-, CD11c-; B cells: CD45+, CD19+ (1D3). cDC1: CD45+, MHCII+, CD11c+, XCR1+ (ZET), CD19-, CD64- (10.1), F4 / 80-, SIRPα- (P84). cDC2: CD45+, MHCII+, CD11c+, SIRPα+, CD19-, CD64-, F4 / 80-, XCR1-. Liver CDC1, CDC2, Kupffer cell, and non-Kupffer cell macrophages were gated using the following surface markers as previously described (Sierro et al., 2017): CD45, MHCII, CD11b, CD11c, CD64 (X54-5 / 7.1), F4 / 80, Ly6C, Tim4 (RMT4-54), and CX3CR1 (SA011F11).For CD40, CD86, CD80 and DEC-205 expression, the following clones were used: CD40 (3 / 23), CD86 (GL-1), CD80 (16-10A1) and DEC-205 (NLDC-145).

[0221] DC preferential binding assay Spleens were removed from humanized CD40 / FcγR mice, and single-cell suspensions were prepared as described above. Splenocytes were stained with CD40 mAb or CD40 / CD11c bsAb using the surface CD19 or CD11b markers. Cells were washed twice with FACS buffer and stained with PE-conjugated anti-human IgG (Jackson ImmunoResearch) in FACS buffer on ice before analysis by flow cytometry.

[0222] Human DC activation assay PBMCs were isolated from fresh whole blood of healthy donors by Ficoll separation (GE Healthcare). Human monocytes (CD14) were isolated using positive selection with CD14 microbeads according to the manufacturer's instructions (Miltenyi Biotec). + Monocytes were isolated at 4 × 10 per well of a 6-well plate in RPMI medium supplemented with 10% heat-inactivated FBS, 1% Pen-Strep, 100 ng / mL GM-CSF (Peprotech), and 100 ng / mL IL-4 (Peprotech). 6 The cells were cultured at 1 × 10 cells / well. The medium was replenished on days 2 and 5. Monocyte-derived immature DCs were harvested on day 7. For analysis of CD54 and CD86 upregulation, monocyte-derived immature DCs were plated at 1 × 10 cells / well in a U-bottom 96-well tissue culture plate (ThermoFisher). 5 Cells were seeded at 1000 cells / well. Antibodies shown in Figure 3A and Figure 4B were added to the wells and incubated overnight at 37°C. Cells were harvested and stained for the following markers: CD86 (BU63), CD54 (HA58). Samples were analyzed by flow cytometry.

[0223] OVA-specific T cell responses Mice (WT or BATF3 in Figure 8(A) and Figure 8(B)) - / - Mice were immunized with 50 mg / kg ovalbumin (Sigma) intraperitoneally in the presence or absence of 5 mg / kg rat anti-mouse CD40 mAb (FGK4.5) (BioXCell) or 0.1 mg / kg to 10 mg / kg anti-human CD40 monospecific or bispecific Abs. Seven days later, peripheral blood was collected and stained with APC-labeled anti-CD8α (53-6.7) (BioLegend) and PE-labeled OVA peptide SIINFEKL H-2b trimer (Tet-OVA, MBL International Corporation) and analyzed by flow cytometry. For specific T cell responses in the OVA-expressing B16 tumor model (B16-OVA), mice were injected with 2 × 10 5 B16-OVA cells were subcutaneously implanted. Tumors were established (total length and width of the tumor was approximately 50 mm). 3 When the CD40 expression level reached 100%, mice were treated with 5 mg / kg of rat anti-mouse CD40 mAb, and the treatment was repeated 3 days later. Peripheral blood was collected on day 7 and processed as described above.

