Agonistic CD40 antibodies

Abstract

To provide humanized monoclonal antibodies or antigen-binding fragments thereof that specifically bind to human CD40 receptor and induce CD40 signaling independent of Fcy-mediated CD40 receptor crosslinking.SOLUTION: The antibodies of the present invention bind to a CD40 epitope that overlaps with the epitope of the CD40 ligand and can activate human APCs. The present invention also provides compositions comprising the antibodies and uses of the antibodies and compositions in the treatment of patients suffering from cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to humanized agonistic monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to the human CD40 receptor and can induce CD40 signaling independent of Fcγ-mediated CD40 cross-linking. The present invention also relates to uses of such antibodies and pharmaceutical compositions containing them. [Background technology]

[0002] Recent successes in cancer immunotherapy have revived the hypothesis that the immune system can control many, if not most, cancers, in some cases generating durable responses in ways not seen with many small molecule drugs. Agonistic CD40 monoclonal antibodies (mabs) offer a new therapeutic option with the potential to generate anticancer immunity through multiple mechanisms.

[0003] CD40 is a cell surface molecule and a member of the tumor necrosis factor (TNF) receptor superfamily. It is widely expressed on antigen-presenting cells (APCs), such as dendritic cells, B cells, and monocytes, as well as on many non-immune cells and a wide range of tumors.

[0004] The natural ligand for CD40 is CD154, which is expressed primarily on the surface of activated T lymphocytes and provides a key component of T cell "help" for immune responses. Signaling through CD40 on APCs mediates much of the ability of helper T cells to license APCs. Ligation of CD40 on DCs induces, for example, increased surface expression of costimulatory and MHC molecules, production of proinflammatory cytokines, and enhanced T cell triggering. Ligation of CD40 on resting B cells increases antigen-presenting function and proliferation.

[0005] The outcomes of CD40 signaling are multifaceted and depend on the type of cell expressing CD40 and the microenvironment in which the CD40 signal is provided. As with several other members of the TNF receptor family, CD40 signaling is mediated by adaptor molecules rather than by the intrinsic signaling activity of the CD40 cytoplasmic tail. Upon receptor assembly, downstream kinases are activated, resulting in the formation of multicomponent signaling complexes. translocates from CD40 to the cytosol and expresses several well-characterized signals. The nucleus transduction pathway is activated.

[0006] Antagonistic human CD40 antibodies are known in the prior art. The respective antagonistic antibodies may be silent Fc mutants that exhibit reduced Fcγ-mediated CD40 receptor cross-linking. Mutations in the Fc region of human IgG1 are described, for example, in U.S. Publication No. 2018 / 0118843.

[0007] Recently designed immunomodulatory approaches have used agonistic monoclonal antibodies (mAbs) targeting CD40 to enhance the immune system's ability to recognize and destroy cancer cells. Preclinical studies have shown that agonistic CD40 mAbs can activate APCs, promote antitumor T cell responses, and cultivate cytotoxic myeloid cells with the potential to control cancer in the absence of T cell immunity. Thus, agonistic CD40 mAbs are fundamentally different from mAbs that achieve immune activation by inhibiting negative checkpoint molecules, such as CTLA-4 or PD-1.

[0008] CP-870,893 acts as a potent and selective agonist of CD40, the first CP-870,893 is a fully human IgG2 mAb. Interestingly, CP-870,893 binding does not compete with CD154 for CD40 binding. In preclinical studies, CP-870,893 has been shown to mediate both immune-dependent and immune-independent effects on tumor cell survival. In initial in-human studies, promising antitumor activity was observed, particularly in melanoma patients. Pharmacodynamically, administration of CP-870,893 results in a transient depletion of peripheral blood B cells and the upregulation of activating markers on APCs.

[0009] Agonistic CD40 mAbs therefore represent a promising strategy for novel cancer therapy. However, concerns have also been raised about their potential cytotoxic side effects. Agonistic monoclonal CD40 antibodies can induce cytokine release syndrome, autoimmune reactions, thromboembolic syndromes (due to CD40 expression by platelets and endothelial cells), excessive immune stimulation leading to activation-induced cell death or tolerance, and tumor angiogenesis. These effects may result in undesirable toxicity or promotion of tumor growth. Mechanistically, the ability of agonistic CD40 and other TNF receptor family targeting antibodies to interact with Fcγ receptors has been associated with the development of toxicity in animal studies (Li & Ravetch 2012, Xu et al., 2003, Byrne et al., 2016).

[0010] The most common side effects reported for CP-870,893, the strongest agonist tested, are cytokine release syndrome manifesting as chills, fever, rigors, and other symptoms shortly after infusion. Several cases of thromboembolic events have also been observed with CP-870,893. Noninfectious inflammatory eye disease has been observed with dacetuzumab.

[0011] Thus, there is a need to provide agonistic CD40 mAbs that exhibit reduced cytotoxicity and fewer clinical side effects while maintaining their potency and clinical effectiveness. The agonistic CD40 mAbs of the present invention can fulfill this need and maximize the immunomodulatory potential of agonistic CD40 antibodies. Summary of the Invention

[0012] The present invention provides monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to the human CD40 receptor and induce CD40 signaling independent of Fcγ-mediated CD40 receptor cross-linking. More specifically, the antibodies of the present invention bind to CD40 epitopes that overlap with epitopes on the CD40 ligand and can activate human APCs. The present invention also provides compositions comprising the antibodies and uses of the antibodies and compositions in the treatment of conditions or diseases in which immune system stimulation is desired, such as the treatment of patients suffering from cancer.

[0013] definition The term "antibody" includes, but is not limited to, whole antibodies and antibody fragments, so long as they exhibit the properties according to the present invention, and encompasses various types of antibody structures.

[0014] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, These include, but are not limited to, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0015] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0016] The term "humanized antibody" or "humanized version of an antibody" refers to an antibody in which both the heavy and light chains have been humanized as a result of antibody engineering. Humanized chains are typically chains in which the amino acid sequence of the V region has been altered so that, when analyzed as a whole, they are closer in homology to the human germline sequence than to the germline sequence of the original species. Assessment of humanization is based on the resulting amino acid sequence, not on the methodology itself.

[0017] As used herein, the term "specifically binds to a target or anti-target antibody" refers to the binding of an antibody to the respective antigen (target) or antigen-expressing cells as measured by ELISA, which preferably comprises coating the respective antigen to a solid support, adding the antibody under conditions that allow the formation of an immune complex with the respective antigen or protein, and detecting the immune complex by measuring the optical density value (OD) using a secondary antibody that binds to an antibody according to the invention and using peroxidase-mediated color development.

