CD40 binding protein

Abstract

Provided herein are cluster of differentiation 40 (CD40) monoclonal antibodies, and conditionally active variants thereof; and therapeutic and diagnostic methods using the anti-CD40 monoclonal antibodies.

Description

[Technical Field]

[0001] This disclosure relates to novel binding proteins that bind to CD40, and conditionally active variants thereof. Also provided are therapeutic and diagnostic methods using the binding proteins. Such CD40 binding proteins are useful, inter alia, in the field of cancer immunology. [Background technology]

[0002] CD40 is a cell surface member of the TNF (tumor necrosis factor) receptor superfamily. It is widely expressed at steady state on many immune cells, including B cells, monocytes, dendritic cells (DCs), and macrophages (most of which are antigen-presenting cells (APCs)). CD40 is also expressed to varying degrees on non-immune cells, such as endothelial cells, epithelial / glandular cells, hepatocytes, and fibroblasts. In the blood, 15–20% of circulating cells express CD40. Finally, tumor cells in multiple indications express CD40 at various antigen density levels. CD40 binds to its ligand, CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions.

[0003] A wide range of molecular and cellular processes, including the initiation and progression of cellular and humoral adaptive immunity, are regulated by CD40 binding. CD40 clustering on the cell membrane induces the recruitment of TNF receptor-associated factors (TRAFs), triggering several downstream signaling pathways: clustering induces macrophages to secrete proinflammatory mediators and enhance antigen-presenting capacity; induces dendritic cell maturation, regulating antigen-presenting function and immune activation; and supports antibody production, class switching, and affinity maturation in B cells. Immediately after activation, CD40 can license dendritic cells to promote antitumor T cell activation and reeducate macrophages to destroy tumor stroma. Thus, the CD40 / CD40L axis plays a central role in APC proliferation and antigen presentation.

[0004] Agonistic anti-CD40 antibodies have been shown to suppress tumor growth in several mouse models and clinical trials through the activation of several cell types, including B cells, macrophages, and DCs. However, there remains a need for agonistic anti-CD40 antibodies with improved agonistic properties.

[0005] In addition, the occurrence of treatment-related adverse events (TRAEs), including cytokine release syndrome (CRS) and hepatotoxicity, has been reported after agonist anti-CD40 antibody treatment in clinical trials. To address this issue, local intratumoral administration of agonist anti-CD40 antibodies has been explored as an alternative option. However, this solution may not be optimal because it limits the dose of agonist anti-CD40 antibody treatment. This solution also poses technical challenges, especially when tumor lesions are not sufficiently accessible, because the targeted tumor must be large enough to ensure needle injectability. Therefore, there is still a need for agonist anti-CD40 antibodies that can be administered systemically and have a better safety profile. Summary of the Invention [Means for solving the problem]

[0006] Provided herein are anti-CD40 binding proteins. In exemplary embodiments, the anti-CD40 binding proteins of the present disclosure preferentially target tumors.

[0007] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD40, preferably human CD40, comprising (i) three light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NO: 1, and (ii) three heavy chain CDR sequences set forth in SEQ ID NO: 7 or 8.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises the three heavy chain CDR sequences set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment comprises the three heavy chain CDR sequences set forth in SEQ ID NO: 8.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region having SEQ ID NO: 1, or a light chain variable region that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1; and (ii) a heavy chain variable region having SEQ ID NO: 7 or 8, or a heavy chain variable region that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 8.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises: (i) a light chain variable region having SEQ ID NO: 1; and (ii) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8.

[0011] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD40, preferably human CD40, comprising: (i) a light chain variable region comprising the three following CDR sequences: VL-CDR1: QGIYSW (SEQ ID NO: 9); VL-CDR2: TAS; and VL-CDR3: QQANIFPLT (SEQ ID NO: 10); and (ii) a heavy chain variable region comprising the three following CDR sequences: VH-CDR1: GYTFTGX1Y (SEQ ID NO: 11) (wherein X1 is selected from the group consisting of lysine (Lys, K) and arginine (Arg, R)); VH-CDR2L: INPDSGGT (SEQ ID NO: 12); and VH-CDR3: ARDQPLGYCTNGVCSYFDY (SEQ ID NO: 13).

[0012] In some embodiments, X1 is lysine (Lys, K). In some embodiments, X1 is arginine (Arg, R).

[0013] In some embodiments, the light chain variable region further comprises the four following framework region sequences: VL-FR1: DIQMTQSPSSVSASVGDRVTITCRAS (SEQ ID NO: 14); VL-FR2: LAWYQQKPGKAPNLLIY (SEQ ID NO: 15); VL-FR3: TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 16); and VL-FR4: FGGGTKVEIK (SEQ ID NO: 17).

[0014] In some embodiments, the heavy chain variable region further comprises the four following framework region sequences: VH-FR1: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 18); VH-FR2: MHWVRQAPGQGLEWMGW (SEQ ID NO: 19); VH-FR3: NYAQKFQGRVTMTRDTSIX2TAYMELNRLRSDDTAVYYC (SEQ ID NO: 20) (wherein X2 is selected from the group consisting of alanine (Ala, A) and proline (Pro, P); and VH-FR4: WGQGTLVTVSS (SEQ ID NO: 21).

[0015] In one embodiment, the antibody or antigen-binding fragment specifically binds to CD40; preferably human CD40.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is coupled to at least one masking moiety.

[0017] In another aspect, provided herein is a conditionally active antibody or antigen-binding fragment thereof capable of specifically binding to CD40, comprising: (i) the three light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NO: 1, and (ii) the three heavy chain CDR sequences set forth in SEQ ID NO: 7, 8, or 2; and further coupled to at least one masking moiety.

[0018] In some embodiments, at least one masking moiety reduces or inhibits binding of the antibody or antigen-binding fragment thereof to CD40.

[0019] In some embodiments, the conditionally active antibody or antigen-binding fragment comprises the three heavy chain CDR sequences set forth in SEQ ID NO:7.

[0020] In some embodiments, the conditionally active antibody or antigen-binding fragment comprises the three heavy chain CDR sequences set forth in SEQ ID NO:8.

[0021] In some embodiments, the conditionally active antibody or antigen-binding fragment comprises the three heavy chain CDR sequences set forth in SEQ ID NO:2.

[0022] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region having SEQ ID NO: 1, or a light chain variable region that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1; and (ii) a heavy chain variable region having SEQ ID NO: 7, 8, or 2, or a heavy chain variable region that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 7, 8, or 2.

[0023] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region having SEQ ID NO: 1; and (ii) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, and 2.

[0024] In some embodiments, at least one masking moiety is coupled to the N-terminus of the light chain variable region of the antibody or antigen-binding fragment thereof.

[0025] In some embodiments, at least one masking moiety comprises or consists of an amino acid sequence having SEQ ID NO: 22 or 23, or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO: 22 or 23.

[0026] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

[0027] In some embodiments, at least one cleavable linker is cleavable by at least one tumor-specific protease.

[0028] In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase 3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

[0029] In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0030] In some embodiments, at least one cleavable linker comprises or consists of an amino acid sequence having SEQ ID NO: 24 or 25, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 24 or 25.

[0031] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof is unmasked and capable of binding to CD40 upon cleavage of at least one cleavable linker.

[0032] In another aspect, provided herein is a conditionally active antibody or antigen-binding fragment thereof capable of specifically binding to CD40, wherein the antibody or antigen-binding fragment thereof is coupled to at least one masking moiety.

[0033] In some embodiments, at least one masking moiety reduces or inhibits binding of the antibody or antigen-binding fragment thereof to CD40.

[0034] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

[0035] In some embodiments, at least one cleavable linker is cleavable by at least one tumor-specific protease.

[0036] In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

[0037] In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0038] In some embodiments, at least one cleavable linker comprises or consists of an amino acid sequence having SEQ ID NO: 24 or 25, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 24 or 25.

[0039] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof is unmasked and capable of binding to CD40 upon cleavage of at least one cleavable linker.

[0040] In another aspect, provided herein is a conditionally active antibody or antigen-binding fragment thereof capable of specifically binding to CD40, wherein the antibody or antigen-binding fragment thereof is coupled to at least one masking moiety comprising or consisting of an amino acid sequence having SEQ ID NO: 22 or 23, or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO: 22 or 23.

[0041] In some embodiments, at least one masking moiety reduces or inhibits binding of the antibody or antigen-binding fragment thereof to CD40.

[0042] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

[0043] In some embodiments, at least one cleavable linker is cleavable by at least one tumor-specific protease.

[0044] In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase 3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

[0045] In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0046] In some embodiments, at least one cleavable linker comprises or consists of an amino acid sequence having SEQ ID NO: 24 or 25, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 24 or 25.

[0047] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof is unmasked and capable of binding to CD40 upon cleavage of at least one cleavable linker.

[0048] In some embodiments applicable to all of the above aspects, the CD40 is human CD40.

[0049] In some embodiments applicable to all of the above aspects, the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, has pure agonist activity. In some embodiments, pure agonist activity means that the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, can cluster CD40 on the surface of a cell and activate the CD40 signaling pathway in an FcγR-independent manner or independent of any other type of target-mediated cross-linking. In some embodiments, pure agonist activity means that the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, can activate the CD40 signaling pathway (i) under soluble conditions, and / or (ii) in the absence of cross-linking reagents, and / or (iii) in an FcγR-independent manner, and / or (iv) in the absence of target-mediated cross-linking of CD40.

[0050] In a further aspect, provided herein is a composition comprising an antibody or antigen-binding fragment thereof, or a conditionally active antibody or antigen-binding fragment thereof described herein. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or excipient.

[0051] In a further aspect, provided herein is a method of treating a subject in need thereof, the method comprising an effective amount of an antibody or antigen-binding fragment thereof, or a conditionally active antibody or antigen-binding fragment thereof described herein, or a composition described herein. In some embodiments, the subject has cancer.

[0052] In a further aspect, the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof described herein, or the composition described herein is used to treat cancer.

[0053] In a further aspect, the antibodies or antigen-binding fragments thereof, or conditionally active antibodies or antigen-binding fragments thereof described herein, or the compositions described herein can be used in the manufacture of a medicament for treating cancer.

[0054] In a further aspect, provided herein is an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof, or a conditionally active antibody or antigen-binding fragment thereof described herein; a vector comprising a polynucleotide described herein; or a host cell comprising a polynucleotide described herein.

[0055] In a further aspect, provided herein is a method of producing an antibody or antigen-binding fragment thereof, or a conditionally active antibody or antigen-binding fragment thereof described herein, the method comprising expressing in a cell a polynucleotide or vector described herein.

[0056] In a further aspect, provided herein is a method of producing an antibody or antigen-binding fragment thereof, or a conditionally active antibody or antigen-binding fragment thereof described herein, the method comprising culturing a host cell described herein in a culture medium; and recovering the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof produced by the host cell by culturing.

[0057] The summary of the disclosure set forth above is non-limiting, and other features and advantages of the disclosed antigen binding proteins and methods will be apparent from the following brief description of the drawings, detailed description of the disclosure, and claims. [Brief explanation of the drawings]