[0224] Serum transaminases and H&E staining Mice (WT, BATF3 - / -Mice (human CD40 / FcγR, CD11c-DTR, or hCD40) were treated intraperitoneally with 5 mg / kg of rat anti-mouse CD40 mAb or 0.1 mg / kg–10 mg / kg of anti-human CD40 monospecific or bispecific Ab. After 24 hours, peripheral blood was collected into clotting factor serum tubes (Becton Dickinson). Blood was allowed to clot for 30 minutes at room temperature and then centrifuged at 3500 rpm for 10 minutes. Serum liver transaminase (ALT / AST) levels were measured by a commercial laboratory (American Medical Research Institute, Israel). Livers were removed from treated mice, placed in 4% paraformaldehyde (PFA) overnight, then paraffinized and stained with hematoxylin and eosin (H&E) at the Histology and Pathology Unit of the Weizmann Institute of Science. Slides were scanned using a Pannoramic scan II scanner (3DHISTECH), and images were acquired with CaseViewer software.

[0225] Serum and intracellular cytokine analysis hCD40 / FcγR mice were intraperitoneally injected with 0.1 mg / kg to 10 mg / kg of anti-human CD40 monospecific or bispecific Ab, and blood was collected 3 hours later to collect serum. IL-6 and TNF-α levels were quantified using an ELISA MAX™ Deluxe kit according to the manufacturer's instructions (BioLegend). For intracellular IL-6 detection, mice were administered 2.5 mg / kg or 0.5 mg / kg of anti-human CD40 mAb, and 2.5 hours later, blood was collected for intracellular IL-6 staining. Samples were evaluated by flow cytometry as described above.

[0226] Tumor Challenge and Treatment Tumor cell lines were maintained in a humidified incubator at 37°C and 5% CO2 and cultured in complete RPMI medium containing 25 mM HEPES, 1% L-glutamine, 10% FBS, 1% Pen-Strep, 1% non-essential amino acids, and 1% pyruvate. MC38 (2 × 10 6 ), B16-F10(4×10 5 ), B16-OVA (2 × 105 ), and MCA-205 (5 × 10 5 ) were implanted subcutaneously into the right flank of mice, and tumor volumes were measured blindly every 2–3 days with an electronic caliper. Volumes are reported using the formula (L22*L1) / 2, where L1 is the longest diameter and L2 is the shortest diameter. Seven to 10 days after tumor inoculation, tumors should reach a total length and width of approximately 50 mm. 3 When tumor size reached 100 μg, mice were randomized based on tumor size (day 0) and treated by intraperitoneal injection as described for each experiment. WT and BATF3- / - mice were treated with 100 μg of rat anti-mouse CD40 mAb or control PBS on days 0, 3, and 6. hCD40 / FcγR mice were treated with CD40 mAb or CD40 / CD11c bsAb at their respective MTDs (0.175 mg / kg and 2.5 mg / kg, respectively) on days 0, 2, 4, and 6, and / or with 10 mg / kg of PD-1 IgG1-N297A mAb (RMP-1-14 clone) or control PBS on days 0, 3, and 6. Mice were monitored for 8 to 20 days after the start of treatment or until tumor size restrictions imposed by the Weizmann Institute of Science IACUC forced the sacrifice of most of the untreated control group.

[0227] Platelet count Platelet analysis was performed on peripheral blood collected from mice in K2E EDTA tubes (Becton Dickinson). Samples were analyzed using a Sysmex XP-300™ automated hematology analyzer (Sysmex).

[0228] Cell depletion studies Twenty-four hours before CD40 treatment, mice were intravenously injected via the lateral tail vein with 10 μL of clodronate liposomes (or control PBS liposomes) (Liposoma BV) per gram of body weight for macrophage depletion, or 2 μg of anti-CD42b Ab (R300) (EMFRET Analytics) per gram of body weight for platelet depletion. +To deplete DCs, diphtheria toxin (DT) (Sigma) was injected intraperitoneally at a dose of 4 μg / kg 4 and 2 days before CD40 treatment. Depletion efficiency was assessed by either flow cytometry analysis or platelet counts.