[0018] The term "antigen" as used herein refers to an antigen used for immunization or a protein that contains the antigen as part of its protein sequence. For example, a fragment of the extracellular domain of a protein (e.g., the first 20 amino acids) can be used for immunization, and the extracellular domain or the full-length protein can be used for detection / assay, etc.

[0019] By "specifically bind" or "specifically recognized" herein is meant an antibody that exhibits adequate affinity for an antigen, preferably an antibody that does not exhibit significant cross-reactivity.

[0020] An antibody that "does not exhibit significant cross-reactivity" is one that does not (appear to) significantly bind to other undesired proteins. Specific binding can be determined by any art-recognized means for measuring such binding, e.g., competitive binding assays such as ELISA.

[0021] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in a competitive assay.

[0022] As used herein, the term "variable region (or domain) of an antibody according to the present invention" (light chain variable region (VL) and heavy chain variable region (VH)) refers to each of a pair of light and heavy chain regions that are directly involved in binding an antigen to the antibody. The variable light and heavy chain regions have the same general structure, and each region contains four framework (FR) regions, the sequences of which are widely conserved, and are connected by three complementarity-determining regions (CDRs).

[0023] As used herein, the term "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody preferably comprises amino acid residues from the "complementarity-determining region" or "CDR." CDR sequences are defined by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual The linear amino acid sequence of a heavy chain may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or CDRs of the variable region. For example, a heavy chain variable region may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues for a given antibody is determined by alignment of the homologous regions of that antibody sequence with the "standard" Kabat numbered sequence. It may be determined by

[0024] The "constant region (constant portion)" is not directly involved in binding of an antibody to an antigen, but also exhibits, for example, effector functions. The heavy chain constant region gene fragment corresponding to human IgG1 is called the γ1 chain. The heavy chain constant region gene fragment corresponding to human IgG3 is called the γ3 chain. The human constant γ heavy chain is described in detail by Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and by Brueggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T.W. et al., Methods Enzymol. 178 (1989) 515-527.

[0025] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0026] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0027] A "variant Fc region" comprises an amino acid sequence that differs from that of a "native" or "wild-type" Fc region sequence by virtue of at least one "amino acid modification" as defined herein.

[0028] As used herein, the term "Fc variant" refers to a polypeptide containing a modification in the Fc domain. The modification can be an addition, deletion, or substitution. The substitution can include naturally occurring and non-naturally occurring amino acids. The variant may also include non-naturally occurring amino acids.

[0029] The term "Fc region-containing polypeptide" refers to a polypeptide that includes an Fc region, such as an antibody.

[0030] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. FcRs (gamma receptors) that bind IgG antibodies include receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. The activating receptor, FcγRIIA, contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor, FcγRIIB, contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcR are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492; Capel et al., Immunomethods 4 (1994) 25-34; and de Haas et al., J. Lab. Clin. Med. 126 (1995) 330-41. Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transport of maternal IgG to the fetus (Guyer et al., J. Immunol. 117 (1976) 587 and Kim et al., J. Immunol. 24 (1994) 249).

[0031] As used herein, the term "IgG Fc ligand" refers to a molecule, preferably a polypeptide, derived from any organism, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include FcγR, FcRn, C1q, C3, mannan binding, and the like. Fc ligands include, but are not limited to, lectins, mannose receptors, staphylococcal protein A, streptococcal protein G, and viral FcγRs. Fc ligands also include Fc receptor homologs (FcRHs), which are a family of Fc receptors that share homology with FcγRs (Davis et al., Immunological Reviews 190 (2002) 123-136, see herein for all references). (Incorporated by reference thereto). Fc ligands may also include as yet undiscovered molecules that bind to Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, "Fc ligand" refers to a molecule, preferably a polypeptide, derived from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0032] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes FcγRI (CD64), which contains the isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32), which contains the isoforms FcγRIIA (including allotypes H131 and R131), FcγRIIB (including FcγRIIB-1 and FcγRIIB-2), and FcγRIIc; and FcγRIII (CD16), which contains the isoforms FcγRIIIA (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIB-NA1 and FcγRIIB-NA2) (Jefferis et al., Immunol Lett 82 (2002)), as well as any unidentified human FcγRs or FcγRs include, but are not limited to, FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0033] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is encoded, at least in part, by the FcRn gene. FcRn may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin.

[0034] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

[0035] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells that express FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI and FcγRII. and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.

[0036] The terms "antibody-dependent cellular phagocytosis" and "ADCP" refer to the process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0037] As used herein, the term "antibody effector function(s)" or "effector function" refers to a function contributed by the Fc effector domain(s) of an IgG (e.g., the Fc region of an immunoglobulin). Such a function is mediated, for example, by binding of the Fc effector domain(s) to Fc receptors on immune cells with phagocytic or lytic activity, or by binding of the Fc effector domain to components of the complement system. Exemplary effector functions are ADCC, ADCP, and CDC.

[0038] "C1q" is a polypeptide that contains a binding site for the Fc region of immunoglobulins. C1q, together with two serine proteases, C1r and C1s, forms the complex C1, which is the first component of the complement-dependent cytotoxicity (CDC) pathway.

[0039] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0040] An "effective amount" of an agent, such as a pharmaceutical formulation, refers to an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0041] The term "cancer" as used herein includes, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, advanced pancreatic carcinoma, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, and the like. cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwanomas, ependymomas, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia Hematologic diseases (including refractory versions of any of the above cancers) or a combination of one or more of the above cancers It may also be a combination. [Brief explanation of the drawings]