[0058] [Figure 1A-1B] Graphical representation of the affinity of the parent antibody [AbC1] and mutant antibody constructs [mAb1; mAb2; mAb3; mAb4] versus isotype control in B cell binding assays on human primary naive B cells (Figures 1A-1B) and cynomolgus monkey CD40-expressing HEK293 cells (Figure 1C). MFI: mean fluorescence intensity. [Figure 1C] Same as above. [Figure 2A] The graphs show the activity of the parent antibody [AbC1] and mutant antibody constructs [mAb1; mAb2; mAb3] versus isotype control in reporter assays in human NFkB-Luc2P / U-2 OS cells (Figure 2A) and rhesus CD40-expressing HEK293 cells (Figure 2B). Results are expressed as the ratio of luciferase activity of each sample to that of the negative serum (signal-to-noise [S / N] ratio). [Figure 2B] The graphs show the activity of the parent antibody [AbC1] and mutant antibody constructs [mAb1; mAb2; mAb3] versus isotype control in reporter assays in human NFkB-Luc2P / U-2 OS cells (Figure 2A) and rhesus CD40-expressing HEK293 cells (Figure 2B). Results are expressed as the ratio of luciferase activity of each sample to that of the negative serum (signal-to-noise [S / N] ratio). [Figure 3A] Expression of activation markers and costimulatory molecules CD86 (Figure 3A), CD69 (Figure 3B), and CD267 ("TACI"; Figure 3C) in B cell activation assays using various concentrations of positive control [hexameric CD40L], isotype control, parental antibody [AbC1], and mutant antibody constructs [mAb1; mAb2; mAb3; mAb4] is shown. MFI: mean fluorescence intensity. [Figure 3B-3C] Same as above. [Figure 4A-4B]Expression of monocyte-derived dendritic cell maturation markers CD80 (Figures 4A-4B), CD83 (Figures 4C-4D), and CD86 (Figures 4E-4F) in primary monocyte-derived dendritic cell activation assays using various concentrations of positive control [hexameric CD40L], isotype control, parental antibody [AbC1], and mutant antibody constructs (Figures 4A, 4C, and 4E: [mAb1; mAb2]; Figures 4B, 4D, and 4F: [mAb3; mAb4]). MFI: mean fluorescence intensity. [Figure 4C-4D] Same as above. [Figures 4E-4F] Same as above. [Figure 5] Shown are the results of binding assays to Raji cells using a mutant antibody construct [mAb1], four masked compounds [MC1-MC4], two protease-activating compounds [MMP9-activating; uPa-activating], and an isotype control [Isotype]. [Figure 6A] Figure 1 shows the results of binding assays against U-2 OS / NFκB cells using the parent antibody [AbC1], the mutant antibody construct [mAb1], four masked compounds [MC1–MC4], their protease-activated versions [MMP9-activated; uPa-activated], and an isotype control. [Figure 6B] Figure 1 shows the results of binding assays against U-2 OS / NFκB cells using the parent antibody [AbC1], the mutant antibody construct [mAb1], four masked compounds [MC1–MC4], their protease-activated versions [MMP9-activated; uPa-activated], and an isotype control. [Figure 7A] Figure 1 shows the results of a luciferase-based reporter assay in U-2 OS / NFκB cells using the parent antibody [AbC1], the mutant antibody construct [mAb1], four masked compounds [MC1-MC4], their protease-activated versions [MMP9-activating; uPa-activating], and an isotype control. RLU: relative luminescence units. S / N: signal-to-noise ratio. [Figure 7B]Figure 1 shows the results of a luciferase-based reporter assay in U-2 OS / NFκB cells using the parent antibody [AbC1], the mutant antibody construct [mAb1], four masked compounds [MC1-MC4], their protease-activated versions [MMP9-activating; uPa-activating], and an isotype control. RLU: relative luminescence units. S / N: signal-to-noise ratio. [Figure 8] Results of a primary human B cell activation assay using a mutant antibody construct (mAb1), four masked compounds (MC1-MC4), two protease-activating compounds (MMP9-activating; uPa-activating), and an isotype control (isotype) are shown. MFI: mean fluorescence intensity. [Figure 9A-9B] Figure 9 shows the results of a mouse B cell activation assay using the parent antibody [AbC1], a positive control [hexameric CD40L], a mutant antibody construct [mAb1], four masked compounds [MC1-MC4], their protease-activated versions [MMP9-activated], and an isotype control. Figure 9A shows CD83 expression from activated mouse B cells; Figure 9B shows CD86 expression from activated mouse B cells. MFI: Mean Fluorescence Intensity. [Figures 10A-10B] Figure 10 shows the activity of the parent antibody [AbC1], positive control [hexameric CD40L], mutant antibody construct [mAb1], four masked compounds [MC1-MC4], their protease-activated versions [MMP9-activated], and isotype controls in a monocyte-derived dendritic cell maturation assay. Figure 10A shows CD83 expression (mean fluorescence intensity, MFI) from mature monocyte-derived dendritic cells; Figure 10B shows interleukin-8 (IL-8) release from mature monocyte-derived dendritic cells; Figure 10C shows donor-dependent activity expressed as EC50 for CD86 expression; Figure 10D shows donor-dependent activity expressed as EC50 for CD83 expression; Figure 10E shows donor-dependent activity expressed as EC50 for CD80 expression; and Figure 10F shows donor-dependent activity expressed as EC50 for interleukin-8 (IL-8) release. [Figures 10C-10D] Same as above. [Figures 10E-10F] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0059] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0060] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] The term "about," when used in reference to a particular recited numerical value, means that the value may vary by no more than 5%, preferably no more than 2%, and more preferably no more than 1% from the recited value. For example, the expression "about 100," as used herein, includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0062] Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, exemplary methods and materials are now described. All publications mentioned herein are incorporated by reference as if set forth in their entirety.

[0063] The term "CD40" refers to a cell surface member of the TNFR superfamily, also known as "tumor necrosis factor receptor superfamily member 5" or "TNFRSF5." CD40 is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds to its ligand CD40L (also known as TNFSF5), which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. Upon activation, CD40 can license dendritic cells to promote anti-tumor T cell activation and reeducate macrophages to destroy tumor stroma. An exemplary amino acid sequence of CD40 is set forth in SEQ ID NO: 30, which corresponds to human CD40 (hCD40) having UniProt accession number P25942-1 (last updated May 1, 1992). [ka]

[0064] As used herein, the term "antibody" is intended to refer to an immunoglobulin (Ig) molecule (i.e., "intact antibody molecule") composed of four polypeptide chains (two heavy (H) chains and two light (L) chains) inter-connected by disulfide bonds, and multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region ("HCCR" or "C H "; Domain C H 1. C H 2, and C H Each light chain is composed of a light chain variable region ("LCVR" or "V"). L ") and a light chain constant region ("LCCR" or "C L "). V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. By default, CDR and FR sequences are given herein as defined by IMGT numbering, unless otherwise specified.

[0065] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies that can omit one or two CDRs for binding have been described in the scientific literature. Padlan et al. (FASEB J. 1995;9(1):133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al., J Mol Biol. 2002;320(2):415-428). Based on previous studies, CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically from regions of the Kabat CDRs outside the Chothia CDRs (e.g., V H (Residues H60 to H65 in CDR2 are often not required.) If omitted, a CDR or its residues are usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence, or a consensus of such sequences. Alternatively, the positions for substitution within the CDR and the amino acids to be substituted can be selected empirically. Experimental substitutions can be conservative or non-conservative.

[0066] The anti-CD40 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and / or light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within a domain are backmutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0067] The present disclosure also includes anti-CD40 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-CD40 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0068] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the disclosure may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences. However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

[0069] As used herein, the term "recombinant" refers to an antibody or antigen-binding fragment thereof of the present disclosure that is made, expressed, isolated, or otherwise obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to an antibody or antigen-binding fragment thereof that is expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or cell (e.g., a CHO cell) expression system, or that is isolated from a recombinant combinatorial human antibody library.

[0070] The term "multispecific antigen-binding molecule" as used herein refers to bispecific, trispecific, or multispecific antigen-binding molecules and antigen-binding fragments thereof. Multispecific antigen-binding molecules may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for epitopes of multiple target polypeptides. Multispecific antigen-binding molecules may be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides covalently or noncovalently linked to each other. The term "multispecific antigen-binding molecule" includes antibodies or antigen-binding fragments thereof of the present disclosure that may be linked to or coexpressed with another functional molecule, e.g., another peptide or protein. For example, antibodies or antigen-binding fragments thereof may be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent binding, or another method) to one or more other molecular entities, e.g., proteins or fragments thereof, to generate bispecific or multispecific antigen-binding molecules with second binding specificities. In accordance with the present disclosure, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies, or antigen-binding fragments thereof. In certain exemplary embodiments, an antibody or antigen-binding fragment thereof of the present disclosure is operably linked to another antibody or antigen-binding fragment thereof to generate a bispecific antibody having a second binding specificity.

[0071] The terms "specifically bind," "specifically binds to," and all conjugations thereof, mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as binding to at least about 1 x 10 -8 The equilibrium dissociation constant (K D ") can be characterized by D (A smaller σ indicates tighter binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, but are not limited to, equilibrium dialysis, surface plasmon resonance, and biolayer interferometry.

[0072] The term "high affinity" with respect to an antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that has a K D a binding affinity for an antigen, e.g., CD40, of at least 10 - 7M, at least 10 -8 M, at least 10 -9 M, at least 10 -10 M, or at least 10 -11 M refers to a mAb.

[0073] The term "off rate" or "K off " refers to a constant used to characterize how quickly an antibody or antigen-binding fragment thereof dissociates from its antigen, e.g., CD40. A "slow off-rate" refers to an antibody or antigen-binding fragment thereof that dissociates from an antigen, e.g., CD40, with a rate constant of 1 x 10, as determined by surface plasmon resonance, e.g., BIACORE™, or biolayer interferometry. -3 s -1 or less or 1×10 -4 s -1 This means that:

[0074] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0075] As used herein, the terms "antigen-binding portion" of an antibody and "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to CD40.

[0076] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure may be conjugated to a moiety such as a ligand or therapeutic moiety (an "immunoconjugate") (e.g., an antibiotic, a second anti-CD40 antibody, or an antibody against another antigen such as a tumor-specific antigen, an autoimmune tissue antigen, a virally infected cell antigen, an Fc receptor, a T cell receptor, or a T cell co-inhibitor, or an immunotoxin), or any other therapeutic moiety useful in treating a disease or condition, including cancer, an autoimmune disease, or a chronic viral infection.

[0077] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) with different antigen specificities (e.g., an isolated antibody that specifically binds to CD40, or an antigen-binding fragment thereof, is substantially free of Abs that specifically bind to antigens other than CD40).

[0078] As used herein, the terms "activating antibody," "enhancing antibody," and "agonist antibody" are intended to refer to an antibody or antigen-binding fragment thereof that, upon binding to CD40, increases or stimulates at least one biological activity of CD40. For example, an "activating antibody," "enhancing antibody," or "agonist antibody" can refer to an antibody or antigen-binding fragment thereof that mimics the action of CD40L, the natural ligand of CD40 (e.g., by triggering CD40 clustering), promoting the maturation of dendritic cells (DCs), improving their antigen-presenting capacity, and ultimately leading to the proliferation of tumor antigen-specific cytotoxic T cells.

[0079] The term "pure agonist," as used herein, is intended to refer to an activating antibody, or antigen-binding fragment thereof, as defined above, whose binding to an antigen increases or stimulates at least one biological activity of the antigen (e.g., by activating the CD40 signaling pathway) (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner (i.e., independent of Fcγ receptor binding), and / or (iv) in the absence of target-mediated cross-linking of the antigen.

[0080] As used herein, the term "Fc-mediated cross-linking" refers to cross-linking of a protein (e.g., an antibody) that comprises an Fc domain via binding of the Fc domain to an Fc-binding moiety, e.g., an anti-Fc antibody or an Fc receptor.

[0081] As used herein, the term "Fc receptor" refers to a surface receptor protein found on immune cells, including B lymphocytes, natural killer cells, macrophages, basophils, neutrophils, and mast cells, that has binding specificity for the Fc region of an antibody. The term "Fc receptor" includes, but is not limited to, Fcγ receptors [e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b)], Fcα receptors (e.g., FcαRI or CD89), and Fcε receptors [e.g., FcεRI and FcεRII (CD23)].

[0082] As used herein, the term "target-mediated crosslinking" refers to the crosslinking of an antibody or antigen-binding fragment thereof through the interaction of the antibody or antigen-binding fragment thereof with, for example, a tumor-associated antigen (TAA), an immune cell surface marker, a stromal antigen, or any other target expressed in cis or trans by tumor cells, immune cells, and / or normal cells.

[0083] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody, known as the "paratope." A single antigen can have multiple epitopes. Thus, different antibodies may bind to different regions of the same antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0084] The terms "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity over at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0085] The terms "substantial similarity" or "substantially similar," as used with polypeptides, mean that two peptide sequences share at least 90% sequence identity, or at least 95%, 96%, 97%, 98%, or 99% sequence identity when optimally aligned, such as by programs like GAP or BESTFIT using default gap weights. In exemplary embodiments, non-identical residue positions may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., with respect to charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (Methods Mol Biol. 1994;24:307-31) (incorporated herein by reference). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine. Representative conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al., Science. 1992 Jun 5;256(5062)1443-5 (incorporated herein by reference). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0086] Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; GCG version 6.1. FASTA programs (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson, Methods Mol Biol. 2000;132:185-219). Another exemplary algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from various organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al., J Mol Biol. 1990 Oct 5;215(3):403-10 and Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402 (each of which is incorporated herein by reference).

[0087] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be conditionally active or activatable. "Conditionally active" or "activatable," as used herein, means that an antibody or antigen-binding fragment thereof can bind to its antigen (i.e., is active) only under certain conditions. In some exemplary embodiments, a conditionally active antibody or antigen-binding fragment thereof includes a masking moiety.

[0088] As used herein, the terms "mask," "masking domain," or "masking moiety" refer to a moiety added to an antibody or antigen-binding fragment thereof to reduce the antibody's ability to bind to its antigen. The mask serves to prevent or reduce antigen binding by one or more CDR sequences of the antibody or antigen-binding fragment thereof. Masking moieties include, but are not limited to, self-hinge domains, coiled-coil domains, non-antibody protein fragments, antibody fragments, affinity peptides, cross-masking antibodies, and bivalent peptide-double-stranded DNA conjugates. For a review of suitable antibody masking moieties, see Lin et al. (J Biomed Sci. 2020 Jun 25;27(1):76).