[0229] Example 2 result Type 1 classical dendritic cells (cDC1) express tumor-specific CD8 + They are specialized to prime T cells, and their frequency and functional status in tumors are associated with increased survival rates in cancer patients and response to checkpoint blockade. Because the CD40 pathway plays an important role in T cell priming by cDC1s and cDC1s are suggested to be the primary target of CD40 mAbs, we investigated the effects of cDC1-deficient Batf3 - / - Mice were used to evaluate the role of cDC1s in various in vivo activities of CD40 agonist mAbs. Immunization of wild-type mice with ovalbumin (OVA) and CD40 mAb induced CD8+ specific for peptides derived from the OVA antigen. + This results in strong activation and systemic proliferation of T cells. - / - In mice, the proliferation of OVA-specific T cells was significantly impaired, suggesting a major role for cDC1s in the in vivo T cell priming activity of the CD40 agonist mAb adjuvant (Fig. 8(A)). To model responses to tumor antigens, mice were inoculated with OVA-expressing B16 melanoma cells and treated with CD40 mAb (Fig. 8(B)). When examined 7 days after the start of treatment, we found that there were no OVA-specific CD8 cells in the blood of wild-type mice. + Although T cells were observed, Batf3 - / - This was not observed in mice treated with CD40 mAb, suggesting that tumor-specific CD8 +These results suggest that cDC1s are required for T cell priming. TME DCs upregulate CD40 surface expression compared with DCs in peripheral tissues and compared with other TME cell types, such as macrophages and monocytes (Figure 8(I)), further implicating tumor cDC1s as a primary target for CD40 mAb treatment. To assess the impact of cDC1s on the overall antitumor efficacy of CD40 mAb treatment, we utilized two tumor models that respond well to anti-CD40 monotherapy. Mice bearing MC38 colon adenocarcinoma or MCA-205 fibrosarcoma were treated with CD40 mAb, and then their tumor growth was followed over time. In wild-type mice, treatment resulted in significant tumor volume reduction, whereas Batf3 mAb treatment resulted in significant tumor volume reduction. - / - No such reduction was achieved in mice (Figure 8(C)). Thus, cDC1s were identified as an essential cell population required for tumor-specific CD8+ T cell priming and mediating the overall antitumor effect of CD40-targeted immunotherapy.

[0230] We then evaluated the role of DCs in hepatotoxicity associated with CD40 mAb treatment. Liver damage after CD40 mAb injection was significantly reduced in wild-type, cDC1-deficient Batf3 - / - , and further CD11c before CD40 mAb injection. + In mice depleted of DCs (pan-DC-deficient CD11c-DTR mice), significant increases in blood levels of liver transaminases were detected (Figure 8(D)). Therefore, DCs are not the cell population that contributes to the hepatotoxicity associated with CD40 mAb therapy. Therefore, it is likely that other liver-specific cell populations expressing CD40 mediate the hepatotoxicity induced by CD40 mAb. The lack of CD11c expression in Kupffer cells and non-Kupffer liver macrophages (Figure 8(J)), the lack of depletion in CD11c-DTR mice, and the fact that Kupffer cells express Batf3 (Batf3) are not consistent with previous findings. - / -Since macrophages were not depleted in mice (Figure 8(L)), they were present in all these mouse models and may be responsible for the observed toxicity. Therefore, we decided to further explore the role of these macrophages in liver toxicity.