[0042] [Figure 1]Cell Binding The anti-CD40 IgG1-LALA monoclonal antibody was tested for binding to CD40 antigen expressed on HEK-Blue-CD40L™ cells (Invivogen). EC50 values ​​demonstrate strong binding of the tested antibodies. [Figure 2] HEK-Blue EC50 Measurement in an Eight-Point Analysis The agonist activity of the humanized anti-CD40 IgG1-LALA monoclonal antibody was tested in a cell-based NF-κB gene reporter assay. HEK-Blue-CD40L™ cells (Invivogen) were incubated with various concentrations of antibody for 24 hours. The EC50 value demonstrates the potency of the antibody to induce NF-κB signaling. [Figure 3] CD40-Ligand Epitope Competition To test whether the humanized anti-CD40 IgG1-LALA monoclonal antibody binds to an epitope overlapping with the CD40L binding site, a CD40L competition ELISA was performed. Various concentrations of anti-CD40 antibody were preincubated with recombinant CD40 protein to form binding complexes. The complexes were then added to a microtiter plate coated with recombinant CD40L. After washing, the bound CD40-anti-CD40 complexes were detected using a peroxidase-conjugated anti-human F(ab)2 antibody. The ELISA signal for the reference CP-870,893 antibody did not compete with CD40L, suggesting binding to an epitope distinct from the CD40L binding site. The data demonstrate that the tested humanized anti-CD40 IgG1-LALA monoclonal antibody binds to an epitope overlapping with the CD40L binding site. [Figure 4] Cynomolgus monkey CD40 binding activity The binding activity of the humanized anti-CD40 IgG1-LALA monoclonal antibody to cynomolgus monkeys (Macaca fascicularis) was tested by ELISA using recombinant cynomolgus monkey CD40 (Acro Biosystems). The EC50 value indicates the binding potency of the antibody. nd = not detectable within the range of concentrations tested. [Figure 5A]Induction of dendritic cell maturation. To test the agonistic activity of the humanized anti-CD40 IgG1-LALA monoclonal antibody on primary target cells, we analyzed the maturation of monocyte-derived immature dendritic cells. Immature dendritic cells differentiated in vitro from monocytes were incubated with agonistic anti-CD40 antibodies at a concentration of 5 μg / ml for 48 hours. Subsequently, dendritic cell-derived secreted IL-12p40 was quantified in the culture supernatant by biochemical ELISA. [Figure 5B] Induction of dendritic cell maturation. To test the agonistic activity of the humanized anti-CD40 IgG1-LALA monoclonal antibody on primary target cells, we analyzed the maturation of monocyte-derived immature dendritic cells. Immature dendritic cells differentiated in vitro from monocytes were incubated with agonistic anti-CD40 antibodies at a concentration of 5 μg / ml for 48 hours. Subsequently, dendritic cell-derived secreted IL-12p40 was quantified in the culture supernatant by biochemical ELISA. [Figure 6] Dendritic Cell Maturation. The ability of the humanized anti-CD40 IgG1-LALA monoclonal antibody to stimulate IL12p40 secretion by dendritic cells was measured at various antibody concentrations and compared with that of CP-870,903 antibodies with various Fc portions (IgG1, IgG1-LALA, IgG2, and IgG1-V11). The antibodies were incubated with immature dendritic cells differentiated in vitro for 48 hours. IL12p40 release was measured by ELISA. [Figure 7] Measurement of anti-CD40 antibody EC50 on dendritic cell maturation. The EC50 value of the humanized anti-CD40 IgG1-LALA monoclonal antibody on dendritic cell-mediated IL12p40 secretion was determined by testing antibody concentrations ranging from 10 to 0.005 μg / ml. The antibody was incubated with immature dendritic cells differentiated in vitro for 48 hours. IL12p40 release was measured by ELISA. [Figure 8]Cytokine Release Assay The humanized anti-CD40 IgG1-LALA monoclonal antibody was tested at 10 μg / ml in a high-density PBMC cytokine release assay to measure the general induction of proinflammatory cytokines, such as TNF-alpha. The data suggest that, in contrast to anti-CD3 (OKT3) antibody, the anti-CD40 antibody does not induce significant TNF-alpha secretion. [Figure 9] Cellular antibody pulse-chase assay. Antibody binding kinetics and internalization were examined in a cellular pulse-chase assay. Antibodies were incubated with HEK-Blue-CD40L™ cell culture medium at a concentration of 0.8 μg / ml for 15 minutes. After washing, the antibodies were allowed to internalize for 60 minutes before cells were washed again and the remaining cell surface-localized anti-CD40 antibodies were detected with an Alexa-488-labeled secondary antibody. Under conditions that do not permit internalization, cells were treated identically, but the antibodies were incubated for only 15 minutes, followed by washing and incubation with the secondary antibody. The data show that under conditions that permit internalization, signal from surface-localized antibodies is reduced to various degrees for the humanized anti-CD40 IgG1-LALA monoclonal antibodies tested. A strong signal reduction is observed for all CP-870,893 antibody isoforms. [Figure 10] Correlation of gene reporter induction and dendritic cell maturation activity of humanized anti-CD40 antibodies. 88 humanized anti-CD40 IgG1-LALA antibodies were tested for their activity in the HEK-Blue gene reporter and dendritic cell maturation assays. HEK-Blue gene reporter activity was quantified by OD at 655, corresponding to induced SEAP secretion, and dendritic cell maturation was quantified by IL12p40 release (ELISA). [Figure 11]Stimulation of costimulatory receptors on dendritic cells by agonistic anti-CD40 antibodies. Immature iDCs differentiated in vitro were stimulated with agonistic CD40 antibodies, isotype control antibodies, or CD40L for 48 hours. Expression of costimulatory receptor molecules was measured by flow cytometry. Mean fluorescence intensity was normalized to isotype control antibody treatment or, in the case of CD40L, to untreated samples. Induction of expression is expressed as fold induction (FOI) relative to control treatment. [Figure 12] Cytokine release by anti-CD40-treated dendritic cells. In vitro differentiated immature iDCs were stimulated with an agonistic CD40 antibody, an isotype control antibody, or CD40L for 48 hours. Cytokine release was measured by ELISA (IL-12p40) or flow cytometry using BD cytometric bead arrays. [Figure 13] Dose-dependent stimulation of costimulatory receptors on dendritic cells by agonistic anti-CD40 antibodies. Immature iDCs differentiated in vitro were stimulated with agonistic CD40 antibodies or isotype control antibodies at concentrations ranging from 10,000 to 5 ng / ml for 48 hours. Expression of costimulatory receptor molecules was measured by flow cytometry. Mean fluorescence intensity was normalized to isotype control treatment. Induction of expression is expressed as fold induction (FOI) relative to control treatment. Calculated EC50 values ​​are shown in the table. [Figure 14] Dose-dependent cytokine release by anti-CD40-treated dendritic cells. In vitro differentiated immature iDCs were stimulated with agonistic CD40 antibodies and isotype control for 48 hours. Cytokine release was measured by flow cytometry using BD Cytometric Bead Arrays. [Figure 15]Dose-dependent stimulation of costimulatory receptors on B cells by agonistic anti-CD40 antibodies. B cells were stimulated with agonistic CD40 antibodies or isotype control antibodies at concentrations ranging from 500 to 0.2 ng / ml for 48 hours. Expression of costimulatory receptor molecules was measured by flow cytometry. Mean fluorescence intensity was normalized to isotype control treatment. Induction of expression is expressed as fold induction (FOI) relative to control treatment. Calculated EC50 values ​​are shown in the table. [Figure 16] Competition between CD40L and CD40 expressed on cells with anti-CD40 antibodies. HEK-Blue-CD40L™ cells were preincubated with anti-CD40 IgG1-LALA antibody at an EC90 concentration before adding CD40L at concentrations ranging from 10,000 to 9.8 ng / ml. Binding of anti-CD40 antibodies and CD40L to cell surface-expressed CD40 was detected using various fluorophore-conjugated secondary antibodies. [Figure 17] Induction of FasR (CD95) death receptor expression by agonistic CD40 antibodies in combination with CD40L. Ramos B lymphoma cells were stimulated overnight with CD40L alone or in combination with agonistic anti-CD40 antibodies or isotype control antibodies. CD95 expression was quantified by flow cytometry. Upregulation of expression is expressed as fold induction (FOI) over control treatment. [Figure 18] Affinity of the humanized, agonistic anti-CD40 IgG1-LALA antibody. Biochemical affinity was measured by surface plasmon resonance on a Biocore T200 SPR instrument. Kinetic data were measured using a Langmuir 1:1 binding model. [Figure 19] Antibody competition for CD40 binding: Binding of a mixture of HIS-tagged CD40 protein and various anti-CD40 antibodies preincubated to plates coated with various anti-CD40 antibodies. Sequential binding of both anti-CD40 antibodies is detected with an anti-HIS POD-labeled antibody. [Figure 20]Antibody-Mediated Effector Function of IgG1-LALA Anti-CD40 Antibody Antibody-mediated effector function was analyzed using the Jurkat effector luciferase gene reporter cell line and HEK-Blue-CD40L™ as target cells. IgG1-LALA or IgG1 anti-CD40 antibodies were incubated with target and effector cells at doses ranging from 10,000 to 0.002 ng / ml for 6 hours. The measured fold induction of luciferase activity indicates effector cell activation mediated by the anti-CD40 antibody. [Figure 21-1] Safety of agonistic anti-CD40 antibody therapy in a humanized mouse model. Nod / Scid / gamma(c)(null)FcRg- / - mice were injected with 3 μg / g of MAB-16-0451 or CP-870,893 anti-CD40 antibody on day 0. Body temperature was measured before and at various time points after injection. Three mice treated with CP-870,893 showed significant hypothermia and had to be sacrificed after 3 days. [Figure 21-2] Safety of agonistic anti-CD40 antibody therapy in a humanized mouse model. Nod / Scid / gamma(c)(null)FcRg- / - mice were injected with 3 μg / g of MAB-16-0451 or CP-870,893 anti-CD40 antibody on day 0. Body temperature was measured before and at various time points after injection. Three mice treated with CP-870,893 showed significant hypothermia and had to be sacrificed after 3 days. [Figure 22-1] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-2]Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-3] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-4] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-5] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-6]Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-7] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-8] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 [Figure 22-9] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: Complete VH: SEQ ID NOs: 1 to 14 Light chain: Complete VL: SEQ ID NOs: 15 to 28 Complementarity determining regions (CDR): Heavy chain: CDR-H1: SEQ ID NOs: 29 to 42 CDR-H2: SEQ ID NOs: 43 to 56 CDR-H3: SEQ ID NOs: 57 to 70 Light chain: CDR-L1: SEQ ID NOs: 71 to 84 CDR-L2: SEQ ID NOs: 85 to 98 CDR-L3: SEQ ID NOs: 99 to 112 DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description of the Invention The present invention addresses the need to provide agonistic CD40 mAbs that exhibit reduced cytotoxicity and fewer clinical side effects, while at least maintaining, if not increasing, their signaling potency and clinical efficacy compared to prior art agonistic CD40 antibodies.