[0089] In certain exemplary embodiments, the masking moiety is a polypeptide that can be removed from the antibody or antigen-binding fragment thereof by cleavage of a cleavable linker that links the masking moiety to the antibody or antigen-binding fragment thereof, thereby enabling the antibody or antigen-binding fragment thereof to bind to its target antigen. In particularly exemplary embodiments, the masking domain of the antibody or antigen-binding fragment thereof is cleaved at the tumor site, e.g., the tumor bed or lymph node. The cleavable linker can be a linker that is cleavable by a protease. In certain exemplary embodiments, the cleavable linker comprises at least one substrate for a tumor-specific protease.

[0090] The phrase "therapeutically effective amount" means an amount that is administered to produce the desired effect. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0091] As used herein, the term "subject" refers to an animal, e.g., a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as cancer, or a chronic viral infection. In some embodiments, the subject is a human subject in need of amelioration, prevention, and / or treatment of a disease or disorder, such as cancer, or a chronic viral infection.

[0092] As used herein, "anti-cancer drug" means any agent useful in treating cancer, including, but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radioactive agents.

[0093] As used herein, a "cytotoxin" or "cytotoxic agent," also referred to as a "chemotherapeutic agent," refers to any agent that is detrimental to cells. Examples include TAXOL® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0094] As used herein, the term "antiviral drug" refers to any drug or therapy used to treat, prevent, or ameliorate a viral infection in a host subject. The term "antiviral drug" includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids. In the context of this disclosure, viral infections include long-term or chronic infections caused by viruses, including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).

[0095] In some embodiments, the antibodies may be useful for stimulating or enhancing immune responses and / or treating subjects suffering from cancer or chronic viral infections. When administered to a subject in need of the antibodies, the antibodies may reduce chronic infection with viruses such as HIV, LCMV, or HBV in the subject. They may be used to inhibit the growth of tumor cells in a subject. They may be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating cancer or viral infections.

[0096] In a first aspect, there is provided an antibody or antigen-binding fragment thereof that specifically binds to CD40, preferably human CD40, wherein the antibody or antigen-binding fragment thereof: - the three light chain complementarity determining region (CDR) sequences set out in SEQ ID NO: 1, and - the three heavy chain CDR sequences set forth in SEQ ID NO: 7 or 8 Includes.

[0097] In some embodiments, the three heavy chain CDR sequences of the antibody or antigen-binding fragment are as set forth in SEQ ID NO: 7. In some embodiments, the three heavy chain CDR sequences of the antibody or antigen-binding fragment are as set forth in SEQ ID NO: 8.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDR sequences (as defined by the IMGT numbering): - light chain CDR1 (V) having the amino acid sequence QGIYSW L -CDR1) (SEQ ID NO: 9); - light chain CDR2 (V) having the amino acid sequence TAS L -CDR2); and - light chain CDR3 (V) having the amino acid sequence QQANIFPLT L -CDR3) (SEQ ID NO: 10); and - heavy chain CDR1 (V) having the amino acid sequence GYTFTGX1Y H -CDR1) (SEQ ID NO: 11) (wherein X1 is selected from the group consisting of lysine (Lys, K) and arginine (Arg, R); - heavy chain CDR2 (V) having the amino acid sequence INPDSGGT H -CDR2) (SEQ ID NO: 12); and - heavy chain CDR3 (V) having the amino acid sequence ARDQPLGYCTNGVCSYFDY H -CDR3) (SEQ ID NO: 13).

[0099] In some embodiments, V H - X1 in CDR1 is lysine (Lys, K). H - X1 in CDR1 is arginine (Arg, R).

[0100] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDR sequences (as defined by Kabat numbering): - light chain CDR1 (V) having the amino acid sequence RASQGIYSWLA L -CDR1) (SEQ ID NO: 31); - light chain CDR2 (V) having the amino acid sequence TASTLQS L -CDR2) (SEQ ID NO: 32); and - light chain CDR3 (V) having the amino acid sequence QQANIFPLT L -CDR3) (SEQ ID NO: 10); and - heavy chain CDR1 (V) having the amino acid sequence GX1YMH H -CDR1) (SEQ ID NO: 33) (wherein X1 is selected from the group consisting of lysine (Lys, K) and arginine (Arg, R); - Heavy chain CDR2 (V) having the amino acid sequence WINPDSGGTNYAQKFQG H -CDR2) (SEQ ID NO: 34); and - heavy chain CDR3 (V) having the amino acid sequence DQPLGYCTNGVCSYFDY H -CDR3) (SEQ ID NO: 35).

[0101] In some embodiments, V H - X1 in CDR1 is lysine (Lys, K). H - X1 in CDR1 is arginine (Arg, R).

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDR sequences (as defined by Chothia numbering): - light chain CDR1 (V) having the amino acid sequence RASQGIYSWLA L -CDR1) (SEQ ID NO: 31); - light chain CDR2 (V) having the amino acid sequence TASTLQS L -CDR2) (SEQ ID NO: 32); and - light chain CDR3 (V) having the amino acid sequence QQANIFPLT L -CDR3) (SEQ ID NO: 10); and - heavy chain CDR1 (V) having the amino acid sequence GYTFTGX1 H -CDR1) (SEQ ID NO: 36) (wherein X1 is selected from the group consisting of lysine (Lys, K) and arginine (Arg, R); - heavy chain CDR2 (V) having the amino acid sequence NPDSGG H -CDR2) (SEQ ID NO: 37); and - heavy chain CDR3 (V) having the amino acid sequence DQPLGYCTNGVCSYFDY H-CDR3) (SEQ ID NO: 35).

[0103] In some embodiments, V H - X1 in CDR1 is lysine (Lys, K). H - X1 in CDR1 is arginine (Arg, R).

[0104] In some embodiments, the antibody or antigen-binding fragment thereof: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1; and a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 5, 6, 7, or 8 Includes.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof: - a light chain variable region having SEQ ID NO: 1; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, or 8 Includes.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1; and a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 8 Includes.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof: - a light chain variable region having SEQ ID NO: 1; and a heavy chain variable region having SEQ ID NO: 7 or 8 Includes.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a third heavy chain framework region (V H -FR3) comprises a substitution at position S77 of SEQ ID NO: 7 or 8. In some embodiments, the substitution is selected from the group consisting of S77A and S77P.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1; and - a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 5 or 6, wherein position 77 of SEQ ID NO: 5 or 6 holds an alanine (Ala, A) or proline (Pro, P) residue. Includes.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof: - a light chain variable region having SEQ ID NO: 1; and a heavy chain variable region having SEQ ID NO: 5 or 6 Includes.

[0111] In some embodiments, the antibodies or antigen-binding fragments thereof described hereinabove bind to CD40, and in particular specifically bind to CD40. In some embodiments, the CD40 is human CD40, an exemplary amino acid sequence of which is SEQ ID NO:30.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof is coupled to at least one masking moiety.

[0113] In a second aspect, a conditionally active antibody or antigen-binding fragment thereof is provided.

[0114] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - the three light chain complementarity determining region (CDR) sequences set out in SEQ ID NO: 1, and - the three heavy chain CDR sequences set forth in SEQ ID NO: 7, 8, or 2 Includes; The conditionally active antibody or antigen-binding fragment thereof is coupled to at least one masking moiety.

[0115] In some embodiments, at least one masking moiety reduces or inhibits binding of an antibody or antigen-binding fragment thereof to its target antigen.

[0116] In some embodiments, the three heavy chain CDR sequences of the conditionally active antibody or antigen-binding fragment are as set forth in SEQ ID NO: 7. In some embodiments, the three heavy chain CDR sequences of the conditionally active antibody or antigen-binding fragment are as set forth in SEQ ID NO: 8. In some embodiments, the three heavy chain CDR sequences of the conditionally active antibody or antigen-binding fragment are as set forth in SEQ ID NO: 2.

[0117] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises the following CDR sequences (as defined by the IMGT numbering): - light chain CDR1 (V) having the amino acid sequence QGIYSW L -CDR1) (SEQ ID NO: 9); - light chain CDR2 (V) having the amino acid sequence TAS L -CDR2); and - light chain CDR3 (V) having the amino acid sequence QQANIFPLT L -CDR3) (SEQ ID NO: 10); and - heavy chain CDR1 (V) having the amino acid sequence GYTFTGX1Y H -CDR1) (SEQ ID NO: 11) (wherein X1 is selected from the group consisting of lysine (Lys, K), arginine (Arg, R), and tyrosine (Tyr, Y); - heavy chain CDR2 (V) having the amino acid sequence INPDSGGT H -CDR2) (SEQ ID NO: 12); and - heavy chain CDR3 (V) having the amino acid sequence ARDQPLGYCTNGVCSYFDY H -CDR3) (SEQ ID NO: 13).

[0118] In some embodiments, V H - X1 in CDR1 is lysine (Lys, K). H - X1 in CDR1 is arginine (Arg, R). H - X1 in CDR1 is tyrosine (Tyr, Y).

[0119] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises the following CDR sequences (as defined by Kabat numbering): - light chain CDR1 (V) having the amino acid sequence RASQGIYSWLA L -CDR1) (SEQ ID NO: 31); - light chain CDR2 (V) having the amino acid sequence TASTLQS L -CDR2) (SEQ ID NO: 32); and - light chain CDR3 (V) having the amino acid sequence QQANIFPLT L -CDR3) (SEQ ID NO: 10); and - heavy chain CDR1 (V) having the amino acid sequence GX1YMH H -CDR1) (SEQ ID NO: 33) (wherein X1 is selected from the group consisting of lysine (Lys, K), arginine (Arg, R), and tyrosine (Tyr, Y); - Heavy chain CDR2 (V) having the amino acid sequence WINPDSGGTNYAQKFQG H -CDR2) (SEQ ID NO: 34); and - heavy chain CDR3 (V) having the amino acid sequence DQPLGYCTNGVCSYFDY H -CDR3) (SEQ ID NO: 35).

[0120] In some embodiments, V H- X1 in CDR1 is lysine (Lys, K). H - X1 in CDR1 is arginine (Arg, R). H - X1 in CDR1 is tyrosine (Tyr, Y).

[0121] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises the following CDR sequences (as defined by Chothia numbering): - light chain CDR1 (V) having the amino acid sequence RASQGIYSWLA L -CDR1) (SEQ ID NO: 31); - light chain CDR2 (V) having the amino acid sequence TASTLQS L -CDR2) (SEQ ID NO: 32); and - light chain CDR3 (V) having the amino acid sequence QQANIFPLT L -CDR3) (SEQ ID NO: 10); and - heavy chain CDR1 (V) having the amino acid sequence GYTFTGX1 H -CDR1) (SEQ ID NO: 36) (wherein X1 is selected from the group consisting of lysine (Lys, K), arginine (Arg, R), and tyrosine (Tyr, Y); - heavy chain CDR2 (V) having the amino acid sequence NPDSGG H -CDR2) (SEQ ID NO: 37); and - heavy chain CDR3 (V) having the amino acid sequence DQPLGYCTNGVCSYFDY H -CDR3) (SEQ ID NO: 35).

[0122] In some embodiments, V H - X1 in CDR1 is lysine (Lys, K). H - X1 in CDR1 is arginine (Arg, R). H - X1 in CDR1 is tyrosine (Tyr, Y).

[0123] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1; and a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 5, 6, 7, 8, or 2; Includes.

[0124] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 1; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2 Includes.

[0125] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1; and a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 7, 8, or 2; Includes.

[0126] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 1; and a heavy chain variable region having SEQ ID NO: 7, 8, or 2 Includes.

[0127] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises a third heavy chain framework region (V H -FR3) comprises a substitution at position S77 of SEQ ID NO: 7, 8, or 2. In some embodiments, the substitution is selected from the group consisting of S77A and S77P.

[0128] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1; and - a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 5 or 6, wherein position 77 of SEQ ID NO: 5 or 6 holds an alanine (Ala, A) or proline (Pro, P) residue. Includes.

[0129] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 1; and a heavy chain variable region having SEQ ID NO: 5 or 6 Includes.

[0130] In some embodiments, at least one masking moiety is a peptide comprising about 10 to about 50 amino acid residues, about 10 to about 40 amino acid residues, about 10 to about 30 amino acid residues, or about 15 to about 25 amino acid residues.

[0131] In some embodiments, the at least one masking moiety, when coupled to the antibody or antigen-binding fragment thereof, reduces or inhibits binding of the antibody or antigen-binding fragment thereof to its target antigen. The at least one masking moiety may act by partially or completely masking the paratope of the antibody or antigen-binding fragment thereof, by creating steric hindrance between the antibody or antigen-binding fragment thereof and its target, etc.