[0231] CD40 mediates toxicity associated with CD40 immunotherapy + To identify these cell populations, we utilized a mouse model in which CD40 and all Fcγ receptors (hCD40 / FcγR) were fully humanized. This line recapitulates the dose-limiting toxicity and additional biological activity of human CD40 mAbs reported in clinical settings. To assess γCD40-mediated hepatotoxicity, hCD40 / FcγR mice were injected with a fully humanized, Fc-engineered CD40 agonist (2141-V11), a second-generation Fc-optimized version of selicrelumab, in which the human IgG1 Fc is mutated to selectively enhance binding to the human inhibitory FcγR, FcγRIIB, thereby providing the cross-linking required for optimal CD40 agonism. This molecule is currently being evaluated in early-phase clinical trials (ClinicalTrials.gov identification numbers NCT04059588 and NCT04547777). As previously reported, a single injection of CD40 mAb resulted in a rapid increase in serum liver transaminase levels, indicating liver injury characterized by hepatocellular coagulative necrosis and sinusoidal thrombosis (Figure 8E). When clodronate liposomes were used to induce systemic depletion of macrophages and phagocytes, including hepatic macrophages and Kupffer cells but not DCs, prior to CD40 mAb injection (Figure 8M), liver toxicity was abolished (Figures 8E and 8N). We hypothesized that the central role of macrophages in mediating liver toxicity is due either to local cross-linking of CD40 mAb and subsequent FcγRIIB expression by Kupffer cells, leading to platelet activation in the hepatic sinusoids, and / or to direct activation of the CD40 pathway by liver macrophages.

[0232] To clarify these possibilities, we used anti-CD42b mAb to eliminate circulating platelets before CD40 mAb injection (Figure 8(O)). In the absence of platelets, a significant reduction in liver toxicity was observed (Figure 8(F)). Although AST and ALT levels were somewhat elevated in these settings, they were significantly lower than those observed with CD40 mAb injection without platelet clearance. Histopathological analysis of the livers of these mice revealed very rare and relatively minor parenchymal necrotic events in platelet-depleted mice. Such events correlated with significantly reduced AST and ALT levels and very few, small fibrin thrombus foci, likely due to residual platelets (approximately 2%) remaining in the circulation after anti-CD42b injection (Figure 8(O)). These foci were often found in vesicles near or surrounded by necrotic parenchyma (Figure 8(P)), suggesting a causal relationship between thrombus and necrosis. Thus, our data support a critical role for platelets in CD40 mAb-induced hepatotoxicity and correlate platelet frequency with the degree of liver injury. These results support a model in which Kupffer cells and hepatic macrophages, via expression of FcγRIIB, mediate platelet cross-linking of CD40 by anti-CD40 mAbs.

[0233] Next, we evaluated the role of macrophages in anti-CD40-mediated thrombocytopenia. A massive decrease in platelet count was observed 24 hours after CD40 mAb injection, and this phenotype was abolished when macrophages were depleted prior to CD40 mAb injection (Figure 8(G)). These results demonstrate that macrophages play a role in mediating anti-CD40-associated thrombocytopenia and suggest a causal relationship between hepatotoxicity and thrombocytopenia induced by both platelets and macrophages.

[0234] Finally, we sought to assess the identity of the cells contributing to CRS, a third type of CD40 mAb-induced toxicity observed in the clinic. A single injection of CD40 2141-V11 mAb resulted in a rapid increase in serum interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) (Figure 8(Q)), similar to what has been reported for cericlerumab and other CD40 mAbs. Intracellular IL-6 staining identified monocytes in the blood, lymph nodes (LNs), and spleen, along with a rare population of LN macrophages, as cells that rapidly upregulated IL-6 expression after CD40 mAb injection (Figures 8(H) and 8(R)), suggesting a major role for monocytes in mediating CD40 mAb-induced CRS. Collectively, these experiments identify the major cell populations contributing to the dose-limiting toxicity associated with CD40 agonists. The present data suggest a primary role for macrophages and platelets in mediating liver toxicity and thrombocytopenia, and for monocytes in mediating IL-6-associated CRS.