[0044] The antibodies or antibody-binding fragments of the present invention provide these beneficial properties because they can specifically bind to the human CD40 receptor and induce CD40 signaling that is independent of Fcγ-mediated CD40 receptor cross-linking.

[0045] In a preferred embodiment, the antibody of the present invention is a humanized IgG1LALA antibody, a humanized IgG1 type antibody, which has at least two alanine amino acids in the human Fc1 region at positions 234 and 235. Thus, in a preferred embodiment, IgG1LALA comprises L234A and L235A mutations in the human Fc region.

[0046] It is further preferred that the antibodies of the present invention are recombinant molecules.

[0047] The present invention provides agonistic monoclonal antibodies, or antigen-binding fragments thereof, that can bind to the human CD40 receptor and induce CD40 signaling that is independent of Fcγ-mediated CD40 receptor cross-linking (see also Example 5, Figure 6, and text below). Furthermore, the antibodies of the present invention may exhibit reduced or abolished ability to signal through human Fcγ receptors when compared to wild-type IgG Fcγ receptor signaling or Fcγ signaling of prior art antibodies.

[0048] In certain embodiments, the agonistic monoclonal CD40 antibodies, or antigen-binding fragments thereof, of the invention may exhibit reduced or depleted affinity for human Fcγ receptors compared to wild-type IgG Fcγ. According to a preferred embodiment, the antibodies of the invention do not bind to Fcγ receptors, thereby preventing Fcγ-mediated CD40 receptor cross-linking.

[0049] In a preferred embodiment, the antibody of the present invention comprises at least amino acid substitutions at L234A and L235A in the human IgG1 Fc region, or at S228P and L235E in the human IgG4 Fc region.

[0050] It is further preferred that the antibodies of the present invention bind to a CD40 epitope that overlaps with the CD40L binding site. Figures 3 and 16 demonstrate this epitope overlap for the humanized anti-CD40 IgG1-LALA antibody tested. It is also preferred that the CD40 antibodies compete with CD40L for binding to the CD40 receptor. Thus, in an epitope competition assay, the antibodies of the present invention compete with CD40L. Such assays are described in Examples 3 and 11, and experimental results with the antibodies of the present invention are depicted in Figures 3 and 16. Thus, in a further preferred embodiment, the antibodies of the present invention inhibit the binding of CD40L to the CD40 receptor. Figure 19 demonstrates that the antibodies of the present invention do not compete with CP-870,893 for binding to CD40.