[0132] In some embodiments, at least one masking moiety is coupled to the N-terminus of the light chain variable region of the conditionally active antibody or antigen-binding fragment thereof.

[0133] In some embodiments, at least one masking moiety comprises or consists of an amino acid sequence having SEQ ID NO: 22 or 23, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 22 or 23.

[0134] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

[0135] In some embodiments, at least one cleavable linker is cleavable by at least one tumor-specific protease, which may be selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

[0136] In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0137] In some embodiments, at least one cleavable linker comprises an amino acid sequence having SEQ ID NO: 38 and / or 39.

[0138] In some embodiments, at least one cleavable linker comprises or consists of an amino acid sequence having SEQ ID NO: 24 or 25; or an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 24 or 25 and retains the ability to be cleaved by at least one tumor-specific protease.

[0139] Further examples of linkers cleavable by at least one tumor-specific protease are known in the art, and one skilled in the art could readily select other suitable amino acid sequences cleavable by a tumor-specific protease.

[0140] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises: a light chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 26, 27, 28, or 29; and a heavy chain variable region that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NO: 5, 6, 7, 8, or 2; Includes.

[0141] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 26, 27, 28, or 29; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2 Includes.

[0142] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 26; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2 Includes.

[0143] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 27; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2 Includes.

[0144] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 28; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2 Includes.

[0145] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises: - a light chain variable region having SEQ ID NO: 29; and a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2 Includes.

[0146] In some embodiments, the conditionally active antibodies or antigen-binding fragments thereof described herein are capable of binding to CD40, preferably human CD40, upon cleavage of the at least one cleavable linker and release of the at least one masking moiety.

[0147] In a third aspect, a conditionally active antibody or antigen-binding fragment thereof capable of specifically binding to CD40 is provided, wherein the conditionally active antibody or antigen-binding fragment thereof is coupled to at least one masking moiety.

[0148] In some embodiments, the at least one masking moiety, when coupled to the antibody or antigen-binding fragment thereof, reduces or inhibits binding of the antibody or antigen-binding fragment thereof to CD40. The at least one masking moiety may act by partially or completely masking the paratope of the antibody or antigen-binding fragment thereof, by creating steric hindrance between the antibody or antigen-binding fragment thereof and its target, etc.

[0149] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

[0150] In some embodiments, at least one cleavable linker is cleavable by at least one tumor-specific protease.

[0151] The at least one tumor-specific protease may be selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

[0152] In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0153] In some embodiments, at least one cleavable linker comprises an amino acid sequence having SEQ ID NO: 38 and / or 39.

[0154] In some embodiments, at least one cleavable linker comprises or consists of an amino acid sequence having SEQ ID NO: 24 or 25, or an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 24 or 25 and retains the ability to be cleaved by at least one tumor-specific protease.

[0155] Further examples of linkers cleavable by at least one tumor-specific protease are known in the art, and one skilled in the art could readily select other suitable amino acid sequences cleavable by a tumor-specific protease.

[0156] In some embodiments, the conditionally active antibodies or antigen-binding fragments thereof described herein are capable of binding to CD40, preferably human CD40, upon cleavage of the at least one cleavable linker and release of the at least one masking moiety.

[0157] In a fourth aspect, there is provided a conditionally active antibody or antigen-binding fragment thereof capable of specifically binding to CD40, wherein the conditionally active antibody or antigen-binding fragment thereof is coupled to at least one masking moiety comprising or consisting of an amino acid sequence having SEQ ID NO: 22 or 23, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 22 or 23.

[0158] In some embodiments, the at least one masking moiety, when coupled to the antibody or antigen-binding fragment thereof, reduces or inhibits binding of the antibody or antigen-binding fragment thereof to CD40. The at least one masking moiety may act by partially or completely masking the paratope of the antibody or antigen-binding fragment thereof, by creating steric hindrance between the antibody or antigen-binding fragment thereof and its target, etc.

[0159] In some embodiments, the conditionally active antibody or antigen-binding fragment thereof further comprises at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

[0160] In some embodiments, at least one cleavable linker is cleavable by at least one tumor-specific protease.

[0161] The at least one tumor-specific protease may be selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

[0162] In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0163] In some embodiments, at least one cleavable linker comprises an amino acid sequence having SEQ ID NO: 38 and / or 39.

[0164] In some embodiments, at least one cleavable linker comprises or consists of an amino acid sequence having SEQ ID NO: 24 or 25, or an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 24 or 25 and retains the ability to be cleaved by at least one tumor-specific protease.

[0165] Further examples of linkers cleavable by at least one tumor-specific protease are known in the art, and one skilled in the art could readily select other suitable amino acid sequences cleavable by a tumor-specific protease.

[0166] In some embodiments, the conditionally active antibodies or antigen-binding fragments thereof described herein are capable of binding to CD40, preferably human CD40, upon cleavage of the at least one cleavable linker and release of the at least one masking moiety.

[0167] In some embodiments applicable to all of the above aspects, the antibodies or antigen-binding fragments thereof described herein and the conditionally active antibodies or antigen-binding fragments thereof described herein have pure agonist activity. In some embodiments, pure agonist activity means that the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, can cluster CD40 on the surface of a cell and activate the CD40 signaling pathway in an FcγR-independent manner or independent of any other type of target-mediated crosslinking. In some embodiments, pure agonist activity means that the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, can activate the CD40 signaling pathway (i) under soluble conditions and / or (ii) in the absence of crosslinking reagents and / or (iii) in an FcγR-independent manner and / or (iv) in the absence of target-mediated crosslinking of CD40.

[0168] Antigen-binding fragment of an antibody Unless otherwise specified, the term "antibody," as used herein, is understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules"), as well as antigen-binding fragments thereof. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing the CDRs, or isolated CDRs. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0169] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Also encompassed by the term "antigen-binding fragment" as used herein are domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains.

[0170] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V bound to the domain H For antigen-binding fragments containing domains, V H Domain and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of the monomer V. H or V L It may contain domains.

[0171] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L ;(viii)(V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H or V L Domain and ( 例えば The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently associated (by disulfide bonds).

[0172] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0173] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in connection with the present disclosure to generate human antibodies that specifically bind to CD40.

[0174] Antibodies to CD40 can be generated using immunogens comprising any one of the following: In certain embodiments, antibodies of the present disclosure are obtained from mice immunized with full-length, native CD40, or recombinant CD40 peptides. Alternatively, CD40 or a fragment thereof can be generated using standard biochemical techniques, modified, and used as an immunogen. In certain embodiments, the immunogen can be a peptide from the N-terminus or C-terminus of CD40.

[0175] In some embodiments, the immunogen can be a recombinant CD40 peptide expressed in Escherichia coli (E. coli) or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0176] In certain embodiments, antibodies that specifically bind to CD40 can be prepared using fragments of the above regions, or peptides extending beyond the designated regions by about 5 to about 20 amino acid residues from either the N-terminus or C-terminus, or both, of the regions described herein. In certain embodiments, any combination of the above regions or fragments thereof can be used to prepare CD40-specific antibodies.

[0177] Using VELOCIMMUNE® technology (see, e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high-affinity chimeric antibodies against CD40 having human variable regions and mouse constant regions can first be isolated. VELOCIMMUNE® technology involves the generation of transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0178] biological equivalent The anti-CD40 antibodies of the present disclosure include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to CD40. Such variant antibodies and antigen-binding fragments thereof contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit essentially equivalent biological activity to that of the described antibodies. Similarly, the antibodies encoded by DNA sequences of the present disclosure include sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0179] Two antigen-binding proteins, or antibodies, for example, are considered to be pharmaceutical equivalents or pharmaceutical substitutes and bioequivalent if their absorption rates and extents do not differ significantly when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies may be considered equivalents or pharmaceutical substitutes and still be considered bioequivalent if the extent of absorption, but not the rate of absorption, is equivalent because such differences in absorption rates are intentional and reflected in labeling, are not essential to achieving effective body drug concentrations, for example, in chronic use, and are not considered medically significant for the particular drug product being studied.

[0180] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in safety, purity, or potency.

[0181] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biologic product without a clinically meaningful change in immunogenicity or an expected increased risk of adverse effects, including diminished efficacy, compared to continued treatment without switching.

[0182] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mode of action for the state or condition of use (to the extent that such mechanism is known).

[0183] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with and are reasonably predictive of human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0184] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or erroneous intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antibodies can include antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0185] Anti-CD40 antibodies containing Fc variants According to certain embodiments of the present disclosure, there are provided anti-CD40 antibodies comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides H 2 or C HThe present invention also includes anti-CD40 antibodies containing mutations in three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 234 (e.g., A), 235 (e.g., A), 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]); or at positions 250 and / or 428; or at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 234A (e.g., L234A) and 235A (e.g., L235A) modifications, 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0186] For example, the disclosure includes anti-CD40 antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P 257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); 433K and 434F (e.g., H433K and N434F); and 234A and 235A (e.g., L234A and L235A). In one embodiment, the present disclosure includes an anti-CD40 antibody comprising an Fc domain that contains an S108P mutation in the IgG4 hinge region to promote dimer stabilization. All possible combinations of the above Fc domain mutations, as well as other mutations in antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.

[0187] The present disclosure also provides chimeric heavy chain constant (C H ) region, and a chimeric C H The C region is a region of interest for multiple immunoglobulin isotypes. H For example, the antibodies of the present disclosure may comprise segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H Part or all of the C2 domain is derived from a human IgG1, human IgG2, or human IgG4 molecule. H Chimeric C containing some or all of the three domains in combination H According to certain embodiments, the antibodies of the present disclosure may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering). According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. H Antibodies comprising the region may, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. See, e.g., U.S. Patent No. 9,359,437, the disclosure of which is incorporated herein by reference in its entirety.

[0188] Biological properties of antibodies Generally, the antibodies of the present disclosure function by binding to CD40. The present disclosure includes anti-CD40 antibodies and antigen-binding fragments thereof that bind to the CD40 molecule with high affinity. For example, the present disclosure includes antibodies with a K of less than about 50 nM as measured by surface plasmon resonance. D CD40 (e.g., at 25°C or 37°C) antibodies and antigen-binding fragments of antibodies. In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance. D It binds to CD40.

[0189] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind to CD40 with a dissociation half-life (t) of greater than about 1.1 minutes as measured by surface plasmon resonance at 25° C. or 37° C. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind to CD40 with a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes as measured by surface plasmon resonance at 25° C. or 37° C.

[0190] Species selectivity and species cross-reactivity According to certain embodiments of the present disclosure, an anti-CD40 antibody binds to human CD40 but does not bind to CD40 from other species. Additionally, in certain embodiments, an anti-CD40 antibody of the present disclosure binds to human CD40 and CD40 from one or more non-human species. For example, an anti-CD40 antibody of the present disclosure may bind to human CD40 and may or may not bind to CD40 from one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee, depending on the context. In certain embodiments, an anti-CD40 antibody of the present disclosure may bind to human and cynomolgus monkey CD40 with the same or different affinities, but does not bind to rat and mouse CD40.

[0191] Immunoconjugates The present disclosure encompasses anti-CD40 antibodies ("immunoconjugates") conjugated to a therapeutic moiety, such as a cytotoxin or chemotherapeutic agent, for treating cancer. As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide or protein, or therapeutic agent. The antibody can be linked to the cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide, or therapeutic agent at any position along with the molecule, so long as it is capable of binding to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a second, different antibody against CD40. In certain embodiments, the antibody can be conjugated to an agent specific for tumor cells or virally infected cells. The type of therapeutic moiety that can be conjugated to an anti-CD40 antibody will take into consideration the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see, for example, WO 05 / 103081.

[0192] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising the anti-CD40 antibodies of the present disclosure. Therapeutic compositions according to the present disclosure will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to improve transport, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., PDA J Pharm Sci Technol. 1998 Sep-Oct;52(5):238-311.

[0193] The antibody dosage may vary depending on the age and size of the recipient, the target disease, symptoms, and route of administration. When the antibody of the present disclosure is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is generally advantageous to administer the antibody of the present disclosure at a single dose of about 0.1 to about 60 mg / kg body weight, about 5 to about 60 mg / kg body weight, about 10 to about 50 mg / kg body weight, or about 20 to about 50 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the symptoms. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be about the same as or less than the initial dose, where the subsequent doses are separated by at least 1 to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0194] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, or receptor-mediated endocytosis (see, e.g., Wu et al., J. Biol. Chem. 1987 Apr 5;262(10):4429-32). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intratumoral, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer. Science. 1990 Sep 28;249(4976):1527-33).