[0235] Bispecific antibodies (Abs) that selectively target CD40 on dendritic cells (DCs) were generated by combining the anti-CD40 human Ab 2141 with an Ab targeting a DC marker. To clone the desired Abs, the variable regions of the Abs were sequenced from RNA obtained from these hybridoma clones. Once the Ab variable domain sequences were identified (Figure 1A), they were cloned in frame with the desired IgG constant regions into vectors previously prepared for the heavy and light chains of monospecific and bispecific Abs, as described above. DNA constructs containing the translated sequences of anti-CD40 (2141), anti-CD11c (N418), and anti-DEC-205 (HD109) monospecific antibodies, and anti-CD40 / CD11c and anti-CD40 / DEC-205 bispecific antibodies were generated (Figure 2). The DNA vectors were combined and transfected into HEK293 cells to generate monospecific and bispecific Abs. These five Abs were produced and purified. The formation of the correct heterodimer combinations required to form the CD40 / CD11c and CD40 / DEC-205 bsAbs was verified by SDS-PAGE and analytical size-exclusion chromatograms (Figure 3). Simultaneous binding of these bsAbs to both CD40 and CD11c / DEC205 was verified, as well as preferential binding to DCs compared with other cell types negative for DEC-205 / CD11c (Figure 4). Next, we evaluated the agonistic activity of these bsAbs and activation of human DCs in vitro (Figure 5) and mouse T cells in vivo (Figure 7). We determined the requirement for FcγRIIB for bsAb activity by expressing several Fc scaffolds, including WT IgG1, IgG1-V11 (selectively enhanced binding to FcγRIIB), or IgG1-N297A (aglycosylated Fc that does not bind to FcγRs), and comparing the activity of each bsAb. In vitro and in vivo experiments demonstrated that these bsAbs required FcγRIIB engagement to effectively activate DCs and T cells (Figures 6A-6B). Specifically, wild-type IgG1 mediated mild DC activation, with significantly reduced potency compared to IgG1-V11 (Figure 6A).Similarly, in vivo T cell activation induced by CD40 / CD11c-V11 and CD40 / DEC-205-V11 in hCD40 / FcγR mice was significantly reduced or completely abolished by the Fc-silenced versions of these bsAbs, IgG1 or N297A, respectively (Figure 6B). Overall, these results suggest that the activity of CD40 / DC bsAbs is Fc-dependent and demonstrate that activity is increased by Fc-engineered bsAbs with enhanced FcγRIIB binding. Therefore, we selected hIgG1-V11 as the best IgG scaffold for CD40 / DC bsAbs, and further evaluated the in vivo properties of Fc-engineered CD40 / DC bsAbs using this Fc variant. Finally, these bsAbs have an improved toxicity profile compared to that of the monospecific 2141 CD40 Ab, demonstrating that they can be used at the high doses required for optimal antitumor activity. Various doses of bsAb were used in the efficacy experiments described above. Their safety profiles were assessed by administering bsAbs at doses that resulted in activity and evaluating for induction of hepatotoxicity. The therapeutic index of each bsAb was compared to that of the monomeric 2141-V11 parent Ab. We determined the maximum safe dose of the optimal CD40 / DC bsAb and compared the therapeutic efficacy of this safe dose with the defined MTD of the monospecific parent 2141-V11 (Figures 7A-7D). From this study, we can conclude that the DC-targeted bsAb format of 2141 can extend the therapeutic window, at least in terms of hepatotoxicity issues.

[0236] We generated variants of each bsAb based on three different Fc scaffolds that exhibit different binding properties to human FcγR: wild-type hIgG1, hIgG1-N297A (a deglycosylated Fc that does not bind to FcγR), and hIgG1-V11 (an Fc point mutation that enhances binding to the inhibitory hFcγRIIB) (Figures 9A to 9B).

[0237] To determine whether the therapeutic efficacy mediated by CD40 / CD11c bsAb could be improved compared to CD40 mAb when administered at nontoxic doses, we evaluated their MTDs based on their degree of hepatotoxicity. This evaluation identified 0.175 mg / kg and 2.5 mg / kg as the maximum doses for CD40 mAb and CD40 / CD11c bsAb, respectively, that were not accompanied by signs of liver necrosis or thrombosis (Figure 10A) and did not induce elevations in serum ALT / AST levels above normal homeostatic values ​​(Figure 7B). At the next higher dose tested, both antibodies induced significant toxicity, as evidenced by elevated ALT / AST levels, and liver histology revealed extensive hepatocellular necrosis and fibrin clots in treated mice. We evaluated serum IL-6 and TNF-α levels at these MTDs and found that the elevation of these CRS-associated cytokines after injection of the CD40 / CD11c bsAb was significantly lower than that of the CD40 mAb (Figure 10(B)). At the MTD, the CD40 / CD11c bsAb induced significantly enhanced T cell activation compared to the CD40 parental mAb (Figure 10(C)), supporting the increased therapeutic index for bsAbs of this design.