[0051] The antibodies of the present invention have very high binding activity to the CD40 receptor. Thus, in the cell binding assay described in Example 1, the antibodies of the present invention exhibit binding activity with an EC50 of at most 49.5 ng / ml. Preferably, the EC50 is less than 25 ng / ml, more preferably less than 15 ng / ml, 9 ng / ml, 7 ng / ml, 6 ng / ml, 5 ng / ml, or 4 ng / ml. Most preferably, the EC50 is less than 3 ng / ml in the cell binding assay described in Example 1 and depicted in Figure 1.

[0052] Humanized agonistic anti-CD40 antibodies according to the invention may be characterized by their biochemical affinity for soluble human or cynomolgus CD40 trimeric protein (see Example 13, Figure 18). Antibodies of the invention may exhibit a KD value of 15.7 nM or less for human CD40. Antibodies of the invention may have a KD value of 10.3 nM or less and be cross-reactive with cynomolgus CD40 protein.

[0053] Furthermore, the antibodies of the invention are capable of inducing NF-κB signaling in cells with high potency. An experimental outline (see Example 2) is depicted in Figure 2, showing EC50 binding values ​​ranging from 1127 to 6243 ng / ml. The EC50 values ​​demonstrate the high potency of the antibodies to induce NF-κB signaling.

[0054] It will also be understood that the antibodies of the present invention can bind to cynomolgus monkey CD40. The binding activity of the humanized anti-CD40 IgG1-LALA monoclonal antibody to cynomolgus monkeys (Macaca fascicularis) was determined by binding to recombinant cynomolgus monkey CD40 recombinant protein. This is shown in the ELISA experiments used (see Example 4). The EC50 values ​​shown in Figure 4 indicate strong binding of the antibody.

[0055] Another feature of the antibodies of the present invention is that they can activate human APCs. For example, the antibodies can activate cells selected from the group consisting of dendritic cells (DCs), B cells, monocytes, and myeloid cells. Preferably, the antibodies activate DCs.

[0056] This potent CD40 agonist activity in APC activation is not due to Fcγ receptor-mediated cross-linking of the CD40 protein (as shown in Figures 5, 6, and 7 and described in Example 5). (See the experimental results provided.)

[0057] Thus, in one embodiment, the antibodies of the invention induce the release of IL-12p40 in a dendritic cell maturation assay as described in Example 5. In such an assay, the antibodies of the invention The results of the experiments conducted using the body are shown in Figures 5 to 7.

[0058] In a more preferred embodiment, the antibody of the present invention induces the maturation of antigen-presenting cells measured by IL12p70 release, and the release is at least equivalent to the release induced by stimulation with the CP-870,893-IgG2 antibody, with an EC50 value of 208 ng / ml or less (Figures 12 and 14). Furthermore, the antibody of the present invention induces the maturation of antigen-presenting cells measured by the induction of CD86 by at least 7.5-fold, with an EC50 of 148 ng / ml or less (Figures 11 and 13).

[0059] Preferably, the present antibody induces the release of IL12p40 from monocyte-derived DCs, which is at least equivalent to the release induced by stimulation with the CP-870,893-IgG2 antibody as shown in Figure 6. As described above, this potential to induce DC maturation is not due to signal transduction via Fc receptors. The humanized anti-CD40 IgG1-LALA monoclonal antibody of the present invention potently induces the activation of monocyte-derived dendritic cells in a manner independent of Fcγ receptors (see Example 5 and Figure 6). Figure 6 demonstrates that the level of IL12p40 release induced by the CP-870,893 mutant correlates with the ability of the mutant to bind to Fc receptors (IgG1-LALA < IgG2 < IgG1 < IgG1-V11). Notably, stimulation with the humanized anti-CD40 IgG1-LALA monoclonal antibody of the present invention results in an Fc-independent IL12p40 secretion level that covers and even exceeds the range obtained with the CP-870,893 mutant. Thus, the anti-CD40 antibody of the present invention provides potent agonist activity to primary monocyte-derived dendritic cells without cross-linking mediated by the Fcγ receptor of the CD40 protein.

[0060] Furthermore, the antibodies of the present invention are highly specific in their activation. They do not induce the general release of inflammatory cytokines such as TNF-alpha (see Example 6 and Figure 8).

[0061] Another feature of the antibodies of the present invention is their reduced clearance from the cell surface. CP-870,893 is known to be internalized after binding to the CD40 receptor on cells. This may be caused by cell internalization. CP-87, possibly reflecting a large CD40 receptor sink in patients Clinical trials have shown that 0,893 is rapidly cleared from the circulation of patients, with an estimated half-life of less than 6 hours (Ruter et al. 2010).

[0062] The antibodies of the invention are retained on the cell surface under conditions that allow endocytosis and internalization (see Example 7 and Figure 9), whereas the CP-870,893 mutant is not retained on the cell surface under conditions that allow endocytosis and internalization (see Figure 9).

[0063] It will be appreciated that the antibodies of the present invention have an indirect (immune-mediated) effect on tumor cell death, and thus the antibodies exert an immune cell-mediated indirect cytotoxic effect on tumor cells.

[0064] In one particular embodiment, the antibodies of the invention do not result in the depletion of CD40-expressing immune cells by the mechanisms of ADCC, ADCP, or CDC.

[0065] In summary, therefore, the antibody of the present invention further comprises: (a) does not bind to Fcγ receptors; (b) having a CD40 cell-binding affinity with an EC50 value of about 49.5 ng / ml or less, (c) having a KD value of about 15.7 nM or less; (d) cross-reactive with cynomolgus CD40 with a K value of about 10.3 nM or less; (e) inhibits CD40L by binding to CD40; (f) preventing the synergistic and additive effects of CD40L-mediated functions; (g) IL12p70 release that is at least equivalent to the release induced by stimulation with CP-870,893-IgG2 antibody, with an EC50 value of about 208 ng / ml or less; and / or as measured by at least a 7.5-fold induction of CD86 in dendritic cells with an EC50 value of about 148 ng / ml or less. Inducing maturation of antigen-presenting cells; (h) reduces the level of CD40 on the cell surface to a lesser extent than CP-870,893; It may be characterized by:

[0066] In a preferred embodiment, the antibodies of the present invention are characterized by having at least one, two, three, four, five, six, seven, or eight, or all, of the above properties (a to h).

[0067] The antibodies of the present invention have favorable properties that make them capable of inhibiting the growth of human tumors.