[0195] The use of nanoparticles to deliver the antibodies of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo et al., 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155 / 2009 / 439389) (incorporated herein by reference). Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target tumor cells, autoimmune tissue cells, or virus-infected cells. Nanoparticles for drug delivery are also described, for example, in U.S. Pat. No. 8,257,740 or U.S. Pat. No. 8,246,995 (each of which is incorporated herein in its entirety).

[0196] In certain circumstances, the pharmaceutical composition may be delivered by a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed near the target of the composition, so that only a fraction of the systemic dose is required.

[0197] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intratumoral, intracranial, intraperitoneal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous injectable media include, for example, saline, isotonic solutions containing glucose and other adjuvants, and the like. These may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil, soybean oil, and the like, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable preparations prepared in this manner are optionally filled into appropriate ampoules.

[0198] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously, intravenously, or intratumorally using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily used to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0199] Numerous reusable pen and autoinjector delivery devices find use in subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, the AUTOPEN™ (Owen Mumford, Inc. Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™ pen, and the OPTIPEN™ pen. Examples of disposable pen delivery devices that have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.).

[0200] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a dosage form with a unit dose suitable for the dosage of the active ingredient. Examples of such dosage forms in a unit dose include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and particularly about 5 to about 100 mg of antibody in injection forms, and about 10 to about 250 mg in other dosage forms.

[0201] Therapeutic Uses of Antibodies The antibodies of the present disclosure are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by CD40 expression, signaling, or activity, or treatable by mimicking the action of a CD40 ligand (e.g., CD40L) or activating CD40 activity and / or signaling pathways. For example, the present disclosure provides methods of treating cancer (tumor growth inhibition) and / or chronic viral infections by administering an anti-CD40 antibody (or a pharmaceutical composition comprising an anti-CD40 antibody) to a patient in need of such treatment as described herein. The antibodies of the present disclosure are useful for treating, preventing, and / or ameliorating a disease, disorder, or condition, such as cancer or viral infection, and / or ameliorating at least one symptom associated with such disease, disorder, or condition. In connection with the methods of treatment described herein, the anti-CD40 antibody can be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents, examples of which are described elsewhere herein.

[0202] In some embodiments of the present disclosure, the antibodies described herein are useful for treating subjects suffering from primary or recurrent cancer, including, but not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney / renal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0203] The antibodies can be used to treat early or late signs of cancer. In one embodiment, the antibodies or fragments thereof of the present disclosure can be used to treat metastatic cancer. The antibodies are useful for reducing, inhibiting, or shrinking tumor growth in both solid tumors and hematological cancers. In certain embodiments, treatment with the antibodies or antigen-binding fragments thereof of the present disclosure results in greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% regression of tumor in a subject. In certain embodiments, the antibodies can be used to prevent tumor recurrence. In certain embodiments, the antibodies are useful for extending overall survival in subjects with cancer. In some embodiments, the antibodies are useful for reducing toxicity resulting from chemotherapy or radiation therapy while maintaining long-term survival in patients with cancer.

[0204] In certain embodiments, antibodies of the present disclosure are useful for treating subjects suffering from chronic viral infections. In some embodiments, antibodies of the present disclosure are useful for reducing viral titers and / or rescuing exhausted T cells in a host. In certain embodiments, antibodies of the present disclosure or fragments thereof can be used to treat chronic viral infections caused by lymphocytic choriomeningitis virus (LCMV). In some embodiments, antibodies of the present disclosure or antigen-binding fragments thereof can be administered in therapeutic doses to patients with infections caused by human immunodeficiency virus (HIV), human papillomavirus (HPV), or hepatitis B / C virus (HBV / HCV). In a related embodiment, antibodies of the present disclosure or antigen-binding fragments thereof can be used to treat infections caused by simian immunodeficiency virus (SIV) in simian subjects, such as cynomolgus monkeys.

[0205] In certain embodiments, the antibodies of the present disclosure may be administered in a therapeutically effective amount to a subject suffering from cancer or a viral infection.

[0206] One or more antibodies of the disclosure may be administered to alleviate, prevent, or reduce the severity of one or more signs or symptoms of a disease or disorder.

[0207] Also contemplated herein is the prophylactic use of one or more antibodies of the present disclosure in patients at risk of developing a disease or disorder, such as cancer and / or chronic viral infection.

[0208] In a further embodiment of the disclosure, the antibody is used in the preparation of a pharmaceutical composition for treating a patient suffering from cancer and / or a viral infection. In another embodiment of the disclosure, the antibody is used as an adjunct therapy with any other agent or therapy known to those skilled in the art to be useful in the treatment of cancer and / or a viral infection.

[0209] Combination Therapies and Formulations The combination therapy can include an anti-CD40 antibody of the present disclosure and any additional therapeutic agent that can be advantageously combined with an antibody of the present disclosure.

[0210] The antibodies of the present disclosure may be synergistically combined with one or more anti-cancer drugs or therapies used to treat cancers, including, for example, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal / kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and uterine cancer. Contemplated herein is the use of anti-CD40 antibodies of the present disclosure in combination with immunostimulatory and / or immunosupportive therapies to inhibit tumor growth and / or enhance survival of cancer patients. Immunostimulatory therapies include direct immunostimulatory therapies to enhance immune cell activity by either "releasing the brakes" on suppressed immune cells or "stepping on the gas" to activate the immune response. Examples include targeting other checkpoint receptors, adoptive cell therapy, vaccinations, and adjuvants. Immune-supportive modalities may increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that promote anti-tumor immune responses. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

[0211] In various embodiments, one or more antibodies of the disclosure are selected from the group consisting of: an antibody against CD40L; a second antibody against CD40; a LAG-3 inhibitor; a CTLA-4 inhibitor (e.g., ipilimumab); a TIM-3 inhibitor; a BTLA inhibitor; a TIGIT inhibitor; a CD47 inhibitor; an antagonist of a T-cell co-inhibitor or ligand (e.g., PD-1, PD-L1, PD-L2, CEACAM, VISTA, LAIR-1, 2B4, B7-H3, B7-H4, KIR, A2aR, , GAL9, or TGFR); T cell costimulatory agent agonists (e.g., antibodies or ligands against 4-1BB, CD28, ICOS, OX40, CD27, B7, CD226, CRTAM, GITR, HVEM, BAFFR, BAFF, Light); adenosine; indoleamine-2,3-dioxygenase (IDO) inhibitors; vascular endothelial growth factor (VEGF) antagonists (e.g., "VEGF-Trap," e.g., aflibercept or other VEGF-inhibitory fusion proteins (described in U.S. Pat. No. 7,087,411), or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib); Ang2 inhibitors (e.g., nesvacumab); transforming growth factor beta (TGFβ) inhibitors; epidermal growth factor receptor (EGFR) inhibitors; ) inhibitors (e.g., erlotinib, cetuximab); agonists of costimulatory receptors (e.g., agonists of glucocorticoid-inducible TNFR-related proteins); antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen [CEA], vimentin, tumor-M2-PK, prostate-specific antigen [PSA], mucin-1, MART-1, and CA19-9); vaccines (e.g., BCG (Bacillus Calmette-Guerin), cancer vaccines); adjuvants for enhancing antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor); bispecific antibodies (e.g., CD3×CD20 bispecific antibody, PSMA×CD3 bispecific antibody); cytotoxins;chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine); cyclophosphamide; radiation therapy; IL-6R inhibitors (e.g., sarilumab); IL-4R inhibitors (e.g., dupilumab); IL-10 inhibitors; cytokines, e.g., IL-2, IL-7, IL-12, IL-21, and IL-15; antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC) ADC); immunocytokines (e.g., anti-FAP x IL-2v [e.g., RO6874281], anti-tenascin-C x IL-2 [e.g., F16-IL2, also known as teleukin], anti-GD2 x IL-2 [e.g., hu14.18-IL2], anti-EDB x IL-2 [e.g., L19-IL2, also known as darleukin], anti-EDB x TNF [e.g., L19-TNF, also known as fibromun], anti-histone complex x IL-12 [e.g., NHS-IL12], anti-EDB x IL-12 [e.g., L19-IL12, also known as dodekin], anti-CSPG4 x IL-2, anti-EpCAM x IL-2, anti-CD20 x IL-2, anti-PD-1 x IL-2, and anti-TNFα x IL-2); anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs); nutritional supplements such as antioxidants; or any palliative care for treating cancer. In certain embodiments, the anti-CD40 antibodies of the present disclosure may be used in combination with cancer vaccines (including, but not limited to, dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc.) or adoptive cell therapy to enhance anti-tumor responses. Examples of cancer vaccines that can be used in combination with the anti-CD40 antibodies of the present disclosure include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancers (including glioblastoma multiforme), or (CEA; + ALVAC-CEA for cancer.

[0212] In certain embodiments, anti-CD40 antibodies of the present disclosure can be administered in combination with radiation therapy in a method of generating a long-term, durable anti-tumor response and / or enhancing survival of cancer patients. In some embodiments, anti-CD40 antibodies of the present disclosure can be administered before, simultaneously with, or after radiation therapy to a cancer patient. For example, radiation therapy can be administered in one or more doses to a tumor lesion, followed by administration of one or more doses of an anti-CD40 antibody of the present disclosure. In some embodiments, radiation therapy can be administered locally to a tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvinating radiation) and / or to kill tumor cells (ablative radiation), followed by systemic administration of an anti-CD40 antibody of the present disclosure. For example, intracranial radiation can be administered to a brain cancer (e.g., glioblastoma multiforme) patient in combination with systemic administration of an anti-CD40 antibody of the present disclosure. In certain embodiments, the anti-CD40 antibodies of the present disclosure may be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

[0213] In certain embodiments, the anti-CD40 antibodies of the present disclosure may be administered in combination with one or more antiviral drugs to treat chronic viral infections caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids. In some embodiments, the anti-CD40 antibodies of the present disclosure may be administered in combination with any antagonist of a LAG3 inhibitor, a CTLA-4 inhibitor, or another T-cell co-inhibitor to treat chronic viral infections.

[0214] The additional therapeutically active agent / component may be administered prior to, concurrently with, or following administration of an anti-CD40 antibody of the present disclosure. For purposes of this disclosure, such an administration regimen will be considered administration of the anti-CD40 antibody "in combination with" the second therapeutically active ingredient.

[0215] An additional therapeutically active ingredient can be administered to a subject prior to administration of an anti-CD40 antibody of the present disclosure. For example, a first ingredient can be considered to be administered "before" a second ingredient if it is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second ingredient. In other embodiments, an additional therapeutically active ingredient can be administered to a subject after administration of an anti-CD40 antibody of the present disclosure. For example, a first component can be considered to be administered "after" the second component if it is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration of the second component. In yet other embodiments, an additional therapeutically active component can be administered to a subject simultaneously with administration of an anti-CD40 antibody of the present disclosure. "Concurrent" administration, for purposes of the present disclosure, includes, for example, administration of an anti-CD40 antibody and an additional therapeutically active component to a subject in a single dosage form (e.g., co-formulated) or in separate dosage forms that are administered to the subject within about 30 minutes of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-CD40 antibody and the additional therapeutically active ingredient may be administered intravenously, intratumorally, subcutaneously, etc.); or alternatively, each dosage form may be administered via a different route (e.g., the anti-CD40 antibody may be administered intravenously and the additional therapeutically active ingredient may be administered subcutaneously). In any event, administration of the ingredients in a single dosage form, in separate dosage forms via the same route, or in separate dosage forms via different routes are all considered "co-administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-CD40 antibody "before," "concurrently with," or "after" (as these periods are defined herein above) administration of the additional therapeutically active ingredient is considered administration of the anti-CD40 antibody "in combination with" the additional therapeutically active ingredient.

[0216] The present disclosure includes pharmaceutical compositions in which the anti-CD40 antibodies of the present disclosure are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein using various dosage combinations.

[0217] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of an anti-CD40 antibody (or a pharmaceutical composition comprising a combination of an anti-CD40 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. A method according to this aspect of the present disclosure comprises sequentially administering multiple doses of an anti-CD40 antibody of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of an anti-CD40 antibody is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of an anti-CD40 antibody, followed by one or more secondary doses of the anti-CD40 antibody, and optionally, one or more tertiary doses of the anti-CD40 antibody. The anti-CD40 antibody may be administered at a dose of 0.1 mg / kg to 100 mg / kg.

[0218] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of the anti-CD40 antibodies of the present disclosure. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-CD40 antibody but generally may differ from one another with respect to administration frequency. However, in certain embodiments, the amount of anti-CD40 antibody contained in the initial, secondary, and / or tertiary doses differs from one another (e.g., adjusted up or down as needed) during the course of treatment. In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered as "loading doses" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a less frequent frequency.

[0219] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, or more) weeks after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means the dose of anti-CD40 antibody administered to a patient prior to the administration of the very next dose in a multiple dose series, with no intervening doses.