[0238] Next, we sought to determine whether this improvement in T cell priming would translate into increased therapeutic antitumor activity. To this end, tumor-bearing mice were treated with CD40 / CD11c bsAb and CD40 mAb at their respective MTDs (Figure 10(D)). In both the MC38 colon tumor model and the B16-F10 melanoma tumor model, CD40 / CD11c bsAb treatment resulted in significantly improved control of tumor growth compared with CD40 mAb treatment. At the end of the treatment course, no signs of liver toxicity were detected in treated mice, demonstrating the safety profile of these treatment regimens. These results suggest that CD40 / CD11c bsAb has improved antitumor efficacy over CD40 mAb when administered at the maximum safe dose.

[0239] CD40 agonist mAbs have been shown to synergize with PD-1 blockade in several preclinical models

[20]

[21] . This combination demonstrated clinical activity in patients with metastatic pancreatic ductal adenocarcinoma (PDAC) in early-phase clinical trials

[22] and is being evaluated for further clinical indications. Therefore, we assessed whether the CD40 / DC11c bsAb retains such synergistic activity. hFcγR / CD40 mice bearing B16 melanoma tumors were treated with either a PD-1 mAb, a CD40 / CD11c bsAb, or their combination (Figure 10(E)). The combination therapy induced increased antitumor activity compared with either monotherapy, supporting the potential of combining a CD40 / DC bsAb with anti-PD1 / L1 for enhanced therapeutic efficacy.

[0240] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0241] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, such headings shall not be construed as necessarily limiting. Furthermore, any priority document of this application is incorporated herein by reference in its entirety.

[0242] References (Other documents are cited throughout the specification.) [1] P. Sharmea, “The future of immue checkpoint therapy,” Science (80-. )., vol. 348, no. 6230, pp. 56-61, 2014. [2] M. K. Callahan, M. A. Postow, and J. D. Wolchok, “Targeting T Cell Co-receptors for Cancer Therapy,” Immunity, vol. 44, no. 5, pp. 1069-1078, 2016. [3] R. H. Vonderheide, “The Immune Revolution: A Case for Priming, Not Checkpoint,” Cancer Cell, vol. 33, no. 4, pp. 563-569, 2018. [4] A. B. Alexandroff et al., “Role for CD40-CD40 ligand interactions in the immune response to solid tumours,” Mol. Immunol., vol. 37, no. 9, pp. 515-526, 2000. [5] R. H. Vonderheide and M. J. Glennie, “Agonistic CD40 antibodies and cancer therapy,” Clin. Cancer Res., vol. 19, no. 5, pp. 1035-1043, 2013. [6] L. P. Richman and R. H. Vonderheide, “Role of Crosslinking for Agonistic CD40 Monoclonal Antibodies as Immune Therapy of Cancer,” Cancer Immunol. Res., vol. 2, no. 1, pp. 19-26, 2014. [7] A. L. White et al., “Interaction with Fc RIIB Is Critical for the Agonistic Activity of Anti-CD40 Monoclonal Antibody,” J. Immunol., vol. 187, no. 4, pp. 1754-1763, 2011. [8] R. H. Vonderheide et al., “Phase i study of the CD40 agonist antibody CP-870,893 combined with carboplatin and paclitaxel in patients with advanced solid tumors,” Oncoimmunology, vol. 2, no. 1, pp. 1-10, 2013. [9] R. H. Vonderheide et al., “Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody,” J. Clin. Oncol., vol. 25, no. 7, pp. 876-883, 2007.