[0068] In a particular embodiment, the antibody of the present invention may comprise a VH region selected from the group of VH regions comprising CDR regions selected from the group consisting of the CDR1H region of SEQ ID NO: 29+n, the CDR2H region of SEQ ID NO: 43+n, and the CDR3H region of SEQ ID NO: 57+n (where n is a number selected from the group consisting of 0 to 13), and a VL region selected from the group of VL regions comprising CDR regions selected from the group consisting of the CDR1L region of SEQ ID NO: 71+m, the CDR2L region of SEQ ID NO: 85+m, and the CDR3L region of SEQ ID NO: 99+m (where m is a number selected from the group consisting of 0 to 13), wherein the CDRs may comprise any one or more amino acid mutations that do not reduce their activity according to the present invention.

[0069] Preferably, the antibody comprises a VH region selected from the group of VH regions comprising CDR regions selected from the group consisting of the CDR1H region of SEQ ID NO: 29+n, the CDR2H region of SEQ ID NO: 43+n, and the CDR3H region of SEQ ID NO: 57+n (where n is a number selected from the group consisting of 0 to 13), and a VL region selected from the group of VL regions comprising CDR regions selected from the group consisting of the CDR1L region of SEQ ID NO: 71+m, the CDR2L region of SEQ ID NO: 85+m, and the CDR3L region of SEQ ID NO: 99+m (where m is a number selected from the group consisting of 0 to 13).

[0070] In a particular embodiment, the antibody of the present invention may comprise a VH region selected from the group of VH regions comprising CDR regions selected from the group consisting of the CDR1H region of SEQ ID NO: 29+n, the CDR2H region of SEQ ID NO: 43+n, and the CDR3H region of SEQ ID NO: 57+n, and a VL region selected from the group of VL regions comprising CDR regions selected from the group consisting of the CDR1L region of SEQ ID NO: 71+n, the CDR2L region of SEQ ID NO: 85+n, and the CDR3L region of SEQ ID NO: 99+n, wherein n is a number selected from the group consisting of 0 to 13, and wherein the CDRs may comprise any one or more amino acid mutations that do not reduce their activity according to the present invention.

[0071] Preferably, the CDRs have at least 91%, preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with their respective SEQ ID NOs.

[0072] In another aspect, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable (VH) region that is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, and more preferably at least 85% identical to a VH region selected from the group consisting of SEQ ID NOs: 1 to 14.

[0073] Preferably, the antibody comprises a heavy chain variable (VH) sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group of VH sequences of SEQ ID NOs: 1 to 14.

[0074] In certain embodiments, VH sequences having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, whereby the antibodies retain the ability of the invention to specifically bind to their respective antigens.

[0075] The present invention also encompasses antibodies comprising a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14.

[0076] Preferably, the heavy chain variable region (VH) sequence is SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0077] The present invention also relates to antibodies comprising a light chain variable (VL) region selected from the group consisting of VL regions of SEQ ID NOs: 15 to 28 that is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, and more preferably at least 85% identical.

[0078] Preferably, the antibody comprises a VL sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group of VL sequences of SEQ ID NOs: 15 to 28.

[0079] In certain embodiments, VL sequences having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, whereby the antibodies retain the ability of the invention to specifically bind to their respective antigens.

[0080] The present invention also encompasses antibodies comprising a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-28.

[0081] Preferably, the light chain variable region (VL) sequence is SEQ ID NO:15, or SEQ ID NO:16, or SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

[0082] In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the VL sequence. In other embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the VH sequence. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in each of the VH or VL sequences. The substitutions, insertions, or deletions may occur in regions outside the CDRs (i.e., in the FRs).

[0083] The present invention also includes affinity matured antibodies, which can be produced by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describe affinity matured antibodies produced by shuffling VH and VL domains. Random mutagenesis of CDR and / or framework residues has been described in: Barbas et al., Proc Nat. Acad. Sci. USA 91: 3809-3813 (1994); Schier et al., J. Med. Chem. Soc. 2004; et al., Gene 169: 147-155 (1995); Yelton et al., J. Immunol. 1 55:1994-2004 (1995); Jackson et al., J. Immunol. 1 54(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992) and WO 2010 / 108127.

[0084] The present invention also encompasses antibodies comprising the VH and VL regions comprising the respective CDR1, CDR2, and CDR3 regions of an antibody selected from the group comprising the antibodies listed in Figure 10 (i.e., including MAB-16-0283, MAB-16-0377, MAB-16-0267, MAB-16-0386, MAB-16-0451, MAB-16-0346, MAB-16-0325, MAB-16-0388, MAB-16-0464, MAB-16-0262, MAB-16-0406, MAB-16-0484, MAB-16-0400, and MAB-16-0489 antibodies).

[0085] The present invention also encompasses antibodies comprising SEQ ID NOs: 1 and 15, or SEQ ID NOs: 2 and 16. Antibodies according to the invention may also comprise SEQ ID NOs: 3 and 17, or SEQ ID NOs: 4 and 18, or SEQ ID NOs: 5 and 19, or SEQ ID NOs: 6 and 20, or SEQ ID NOs: 7 and 21, or SEQ ID NOs: 8 and 22, or SEQ ID NOs: 9 and 23, or SEQ ID NOs: 10 and 24, or SEQ ID NOs: 11 and 25, or SEQ ID NOs: 12 and 26. Alternatively, antibodies of the invention comprise SEQ ID NOs: 13 and 27, or SEQ ID NOs: 14 and 28.

[0086] In another aspect, the antibodies of the invention are for use in treating a patient suffering from cancer.

[0087] The cancers include pancreatic cancer, advanced pancreatic carcinoma, lung cancer, and non-small cell lung cancer (NSCLC). SCL cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, ovarian The cancer may be one or more types of cancer selected from the group including: colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, kidney cancer, Hodgkin's lymphoma, liver cancer, gallbladder cancer, bladder cancer, prostate cancer, thyroid cancer, salivary gland cancer, or uterine cancer.

[0088] In certain embodiments, the cancer is a solid tumor.

[0089] In some embodiments, the cancer can be a CD40-expressing cancer, although this is not required for the effective function of the antibodies of the invention.

[0090] The antibodies of the invention may be used in patients as the sole treatment for cancer or as part of a combination treatment (wherein the further treatment may be a pharmaceutical cytotoxic or cytostatic agent, radiation therapy, targeted therapy, and / or surgery).

[0091] Thus, the patient may also receive one or more additional treatments for cancer, such as medications (cytotoxic or cytostatic agents, targeted therapies, etc.), radiation therapy, and / or surgery.