[0220] Methods according to this aspect of the disclosure can include administering any number of secondary and / or tertiary doses of anti-CD40 antibody to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0221] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments of the present disclosure, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. Additionally, the administration frequency may be adjusted by the physician during the course of treatment, depending on the needs of the individual patient, following clinical testing.

[0222] The present disclosure includes dosing regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once weekly, once every two weeks, once every three weeks, once monthly, once every two months, etc.), followed by two or more maintenance doses administered to the patient less frequently. For example, according to this aspect of the disclosure, if a loading dose is administered at a frequency of, e.g., once monthly (e.g., two, three, four, or more loading doses administered monthly), then maintenance doses can be administered to the patient once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, etc.

[0223] Diagnostic Uses of Antibodies Anti-CD40 antibodies of the present disclosure can be used to detect and / or measure CD40 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present disclosure in assays to detect diseases or disorders such as cancer, autoimmune disease, or chronic viral infection. An exemplary diagnostic assay for CD40 can include, for example, contacting a sample obtained from a patient with an anti-CD40 antibody of the present disclosure, where the anti-CD40 antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate CD40 from the patient sample. Alternatively, unlabeled anti-CD40 antibodies can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The CD40 may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Certain exemplary assays that can be used to detect or measure CD40 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0224] Samples that can be used in CD40 diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a patient that contains a detectable amount of CD40 protein or a fragment thereof under normal or pathological conditions. Generally, the level of CD40 in a particular sample obtained from a healthy patient (e.g., a patient not suffering from cancer, an autoimmune disease, or a chronic viral infection) will first be measured to establish a baseline or standard level of CD40. This baseline level of CD40 can then be compared to the level of CD40 measured in a sample obtained from an individual suspected of having a cancer-related condition, an autoimmune disease, or a chronic viral infection; or symptoms associated with such conditions.

[0225] Antibodies specific for CD40 may contain no additional label or moiety, or may contain an N- or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if present) may determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal from the surface.

[0226] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference for all purposes.

[0227] Furthermore, in accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. For example, Green & Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985); Oligonucleotide Synthesis (MJ Gait ed.1984); eds.(1985)];Transcription And Translation[BDHames&S.J.Higgins,eds.(1984)];Animal Cell Culture[RIFreshney,ed.(1986)];Immobilized Cells And Enzymes[IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning(1984);FMAusubel et al.(eds.),Current Protocols in Molecular Biology,John See Wiley & Sons, Inc. (1994). [Example]

[0228] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the compositions and methods featured in this disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. While attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0229] Example 1 Agonistic mechanism of anti-CD40 monoclonal antibodies The mechanism of action of a fully human anti-CD40 IgG2 monoclonal antibody (referred to herein as "AbC1") was investigated. AbC1 is a "pure agonist," i.e., its binding to CD40 activates the CD40 signaling pathway in an FcγR-independent manner (i.e., independent of Fcγ receptor binding) or any other type of target-mediated cross-linking. AbC1 contains a light chain variable region (LCVR) having SEQ ID NO:1 and a heavy chain variable region (HCVR) having SEQ ID NO:2. [ka]

[0230] To understand the mechanism of AbC1 agonism, we solved the crystal structure of CD40 complexed with AbC1 Fab. The crystal structure revealed a heterotetramer containing two copies of CD40 and two copies of AbC1. In addition to the recognized interaction between CD40 and AbC1 in cis, two additional interfaces were observed in the crystal structure: (1) a Fab-CD40 interaction in trans, and (2) a Fab-Fab interface. These additional interactions are typically considered artifacts due to crystal packing. However, given the agonistic properties of AbC1, we suspected that these additional interactions might actually exist and contribute to AbC1's agonistic mechanism of action.

[0231] To answer this question, chemical cross-linking experiments were performed to investigate whether this oligomerization state also exists in solution or is an artifact due to crystal packing. After disuccinimidyl suberate (DSS)-mediated cross-linking, CD40-Fab heterotetramers appeared in a concentration-dependent manner, indicating that the CD40-Fab heterotetramers observed in the crystal structure also exist in solution.

[0232] To test whether these additional interactions could contribute to the pure agonist activity of AbC1, several mutants were designed: - G4S loop insertion into CDR1 of Abc1 HCVR (which results in a collision with a second CD40 molecule in trans); - an engineered glycosylation site at position T28 of the HCVR having SEQ ID NO: 2 (which allows for a collision with a second CD40 molecule in trans upon glycosylation); -mutations at the Fab-CD40 interaction interface in trans (T28A / T30A / Y32F and Y27A / T28A / T30A / Y32F, with reference to the numbering in SEQ ID NO: 2); - Mutations at the Fab-Fab interaction interface (L102A / Y104A, with reference to the numbering in SEQ ID NO: 2).

[0233] These mutations, with the exception of two interface mutants (Y27A / T28A / T30A / Y32F and L102A / Y104A of SEQ ID NO: 2), were engineered to reside at the CD40-Fab trans interface or Fab-Fab interface so as not to affect the primary CD40 binding site. This was also confirmed by cell-based binding assays. When assayed, these mutants, with the exception of the Fab-Fab interface mutants, exhibited significantly lower agonistic activity compared to the parent AbC1. Primary CD40 binding was unaffected. This result suggests that secondary Fab-CD40 interactions in trans may be important for the agonistic activity of AbC1.

[0234] Example 2 Mutant anti-CD40 monoclonal antibodies Given that secondary CD40-Fab interface-mediated CD40 clustering appears to be important for AbC1 agonist activity, other mutations at this interface were designed and tested to see if they could result in more potent agonist-binding proteins.

[0235] Through structural analysis, single and double point mutations were designed focusing on residues Y32 and S77 of the HCVR of AbC1, having SEQ ID NO: 2. All mutants contained the same LCVR, having SEQ ID NO: 1; and the same constant regions as AbC1, having SEQ ID NO: 3 for the light chain constant region (LCCR) and SEQ ID NO: 4 for the heavy chain constant region (HCCR). [ka]

[0236] The following single and double mutants were generated and tested: -Y32K / S77A (hereinafter referred to as "mAb1"); -Y32K / S77P (hereinafter referred to as "mAb2"); -Y32K (hereinafter referred to as "mAb3"); and -Y32R (hereinafter referred to as "mAb4").

[0237] Example 3 In vitro assays Materials and Methods Surface plasmon resonance (SPR) binding assay The binding affinity of mutant anti-CD40 mAbs versus parental antibodies to recombinant human CD40 was evaluated using a Biacore 8K+ instrument. Protein A / G was immobilized on the surface of a CM5 series S chip using amine chemistry. Test compounds were then captured on the surface at between 120 and 170 RU. Recombinant hCD40 was diluted 2-fold from 300 nM to 2.34 nM in HBS-EP+ buffer. The association constant (K a ) and dissociation constant (K d) was measured for 300 and 450 seconds, respectively. The chip surface was regenerated using glycine (10 mM) pH 1.5. Binding affinity (K D ) was determined using a 1:1 binding model with a global fit of all concentrations.

[0238] Binding assay About primary naive human B cells Objective: To evaluate the affinity of mutant anti-CD40 mAbs versus parental antibody AbC1 and isotype control for receptors expressed by B cells. Compound binding EC 50 value and E max Calculate the value.

[0239] Naive B cells were isolated from PBMCs of healthy donors using negative selection with microbeads (Miltenyi, Cat. 130-091-150) and cultured in RPMI 1640 medium (ThermoFischer, Cat. 31870-025) supplemented with 10% fetal bovine serum (Biowest, Cat. S181H-100), 1% penicillin / streptomycin (ThermoFischer, Cat. 15140122), 1% sodium pyruvate (ThermoFischer, Cat. 11360), and 1% Glutamax (ThermoFischer, Cat. 35050) using industry-standard aseptic techniques.

[0240] Naive B cells were cultured at 50 x 10 per well in a 96-well plate. 3 Cells were plated at a density of 1000x and incubated with CD40 agonist, hexameric ligand, and anti-CD40 antibody for 1 hour at 4° C. Cells were then harvested and incubated with AF 488-conjugated secondary antibody before flow cytometry analysis using a Fortessa X20.

[0241] Data from the flow cytometer was analyzed using FlowJo (V10.8.1) before generating binding curves, E max , and E.C. 50Values ​​were plotted using GraphPad Prism (V9.1.2).

[0242] cynoCD40-expressing HEK293 cells Objective: To evaluate the affinity of mutant anti-CD40 mAbs versus the parental antibody AbC1 and isotype control for the cynomolgus monkey CD40 receptor and determine the binding EC 50 and E max Calculate the value.

[0243] Binding assays were performed using in-house transfected HEK293 cells expressing cyno CD40. Cyno CD40-expressing HEK293 cells were cultured in DMEM (ThermoFischer, Cat. 31966) supplemented with 10% fetal bovine serum (Biowest, Cat. S181H-100) and 1 mg / mL Geneticin (ThermoFischer, Cat. 10131035) using industry-standard aseptic techniques.

[0244] Cyno CD40-expressing HEK293 cells were cultured at 50 × 10 per well in a 96-well plate. 3 Cells were plated at a density of 100x and incubated with serially diluted concentrations of CD40 agonist, hexameric ligand, and anti-CD40 antibody for 1 hour at 4° C. Cells were then harvested and incubated with AF 488-conjugated secondary antibody before flow cytometry analysis using a Fortessa X20.

[0245] Data from the flow cytometer was analyzed using FlowJo (V10.8.1) before generating binding curves, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0246] Reporter assay Human CD40 reporter cells Objective: Evaluate the activity of mutant anti-CD40 mAbs versus the parental antibody AbC1 and isotype control to determine the EC 50and E max Calculate.

[0247] NFkB-Luc2P / U-2 OS cells (Promega, Cat. J2132) were grown in McCoy's 5A (ThermoFischer, Cat. 26600) supplemented with 10% fetal bovine serum (Biowest, Cat. S181H-100), 1% penicillin / streptomycin (ThermoFischer, Cat. 15140122), and 1 / 250 hygromycin (ThermoFischer, Cat. 10687010) using industry-standard aseptic techniques.

[0248] NFkB-Luc2P / U-2 OS cells were cultured at 25 × 10 cells per well in a 96-well white flat-bottom culture plate. 3 Cells were plated at a density of 1:1 and grown overnight at 37°C in RPMI 1640 medium (ThermoFischer, Cat. 31870-025) supplemented with 1% fetal bovine serum (Biowest, Cat. S181H-100). The next day, the medium was removed and serially diluted concentrations of CD40 agonist or isotype were added to the cell cultures. After 4 hours of incubation at 37°C, Bio-Glo Reagent (Promega, Cat. G7941) was added to each well containing cells. Luminescence was then measured using a GloMax® Discover plate reader.

[0249] Bonding curve, E max , and EC50 values ​​were plotted using GraphPad Prism (V9.1.2).

[0250] Rhesus CD40 reporter cells Objectives: To evaluate the activity of mutant anti-CD40 mAbs versus the parental antibody AbC1 and isotype control in rhesus CD40 reporter cells.

[0251] Reporter assays were performed using in-house transfected HEK293 cells expressing rhesus CD40. HEK293H rhesus CD40 reporter cells were cultured in DMEM (ThermoFischer, Cat. 31966) supplemented with 10% fetal bovine serum (Biowest, Cat. S181H-100), 200 μg / mL hygromycin B (ThermoFischer, Cat. 10687010), and 100 μg / mL zeocin (ThermoFischer, Cat. R25001) using industry-standard aseptic techniques.

[0252] HEK293H rhesus CD40 reporter cells were plated at 25 x 10 cells per well in a 96-well white flat-bottom culture plate. 3 Cells were plated at a density of 1:1 and grown overnight at 37°C in DMEM (ThermoFischer, Cat. 31966) supplemented with 10% fetal bovine serum (Biowest, Cat. S181H-100). Serially diluted concentrations of CD40 agonists or isotypes were added to the cell cultures. After overnight incubation at 37°C, Bio-Glo reagent (Promega, Cat. G7941) was added to each well containing cells. Luminescence was then measured using a GloMax® Discover plate reader.

[0253] Bonding curve, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0254] Human B Cell Activation Assay (BCA) Objectives: To evaluate the activity of mutant anti-CD40 mAbs versus the parental antibody AbC1 and isotype control on human B cells. To monitor B cell activation markers and costimulatory molecules.

[0255] CD20 +B cells were isolated from PBMCs of healthy donors using CD20 microbeads (Miltenyi, Cat. 130-091-104) and cultured in RPMI 1640 medium (ThermoFischer, Cat. 31870-025) supplemented with 10% fetal bovine serum (Biowest, Cat. S181H-100), 1% penicillin / streptomycin (ThermoFischer, Cat. 15140122), 1% sodium pyruvate (ThermoFischer, Cat. 11360), and 1% Glutamax (ThermoFischer, Cat. 35050) using industry-standard aseptic techniques.