[10] R. Dahan, B. C. Barnhart, F. Li, A. P. Yamniuk, A. J. Korman, and J. V. Ravetch, “Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement,” Cancer Cell, vol. 29, no. 6, pp. 820-831, 2016.

[11] D. A. Knorr, R. Dahan, and J. V Ravetch, “Toxicity of an Fc-engineered anti-CD40 antibody is abrogated by intratumoral injection and results in durable antitumor immunity [Immunology and Inflammation],” Proc. Natl. Acad. Sci. U. S. A., pp. 2-7, 2018.

[12] Y. Mazor et al., “Improving target cell specificity using a novel monovalent bispecific IgG design,” MAbs, vol. 7, no. 2, pp. 377-389, 2015.

[13] C. G. Park et al., “Generation of anti-human DEC205 / CD205 monoclonal antibodies that recognize epitopes conserved in different mammals,” J. Immunol. Methods, vol. 377, no. 1-2, pp. 15-22, 2012.

[14] C. Demangel, J. Zhou, A. B. H. Choo, G. Shoebridge, G. M. Halliday, and W. J. Britton, “Single chain antibody fragments for the selective targeting of antigens to dendritic cells,” Mol. Immunol., vol. 42, no. 8, pp. 979-985, 2005.

[15] I. Caminschi et al., “The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement,” Blood, vol. 112, no. 8, pp. 3264-3273, 2008.

[16] D. Sancho et al., “Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin,” J. Clin. Invest., vol. 118, no. 6, pp. 2098-2110, 2008.

[17] A. Bachem et al., “Expression of XCR1 characterizes the Batf3-dependent lineage of dendritic cells capable of antigen cross-presentation,” Front. Immunol., vol. 3, no. JUL, pp. 1-12, 2012.

[18] R. Dahan, B. C. Barnhart, F. Li, A. P. Yamniuk, A. J. Korman, and J. V. Ravetch, “Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcR Engagement,” Cancer Cell, vol. 29, no. 6, pp. 820-831, 2016.

[19] P. Smith, D. J. DiLillo, S. Bournazos, F. Li, and J. V. Ravetch, “Mouse model recapitulating human Fcγ receptor structural and functional diversity,” Proc. Natl. Acad. Sci. U. S. A., vol. 109, no. 16, pp. 6181-6186, 2012.

[20] H. S. Ma et al., “A CD40 Agonist and PD-1 Antagonist Antibody Reprogram the Microenvironment of Nonimmunogenic Tumors to Allow T-cell-Mediated Anticancer Activity,” Cancer Immunol. Res., 2019.

[21] S. F. Ngiow et al., “Agonistic CD40 mAb-driven IL12 reverses resistance to anti-PD1 in a T-cell-rich tumor,” Cancer Res., 2016.

[22] M. H. O. Hara et al., “CD40 agonistic monoclonal antibody APX005M (sotigalimab ) and chemotherapy, with or without nivolumab, for the treatment of metastatic pancreatic adenocarcinoma: an open-label, multicentre, phase 1b study,”vol. 22, no. January, 2021.