[0092] Thus, in some variations, the antibodies of the invention are used in the treatment of cancer in combination with cytotoxic or cytostatic agents, radiation therapy, targeted therapy, and / or immunotherapy.

[0093] The antibodies of the invention may also be used in the treatment of patients who have an inadequate response and / or are resistant to cytotoxic or cytostatic agents, radiotherapy, targeted therapy, and / or immunotherapy.

[0094] The radiotherapy may be selected from the group comprising external beam radiation therapy, contact x-ray brachytherapy, brachytherapy, systemic radiation therapy, or intraoperative radiation therapy.

[0095] The cytotoxic or cytostatic anti-cancer agents of the present invention may be from the group including taxanes, anthracyclines, alkylating agents, histone deacetylase inhibitors, topoisomerase inhibitors, kinase inhibitors, nucleotide analogues, peptide antibiotics, and platinum-based agents.

[0096] Preferably, the targeted anti-cancer agent is used in targeted therapy and is selected from one or a combination of the following: anti-EGFR compounds such as cetuximab, gefitinib, erlotinib, lapatinib, panitumumab; anti-HER2 compounds such as trastuzumab, ad-lastuzumab, emtansine, pertuzumab; VEGF-targeting compounds such as bevacizumab, aflibercept, and pegaptanib; and tyrosine kinase inhibitors such as sunitinib, pazopanib, axitinib, vandetanib, cabozantinib, and regorafinib. can be.

[0097] If the patient is receiving immunotherapy, this may be an immune checkpoint inhibitor, and one or more immune checkpoint inhibitors may be used, which may be selected from the group including anti-PD-L1, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40, and / or anti-GITR.

[0098] The antibodies of the present invention may also be used in combination with antibodies that specifically bind to human PD-L1, CTLA-4, LAG-3, TIM-3, CD137, OX40, or GITR, and / or in combination with the drugs nivolumab, pembrolizumab, urelumab, utomilumab, atezolizumab, avelumab, durvalumab, tremelimumab, or ipilimumab.

[0099] In some embodiments, the present invention provides a weekly to monthly dosing regimen. In certain regimens, the antibodies are used in the treatment of cancer.

[0100] One particular advantage of this invention is that, due to the mutations in their Fc region, the present antibodies exhibit less dose- or treatment-limiting toxicity compared to prior art antibodies. The present antibodies cause typical side effects of CD40 antibodies only to a very limited extent, if at all. Such side effects are conditions selected from the group consisting of cytokine release syndrome, thrombosis, cerebral embolism, elevated transaminases, lymphopenia, fatigue, peripheral neuropathy, alopecia, constipation, nausea, and neutropenia. .

[0101] In another aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody of the invention.

[0102] The pharmaceutical composition of the present invention can be used in the treatment of patients suffering from cancer. Such cancer can be a solid tumor. The cancer can be pancreatic cancer, advanced pancreatic cancer, Lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell ) lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, It may also be selected from the group comprising cutaneous or intraocular melanoma, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, kidney cancer, Hodgkin's lymphoma, liver cancer, gallbladder cancer, bladder cancer, prostate cancer, thyroid cancer, salivary gland cancer, or uterine cancer.

[0103] The compositions may also be used in the treatment of cancer in combination with chemotherapy, radiation therapy, targeted therapy, and / or immunotherapy, which may be an immune checkpoint inhibitor.

[0104] Patients treated with the compositions may be poor responders and / or resistant to chemotherapy, radiation therapy, targeted therapy, and / or immunotherapy.

[0105] The pharmaceutical compositions of the invention may be used in the treatment of cancer in combination with one or more cytotoxic, cytostatic, or targeted anti-cancer compounds.

[0106] It may also be used in combination with one or more immune checkpoint inhibitors in the treatment of cancer, wherein the immune checkpoint inhibitors may be selected from the group including anti-PD-L1, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40, and / or anti-GITR.

[0107] The compositions may also be used in combination with antibodies that specifically bind to human PD-L1, CTLA-4, LAG-3, TIM-3, CD137, OX40, or GITR, and / or in combination with the drugs nivolumab, pembrolizumab, urelumab, utomilumab, atezolizumab, avelumab, durvalumab, tremelimumab, or ipilimumab.

[0108] It can also be used in the treatment of cancer in a weekly to monthly dosing regimen.

[0109] It will be appreciated, particularly by patients, that the antibodies and compositions of the invention exhibit any dose- or treatment-limiting toxicity, if any, to a very limited extent, and importantly, to a lesser extent than prior art antibodies and compositions. In another aspect, the invention also relates to methods of treatment comprising administering to an individual in need thereof an effective amount of an antibody of the invention. Such an individual may be a patient suffering from cancer. Thus, the invention also relates to methods of treating cancer, wherein the cancer may be a solid tumor.

[0110] The step of administering to an individual in need thereof may include local administration, such as local administration to a tumor in a patient (eg, intratumoral or peritumoral administration).

[0111] The antibody-based agents of the invention are suitable for use in the treatment of any type of cancer in which activation of CD40 may provide a therapeutic benefit, and methods involving administration of the antibodies are also suitable for the treatment of any type of cancer in which activation of CD40 may provide a therapeutic benefit.

[0112] For example, cancers include prostate cancer, breast cancer, colorectal cancer, and pancreatic cancer. ovarian cancer; lung cancer; cervical cancer; rhabdomyosarcoma; neuroblastoma; multiple myeloma; leukemia; acute lymphoblastic leukemia, melanoma, bladder cancer, and glioblastoma. stomach.

[0113] It will further be appreciated that the therapeutic methods of the invention may involve administration of an antibody-based agent of the invention to a patient alone or as part of a combination therapy (which further therapy may be a pharmaceutical cytotoxic or cytostatic agent, radiation therapy, targeted therapy, and / or surgery).

[0114] Indeed, all the characteristics and preferred properties of the antibodies of the invention described above are also reflected and included in the methods of treatment and uses of the antibodies according to the invention. [Example]

[0115] The following examples, together with the figures and tables, are used to illustrate the invention.