[0256] CD20 + B cells were cultured in a 96-well plate at 150 × 10 cells per well. 3 Cells were plated at a density of 100x and incubated with CD40 agonist, hexameric ligand, and anti-CD40 antibody under humidified conditions for 68 hours at 37°C. Cells were then harvested for flow cytometric analysis of B cell activation markers and costimulatory molecules, such as CD69, CD86, and CD267 (TACI; transmembrane activator, calcium modulator, and CAML interactor), using a Fortessa X20.

[0257] Flow cytometry results were analyzed using FlowJo (V10.8.1) and then plotted using GraphPad Prism (V9.1.2).

[0258] Monocyte-derived dendritic cell (MoDC) maturation assay Objectives: To evaluate the activity of mutant anti-CD40 mAbs versus parental antibody AbC1 and isotype control on MoDC activation and maturation markers.

[0259] CD14+ monocytes were isolated from PBMCs of healthy donors using CD14 microbeads (Miltenyi, Cat. 130-050-201) and cultured using industry-standard aseptic techniques in 10% fetal bovine serum (Biowest, Cat. S181H-100), 1% penicillin / streptomycin (ThermoFischer, Cat. 15140122), and 1% Glutamax (ThermoFischer, Cat. 15140122). The cells were cultured in RPMI 1640 medium (ThermoFischer, Cat. 31870-025) supplemented with GM-CSF (50 ng / mL, Miltenyi, Cat. 130-093-867) and IL-4 (10 ng / mL, Miltenyi, Cat. 130-093-922) for 5 days at 37°C under humidified conditions. + The cells were allowed to differentiate into immature MoDCs.

[0260] Immature moDCs were plated at 200 × 10 per well in a 96-well plate. 3 Cells were plated at a density of 100x and incubated with CD40 agonist, hexameric ligand, and anti-CD40 antibody under humidified conditions for 24 hours at 37°C. Cells were then harvested for flow cytometric analysis of MoDC maturation markers, such as CD80, CD83, and CD86, using a Fortessa X20.

[0261] Flow cytometry results were analyzed using FlowJo (V10.8.1) and then plotted using GraphPad Prism (V9.1.2).

[0262] result Surface plasmon resonance (SPR) binding assay The results of the SPR binding assay are shown in Table 1.

[0263] [Table 1]

[0264] Binding assay Binding assays against human primary naive B cells and cynomolgus monkey CD40-expressing HEK293 cells demonstrated that binding to hCD40 (Figures 1A-1B) and cyno CD40 (Figure 1C) was preserved by each of mAb1-mAb4 compared to the parent antibody AbC1.

[0265] Reporter assay The results showed that the single mutation Y32K in mAb3 increased the agonist activity of the mutant anti-CD40 mAbs in human CD40 U-2 OS reporter cells (Fig. 2A; Table 2) and in rhesus monkey CD40 reporter cells (Fig. 2B; Table 2). Combinations of Y32K with S77A or S77P in mAb1 and mAb2, respectively, showed slight improvements in activity compared with the single Y32K mutation alone, but these were not statistically significant.

[0266] [Table 2]

[0267] B cell activation assay (BCA) In primary B cell activation assays, all four mutant mAbs 1-4 exhibited activity equivalent to that of the CD40 natural ligand (hexameric CD40L) control and superior to that of the parent AbC1 antibody (Figures 3A-C).

[0268] Monocyte-derived dendritic cell (MoDC) maturation assay Finally, in primary monocyte-derived dendritic cell (MoDC) assays, all four mutant mAbs 1-4 exhibited activity equal to that of the hexameric CD40L control and again superior to that of the parental AbC1 antibody (Figures 4A-F).

[0269] Example 4 Conditionally active anti-CD40 monoclonal antibodies The present inventors generated a conditionally active anti-CD40 monoclonal antibody from the "mAb1" double mutant (Y32K / S77A) described in Example 2.

[0270] Briefly, conditionally active anti-CD40 monoclonal antibodies include: - a "masked" light chain comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable linker (CL), and an anti-CD40 "mAb1" light chain (LCVR having SEQ ID NO: 1 and LCCR having SEQ ID NO: 3); and - anti-CD40 "mAb1" heavy chain (HCVR with SEQ ID NO: 5 and HCCR with SEQ ID NO: 4).

[0271] Cleavable linkers typically contain a short amino acid sequence that is a target for a protease. In the case of cancer treatment, the protease is preferably a tumor-specific protease, i.e., a protease that is found at least predominantly, if not exclusively, in the tumor microenvironment in vivo. Thus, in the absence of a protease, the masking moiety remains fused to the antibody, thereby reducing, inhibiting, or eliminating the antibody's binding to its target (herein, to CD40). However, when the conditionally active antibody colocalizes with a protease that can cleave the cleavable linker, for example, in the tumor microenvironment, the masking moiety is released from the antibody, restoring the latter's binding to the target antigen (e.g., CD40).

[0272] Two alternative masking moieties (hereafter "MM1" and "MM2") were identified according to Adagene's protocol using a synthetic library described in WO 2019 / 149282 A1. MM1 or MM2 was linked at the N-terminus of the LCVR of "mAb1" via one of two alternative cleavable linkers (CL1 or CL2). Combining one masking moiety with one cleavable linker led to four different conditionally active (masked) anti-CD40 monoclonals: -MC1, including MM1 and CL1; -MC2, including MM2 and CL1; MC3, including MM1 and CL2; and -MC4 including MM2 and CL2.

[0273] Materials and Methods MMP9-mediated cleavage For experiments requiring cleavage of the masking moiety by MMP9 protease (a compound hereafter identified as "MMP9 activation").

[0274] In the first step, recombinant human MMP9 protein (R&D Systems, Ref. 911-MP-010) was activated using p-aminophenylmercuric acetate (APMA; Calbiochem, Ref. 164610-700MG) and added to a final concentration of 1 mM in a 100 μg / mL MMP9 solution and incubated for 24 hours at 37° C. The activated MMP9 was then aliquoted and stored at −80° C. until further use.

[0275] Test (masked) compounds were then diluted to 1 mg / mL and incubated with activated MMP9 at a final concentration of 5 nM for 24 hours at 37° C. under gentle shaking (300 rpm).

[0276] If necessary, samples were purified using HiLoad® 26 / 600 Superdex® 200 (GE Healthcare, Ref. 28-9893-36) to remove excess masking peptides.

[0277] uPA-mediated cleavage For experiments requiring cleavage of the masking moiety by uPA protease (compounds hereafter identified as "uPA activated").

[0278] Test (masked) compounds were diluted to 1 mg / mL and incubated with recombinant human uPA protein (Sino Biological, Ref. 10815-H08H-A) at a final concentration of 209 nM at 37° C. under gentle shaking (300 rpm) for 24 h.

[0279] If necessary, samples were purified using HiLoad® 16 / 600 Superdex® 200 (GE Healthcare, Ref. 28-9893-35) to remove excess masking peptides.

[0280] Binding assay About Raji cells The dose-dependent binding activity of the mAb1 double mutant (unmasked), each of MC1 to MC4 (masked), and the protease-activating compound (unmasked) to Raji cells (human B lymphoblastoid cells originally derived from a patient with Burkitt's lymphoma) was measured using flow cytometry.

[0281] Briefly, Raji cells were cultured at 1.0 × 10 in a 96-well plate. 5 Cells were seeded at 1000 kJ / well and incubated with serially diluted test antibodies in 2% FBS / DPBS buffer at 4°C for 60 minutes. Cells were then washed twice with dPBS and further incubated with a secondary allophycocyanin (APC)-conjugated AffiniPure F(ab')2 fragment donkey anti-human IgG (H+L) antibody (Jackson ImmunoResearch, Ref. 709-136-149) (1:500) at 4°C for 30 minutes. Finally, cells were washed twice with dPBS and suspended in FACS buffer for flow cytometry analysis. MFI values ​​vs. concentration were analyzed using FlowJo, and the data were further fitted with a four-parameter nonlinear regression using GraphPad Prism software to determine EC 50 got the value.

[0282] About U-2 OS / NFκB cells The dose-dependent binding activity of the mAb1 double mutant (unmasked), each of MC1 to MC4 (masked), and the protease-activated compound (unmasked) to U-2 OS / NFκB cells was measured using flow cytometry.

[0283] Briefly, cultured U-2 OS / NFκB cells were plated at 1.0 × 10 5 Cells were seeded at 1000 kJ / well and incubated with serially diluted test antibodies in 2% FBS / DPBS buffer at 4°C for 60 minutes. Cells were then washed twice with dPBS and further incubated with a secondary allophycocyanin (APC)-conjugated AffiniPure F(ab')2 fragment donkey anti-human IgG (H+L) antibody (Jackson ImmunoResearch, Ref. 709-136-149) (1:500) at 4°C for 30 minutes. Finally, cells were washed twice with dPBS and suspended in FACS buffer for flow cytometry analysis. MFI values ​​vs. concentration were analyzed using FlowJo, and the data were further fitted with a four-parameter nonlinear regression using GraphPad Prism software to determine EC 50 got the value.

[0284] Reporter assay The mAb1 double mutant (unmasked), each of MC1-MC4 (masked), and the protease-activated compound (unmasked) were characterized by luciferase-based reporter assays.

[0285] Briefly, U-2 OS / NFκB cells (5 × 10 in 96-well plates) containing a luciferase gene under the control of an NFκB response element were cultured. 4 Cells / well) were mixed with serial dilutions of test antibodies. After approximately 5 hours of incubation at 37°C and 5% CO2, ONE-GLO substrate was added and luminescence was measured by a multiplate reader (Molecular Devices SpectraMax I3x).

[0286] B cell activation assay Human The in vitro biological activities of the mAb1 double mutant (unmasked), each of MC1-MC4 (masked), and the protease-activated compound (unmasked) were measured on primary human B cells using flow cytometry.

[0287] Briefly, total human B cells were purified from human peripheral blood mononuclear cells (PBMCs) (D#A10K797075) using the StemCell kit (17954). These cells (5 x 10 in a 96-well plate) were cultured in a 2000-well plate. 4 Cells / well) were incubated with titrated test antibodies for 2 days. CD86 (detected by anti-CD86, BioLegend, 1:200 dilution) expression on human B cells was assessed by flow cytometry.

[0288] mouse Objectives: To evaluate the activity of mutant anti-CD40 mAbs versus the parental antibody AbC1 and isotype control on mouse B cells. To monitor the expression of key activation markers and cytokine release from activated mouse B cells.

[0289] Experiments were performed using hCD40KI mice from CIPHE (Center for Immunophenomics, Marseille, France). Mouse B cells were isolated from splenocytes via negative selection. B cells were plated in 96-flat-bottom well plates at 150 × 10 3 Cells were plated at a density of 100 cells / well and cultured in complete medium containing RPMI 1640 supplemented with 1% penicillin / streptomycin (Gibco 31870-025), 2 mM L-glutamine (Gibco 25030-081), 10% FCS (Eurobio CVFSF00-01), 1x non-essential amino acids (Gibco 11140-050), 1 mM sodium pyruvate (Gibco 11360-070), and 0.05 mM 2-mercaptoethanol (Gibco 31350-010).

[0290] Serially diluted concentrations of test compounds were added to the cultures. After 2 days of incubation, the expression of key activation markers and cytokine secretion were assessed in the collected supernatants by flow cytometry using a Mouse Inflammation Cytometric Bead Array (CBA) kit according to the manufacturer's instructions (BD Bioscience 552364).

[0291] Expression of activation markers was analyzed by FlowJo (V10.9) and then plotted using GraphPad Prism (v9.5.0). Cytokine secretion was analyzed by FCAP array (V3.0.19.2091) for CBA samples and then plotted using GraphPad Prism.

[0292] Monocyte-derived dendritic cell (MoDC) maturation assay For MoDC maturation assay, see protocol in Example 3.

[0293] result Binding assay: About Raji cells As shown in Table 3 and Figure 5, protease-activating (i.e., the masking moiety is cleaved) compounds (MMP9-activating and uPA-activating) inhibited EC 50 Based on the EC values, the mAb1 double mutant shows similar binding activity to Raji cells compared to the mAb1 double mutant (without the masking moiety). Binding was measured using the EC values ​​for all masked compounds MC1 to MC4 against the mAb1 double mutant. 50 The comparison shows a reduction of at least 107-fold (for MC4) and up to 364-fold (for MC2).