Sequence list free text

[0243] Sequence number 2: Human IgG1 V11 amino acid sequence SEQ ID NO: 3: Human IgG1 amino acid sequence with knob mutations (S354C / T366W) SEQ ID NO: 4: Human IgG1 amino acid sequence with hole mutations (Y349C / T366S / L368A / Y407V) SEQ ID NO: 5: 2141 (anti-CD40) heavy chain amino acid sequence SEQ ID NO: 6: 2141 (anti-CD40) light chain amino acid sequence SEQ ID NO: 7: n418 vh amino acid sequence SEQ ID NO: 8: n418 vl amino acid sequence SEQ ID NO: 9: hd109 vh amino acid sequence SEQ ID NO: 10: hd109 vl amino acid sequence SEQ ID NO: 11: cp870 vh amino acid sequence SEQ ID NO: 12: cp870 vl amino acid sequence SEQ ID NO: 13: 19g3 vh amino acid sequence SEQ ID NO: 14: 19g3 vl amino acid sequence SEQ ID NO: 15: 10b4 vh amino acid sequence SEQ ID NO: 16: 10b4 vh amino acid sequence SEQ ID NO: 17: mrx10 vh amino acid sequence SEQ ID NO: 18: mrx10 vh amino acid sequence SEQ ID NO: 19: Complementarity determining region (CDR) SEQ ID NO: 20: Complementarity determining region (CDR) SEQ ID NO: 21: Complementarity determining region (CDR) SEQ ID NO: 22: Complementarity determining region (CDR) SEQ ID NO: 23: Complementarity determining region (CDR) SEQ ID NO: 24: Complementarity determining region (CDR) SEQ ID NO: 25: Complementarity determining region (CDR) SEQ ID NO: 26: Complementarity determining region (CDR) SEQ ID NO: 27: Complementarity determining region (CDR) SEQ ID NO: 28: Complementarity determining region (CDR) SEQ ID NO: 29: Complementarity determining region (CDR) SEQ ID NO: 30: Complementarity determining region (CDR) SEQ ID NO: 31: Complementarity determining region (CDR) SEQ ID NO: 32: Complementarity determining region (CDR) SEQ ID NO: 33: Complementarity determining region (CDR) SEQ ID NO: 34: Complementarity determining region (CDR) SEQ ID NO: 35: Complementarity determining region (CDR) SEQ ID NO: 36: Complementarity determining region (CDR) SEQ ID NO: 37: n418 vh amino acid sequence SEQ ID NO: 38: n418 vl amino acid sequence SEQ ID NO: 39: hd109 vh amino acid sequence SEQ ID NO: 40: hd109 vl amino acid sequence SEQ ID NO: 43: single-stranded DNA oligonucleotide SEQ ID NO: 44: single-stranded DNA oligonucleotide SEQ ID NO: 45: single-stranded DNA oligonucleotide SEQ ID NO: 46: single-stranded DNA oligonucleotide SEQ ID NO: 47: single-stranded DNA oligonucleotide SEQ ID NO: 48: single-stranded DNA oligonucleotide SEQ ID NO: 49: single-stranded DNA oligonucleotide SEQ ID NO: 50: single-stranded DNA oligonucleotide SEQ ID NO: 51: Short synthetic peptide SEQ ID NO: 52: Complementarity determining region (CDR) SEQ ID NO: 53: Complementarity determining region (CDR) SEQ ID NO: 54: Complementarity determining region (CDR) SEQ ID NO: 55: Complementarity determining region (CDR) SEQ ID NO: 56: Complementarity determining region (CDR) SEQ ID NO: 57: Complementarity determining region (CDR) SEQ ID NO: 58: Complementarity determining region (CDR) SEQ ID NO: 59: Complementarity determining region (CDR) SEQ ID NO: 60: Complementarity determining region (CDR) SEQ ID NO: 61: Complementarity determining region (CDR) SEQ ID NO: 62: Complementarity determining region (CDR) SEQ ID NO: 63: Complementarity determining region (CDR)

Claims

1. 1. A multispecific antibody comprising: a first portion that binds to and activates CD40; a second portion that specifically binds to Clec9a or XCR1; and a third portion comprising an altered Fc region of the multispecific antibody to increase the specificity and affinity of binding to FcγRIIb.

2. The multispecific antibody of claim 1, which is a trifunctional antibody.

3. 2. The multispecific antibody of claim 1, wherein the modified Fc region comprises the amino acid sequence of SEQ ID NO:

2.

4. The multispecific antibody of any one of claims 1 to 3, comprising a knob-into-hole mutation.

5. 5. The multispecific antibody of claim 4, wherein the mutations are in the CH3 domain of a first antibody comprising Y349C / T366S / L368A / Y407V of the bispecific antibody and in the CH3 domain of a second antibody comprising S354C / T366W of the multispecific antibody.

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