[0294] [Table 3]

[0295] About U-2 OS / NFκB cells As shown in Table 4 and Figure 6A, protease-activating (i.e., the masking moiety is cleaved) compounds (MMP9-activating and uPA-activating) inhibited EC 50 Based on the EC values, the mAb1 double mutant exhibits similar binding activity to U-2 OS / NFκB cells compared to the mAb1 double mutant (without the masking moiety). Binding was measured using the EC values ​​for all masking compounds MC1 to MC4 against the mAb1 double mutant. 50By comparison, there is a reduction of at least 118-fold (for MC4) and up to 220-fold (for MC1).

[0296] [Table 4]

[0297] In further experiments on U-2 OS cells, mAb1 (unmasked) inhibited EC 50 All four conditionally active anti-CD40 antibodies tested showed EC2 activity against CD40 comparable to that of the parent AbC1 antibody. 50 After MMP9-mediated cleavage of their masking moieties, all four unmasked antibodies had EC values ​​comparable to that of mAb1 (unmasked) and to that of the parent AbC1 antibody. 50 was recovered (Figure 6B).

[0298] Reporter assay As shown in Table 5 and Figure 7A, protease-activating (i.e., the masking moiety is cleaved) compounds (MMP9-activating and uPA-activating) inhibited EC 50 Based on the EC values ​​and maximum signal, this shows similar reporter gene activity compared to the mAb1 double mutant (without the masking moiety). 50 The values ​​are shifted by at least 193-fold (for MC4) and up to 753-fold (for MC1) for masked compounds MC1–MC4 relative to the mAb1 double mutant (without masking moiety).

[0299] [Table 5]

[0300] In further experiments, mAb1 (unmasked) showed clear activity superior to that of the parent AbC1 antibody. Conditionally active anti-CD40 antibodies with cleavable linker CL1 (MC1 and MC2) showed lower EC values ​​in the CD40 reporter assay than those with cleavable linker CL2 (MC3 and MC4), although in a comparable nM range. 50 After MMP9-mediated cleavage of their masking moieties, all four unmasked antibodies had EC values ​​comparable to that of mAb1 (unmasked). 50 was recovered (Figure 7B).

[0301] B cell activation assay Human As shown in Table 6 and Figure 8, protease-activated (i.e., the masking moiety is cleaved) compounds (MMP9-activating and uPA-activating) inhibited EC 50 Based on the EC values ​​and maximum CD86 signal, the mAb1 double mutant shows similar B cell activating activity compared to the mAb1 double mutant (without the masking moiety). 50 The values ​​are shifted by at least 84-fold (for MC4) and up to 230-fold (for MC1) for masked compounds MC1 to MC4 relative to the mAb1 double mutant (without the masking moiety).

[0302] [Table 6]

[0303] mouse As shown in Table 7 and Figures 9A-9B, protease-activated (i.e., the masking moiety is cleaved) compounds (MMP9 activation) inhibited EC 50 Based on the values ​​and the maximum CD83 and CD86 signals, it shows similar activation activity in mouse B cells compared to the mAb1 double mutant (without the masking moiety).

[0304] [Table 7]

[0305] Monocyte-derived dendritic cell (MoDC) maturation assay All test compounds induced CD83 and IL-8 secretion with a dose-dependent effect (Figures 10A-B). The mAb1 double mutant (without the masking moiety) showed an EC20 activity better than the parent antibody AbC1 and closer to that of hexameric CD40L, the natural ligand of CD40. 50 showed.

[0306] After MMP9-mediated cleavage of their masking moieties, all four unmasked antibodies were more active than their masked counterparts MC1–MC4 and inhibited EC 50 was similar to that of mAb1 (unmasked) (Table 8; EC 50 (nM)). Overall, we observed donor- and cytokine-dependent masking shifts (CD86: Figure 10C; CD83: Figure 10D; CD80: Figure 10E; IL-8: Figure 10F).

[0307] [Table 8]

[0308] array All mutant anti-CD40 mAbs described herein share the same parent LCVR having SEQ ID NO:1.

[0309] The HCVR (Y32K / S77A) of "mAb1" comprises or consists of the amino acid sequence having SEQ ID NO:5. [ka]

[0310] The HCVR (Y32K / S77P) of "mAb2" comprises or consists of the amino acid sequence having SEQ ID NO:6. [ka]

[0311] The HCVR(Y32K) of "mAb3" comprises or consists of the amino acid sequence having SEQ ID NO:7. [ka]

[0312] The HCVR(Y32R) of "mAb4" comprises or consists of the amino acid sequence having SEQ ID NO:8. [ka]

[0313] Masking moiety #1 (MM1) has the amino acid sequence set forth as EDEPGIRCYYNDDTCQY (SEQ ID NO: 22).

[0314] Masking moiety #2 (MM2) has the amino acid sequence set forth as EVGSYYVCPVRFQYFCEE (SEQ ID NO: 23).

[0315] Cleavable linker #1 (CL1) is [ka] and PLGLAG (bold; SEQ ID NO: 38) is a matrix metalloproteinase (MMP) substrate.

[0316] Cleavable linker #2 (CL2) is: [ka] and having an amino acid sequence described as follows: SGRSA (underlined; SEQ ID NO: 39) is a urokinase-type plasminogen activator (uPa) substrate, and PLGLAG (bold; SEQ ID NO: 38) is a matrix metalloproteinase (MMP) substrate.

[0317] The LCVR of "MC1" comprises or consists of the amino acid sequence having SEQ ID NO:26. [ka]

[0318] The LCVR of "MC2" comprises or consists of the amino acid sequence having SEQ ID NO:27. [ka]

[0319] The LCVR of "MC3" comprises or consists of the amino acid sequence having SEQ ID NO:28. [ka]

[0320] The LCVR of "MC4" comprises or consists of the amino acid sequence having SEQ ID NO:29. [ka]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD40, preferably human CD40: (i) three light chain complementarity determining region (CDR) sequences set forth in SEQ ID NO: 1; and (ii) the three heavy chain CDR sequences set forth in SEQ ID NO: 7 or 8 An antibody or antigen-binding fragment thereof comprising:

2. The antibody or antigen-binding fragment thereof of claim 1, comprising the three heavy chain CDR sequences set forth in SEQ ID NO:

7.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising the three heavy chain CDR sequences set forth in SEQ ID NO:

8.

4. (i) the three following CDR sequences: a.V Ｌ CDR1: QGIYSW (SEQ ID NO: 9); ｡.V Ｌ -CDR2:TAS; c.V Ｌ - CDR3: QQANIFPLT (SEQ ID NO: 10) a light chain variable region comprising: (ii) the three following CDR sequences: a.V Ｈ CDR1: GYTFTGX １ Y (SEQ ID NO: 11) １ is selected from the group consisting of lysine (Lys, K) and arginine (Arg, R); b.V Ｈ - CDR2: INPDSGGT (SEQ ID NO: 12); c.V Ｈ - CDR3: ARDQPLGYCTNGVCSYFDY (SEQ ID NO: 13) a heavy chain variable region comprising The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

5. V having SEQ ID NO: 11 Ｈ - X in CDR1 １ is lysine (Lys, K).

6. V having SEQ ID NO: 11 Ｈ - X in CDR1 １ is arginine (Arg, R),

7. a) the light chain variable region further comprises the four following framework region sequences: V Ｌ - FR1: DIQMTQSPSSVSASVGDRVTITCRAS (SEQ ID NO: 14); V Ｌ - FR2: LAWYQQKPGKAPNLLIY (SEQ ID NO: 15); V Ｌ FR3: TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 16); and V Ｌ -FR4: FGGGTKVEIK (SEQ ID NO: 17) Including; and b) the heavy chain variable region further comprises the four following framework region sequences: V Ｈ - FR1: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 18); V Ｈ - FR2: MHWVRQAPGQGLEWMGW (SEQ ID NO: 19); V Ｈ -FR3:NYAQKFQGRVTMTRDTSIX ２ TAYMELNRLRSDDTAVYYC (SEQ ID NO: 20) ２ is selected from the group consisting of alanine (Ala, A) and proline (Pro, P); and V Ｈ -FR4: WGQGTLVTVSS (SEQ ID NO: 21) Including, The antibody or antigen-binding fragment thereof according to any one of claims 4 to 6.

8. (i) a light chain variable region having SEQ ID NO: 1, or a light chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1; and (ii) a heavy chain variable region having SEQ ID NO: 5, 6, 7, or 8, or a heavy chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 5, 6, 7, or 8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising:

9. (i) a light chain variable region having SEQ ID NO: 1; and (ii) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising:

10. (i) a light chain variable region having SEQ ID NO: 1; and (ii) a heavy chain variable region having SEQ ID NO: 5 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising:

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, which is coupled to at least one masking moiety.

12. 1. A conditionally active antibody or antigen-binding fragment thereof capable of specifically binding to CD40, preferably human CD40: (i) three light chain complementarity determining region (CDR) sequences set forth in SEQ ID NO: 1; and (ii) the three heavy chain CDR sequences set forth in SEQ ID NO: 7, 8, or 2 Including; Additionally, a conditionally active antibody or antigen-binding fragment thereof coupled to at least one masking moiety.

13. 13. The conditionally active antibody or antigen-binding fragment thereof of claim 12, wherein the at least one masking moiety reduces or inhibits binding of the antibody or antigen-binding fragment thereof to CD40.

14. 14. The conditionally active antibody or antigen-binding fragment thereof of claim 12 or 13, comprising the three heavy chain CDR sequences set forth in SEQ ID NO:

7.

15. 14. The conditionally active antibody or antigen-binding fragment thereof of claim 12 or 13, comprising the three heavy chain CDR sequences set forth in SEQ ID NO:

8.

16. 14. The conditionally active antibody or antigen-binding fragment thereof of claim 12 or 13, comprising the three heavy chain CDR sequences set forth in SEQ ID NO:

2.

17. (i) a light chain variable region having SEQ ID NO: 1, or a light chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1; and (ii) a heavy chain variable region having SEQ ID NO: 5, 6, 7, 8, or 2, or a heavy chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 5, 6, 7, 8, or 2; The conditionally active antibody or antigen-binding fragment thereof of any one of claims 12 to 16, comprising:

18. (i) a light chain variable region having SEQ ID NO: 1; and (ii) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, and 2; The conditionally active antibody or antigen-binding fragment thereof of any one of claims 12 to 17, comprising:

19. 19. The conditionally active antibody or antigen-binding fragment thereof of any one of claims 12 to 18, wherein the at least one masking moiety is coupled to the N-terminus of the light chain variable region of the antibody or antigen-binding fragment thereof.

20. 20. The conditionally active antibody or antigen-binding fragment thereof of any one of claims 12 to 19, further comprising at least one cleavable linker between the at least one masking moiety and the antibody or antigen-binding fragment thereof.

21. 21. The conditionally active antibody or antigen-binding fragment thereof of claim 20, wherein the at least one cleavable linker is cleavable by at least one tumor-specific protease.

22. 22. The conditionally active antibody or antigen-binding fragment thereof of claim 21, wherein the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

23. 23. The conditionally active antibody or antigen-binding fragment thereof of claim 21 or 22, wherein the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

24. 24. The conditionally active antibody or antigen-binding fragment thereof of any one of claims 21 to 23, which is capable of binding to CD40, preferably human CD40, upon cleavage of the at least one cleavable linker and release of the at least one masking moiety.

25. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a conditionally active antibody or antigen-binding fragment thereof according to any one of claims 12 to 24, having pure agonist activity.

26. Pure agonist activity means that the antibody or antigen-binding fragment thereof or the conditionally active antibody or antigen-binding fragment thereof is capable of activating the CD40 signaling pathway (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner, and / or (iv) in the absence of target-mediated cross-linking of CD40.

27. A composition comprising the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, of any one of claims 1 to 26.

28. 28. The composition of claim 27, which is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or excipient.

29. 29. A method of treating a subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, of any one of claims 1 to 26, or the composition of claim 27 or 28.

30. 30. The method of claim 29, wherein the subject has cancer.

31. 29. The antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or the conditionally active antibody or antigen-binding fragment thereof, or the composition of claim 27 or 28, for use in the treatment of cancer.

32. 29. Use of the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, of any one of claims 1 to 26, or the composition of claim 27 or 28, in the manufacture of a medicament for treating cancer.

33. 27. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, of any one of claims 1 to 26.

34. A vector comprising the polynucleotide of claim 33.

35. 35. A host cell comprising the polynucleotide of claim 33 or the vector of claim 34.

36. 36. A method for producing the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, of any one of claims 1 to 26, comprising expressing the polynucleotide of claim 33 or the vector of claim 34 in a cell.

37. A method for producing the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof, of any one of claims 1 to 26, comprising culturing the host cell of claim 35 in a culture medium; and recovering the antibody or antigen-binding fragment thereof, or the conditionally active antibody or antigen-binding fragment thereof produced by the host cell by said culturing.

Images