[0116] Example 1: Cell binding of anti-CD40 antibodies To measure the binding potency of the humanized anti-CD40 IgG1-LALA monoclonal antibody to cell-expressed CD40, HEK-Blue-CD40L™ (Invivogen) cells were seeded at a cell density of 1,000 cells / well in 25 μl of DMEM containing 10% FBS in a 384-well plate treated with cell culture medium and clear-bottomed. Antibody was added in 5 μl of medium to achieve final concentrations ranging from 1.25 μg / ml to 0.01 ng / ml. After 24 hours, cells were washed three times with 25 μl of wash buffer (PBS, 0.05% Tween) before adding Alexa Fluor-488-conjugated goat anti-human IgG (Jackson Laboratories) at a concentration of 0.8 μg / ml in 20 μl of medium. After 4 hours, 5 μl of Hoechst dye in medium was added to a final concentration of 5 μg / ml. Fluorescent cell binding signals were measured using a Cellinsight automated high-content imager (Thermo Fisher Scientific). Fit curves and EC50 calculations were obtained using Excel (Microsoft) and Xfit (IDBS). Figure 1 shows an overview of EC50 binding values ​​ranging from 3 to 49.5 ng / ml.

[0117] Example 2: Induction of intracellular NF-κB signaling by anti-CD40 agonistic antibodies The agonist activity of the humanized anti-CD40 IgG1-LALA monoclonal antibody was tested by stimulating HEK-Blue-CD40L™ (Invivogen) cells carrying an NF-κB-inducible secreted fetal alkaline phosphatase (SEAP) gene construct. 25,000 cells / well were seeded in 20 μl of DMEM containing 10% FBS in a 384-well plate with clear cell culture media and cultured overnight. Antibodies were then added in a volume of 5 μl of medium to final concentrations ranging from 20 to 0.013 μg / ml. After 6 hours of incubation at 37°C and 5% CO2, 5 μl of medium supernatant from each well was transferred to a white, clear-bottom 384-well plate containing 20 μl of 2xQUANT-Blue™ reagent (Invivogen). After 1 hour of incubation at 37°C and 5% CO2, the optical density at a wavelength of 620 nm, which reflects the activation of NF-κB-dependent phosphatase secretion, was measured. Excel (Microsoft) and XLfit (IDBS) were used to generate fitted curves and calculate EC50 values. The EC50 values ​​in Figure 2 indicate the potency of anti-CD40 antibodies to induce NF-κB signaling in the HEK-BlueCD40™ cell line.

[0118] Example 3: Competition with CD40L binding The competition of humanized anti-CD40 IgG1-LALA monoclonal antibody for binding to CD40 with CD40L was tested using an ELISA assay. The surface of a 384-well Nunc™ MaxiSorp™ plate was coated with CD40L at a concentration of 1 μg / ml in PBS in a volume of 25 μl for 1 hour at room temperature. The antibody at a concentration of 5 μg / ml was mixed with recombinant CD40 protein at a concentration of 1.7 μg / ml in a total volume of 40 μl for 1 hour at room temperature. The plates were preincubated in ELISA buffer (PBS, 0.5% BSA, 0.05% Tween) for 1.5 hours at room temperature. The Nunc™ MaxiSorp™ plates were washed three times with wash buffer (PBS, 0.1% Tween) and blocked with PBS, 2% BSA, 0.05% Tween for 1 hour at room temperature. After washing three times with wash buffer, 25 μl of antibody-CD40 complex was added to the wells of the Nunc™ MaxiSorp™ plate and incubated for 1 hour at room temperature. After washing three times with wash buffer, the wells were incubated with 25 μl of a 1:2000 dilution of anti-human peroxidase-conjugated, species-specific F(ab)2 fragment from goat (AbD Serotec) in ELISA buffer for 1 hour at room temperature. The wells were washed six times with wash buffer, and 30 μl / well of TMB substrate solution (Invitrogen) was added. After 10 minutes at room temperature, 30 μl of stop solution (1 M HCl) was added per well, and absorbance was measured at wavelengths of 450 and 620 nm using a Tecan M1000 microtiter plate reader. The ELISA signal of samples incubated with CP-870,893 indicated a lack of competition with CD40L, whereas the binding of the humanized anti-CD40 IgG1-LALA monoclonal antibody of the present invention to CD40 competed with CD40L (see Figure 3).

[0119] Example 4: Cynomolgus monkey CD40 binding activity The binding of the humanized anti-CD40 IgG1-LALA monoclonal antibody to cynomolgus monkey CD40 protein was tested by biochemical ELISA. Recombinant cynomolgus monkey CD40 protein (Acro Biosystems) was incubated in a 384-well Nunc™ MaxiSorp™ plate at a concentration of 0.5 μg / ml in PBS for 1 hour at room temperature. After washing three times with wash buffer (PBS, 0.1% Tween), the plate was blocked with PBS, 2% BSA, 0.05% Tween for 1 hour at room temperature. The plate was washed again three times with wash buffer and incubated with antibody at concentrations ranging from 500 to 0.03 ng / ml in PBS, 0.5% BSA, 0.05% Tween for 1 hour at room temperature. After washing three times with wash buffer, the wells were incubated with 12.5 μl of a 1:3000 dilution of anti-human peroxidase-conjugated, species-specific F(ab)2 fragment from goat (AbD Serotec) in ELISA buffer for 1 hour at room temperature. The wells were washed six times with wash buffer, and 15 μl / well of TMB substrate solution (Invitrogen) was added. After 10 minutes at room temperature, 15 μl of stop solution (1 M HCl) was added per well, and absorbance was measured at wavelengths of 450 nm and 620 nm using a Tecan M1000 microtiter plate reader. Fitting curves and EC50 calculations were obtained using Excel (Microsoft) and XLfit (IDBS). As seen in Figure 4, many antibodies bound to cynomolgus monkey CD40 with EC50 values ​​between 8 and 31.8 ng / ml.

[0120] Example 5: Induction of dendritic cell maturation a. Monocyte-derived dendritic cells were generated and the ability of the humanized anti-CD40 IgG1-LALA monoclonal antibody to stimulate dendritic cell maturation, as measured by IL12p40 cytokine secretion, was tested. Human buffy coat preparations from various donors were used to differentiate dendritic cells from monocytes in vitro. Buffy coats received from the Bavarian Red Cross were diluted 1:4 with DPBS and layered on a Ficoll-Paque (GE Healthcare) density gradient. After centrifugation, the interphase peripheral blood mononuclear cells (PBMCs) were washed three times with DPBS, and monocytes were isolated using magnetic CD14 microbeads (Miltenyi Biotec) according to the manufacturer's instructions. 1.2 × 10 cells were cultured in a T-175 cell culture flask. 6 Monocytes were cultured at a cell density of 1000 cells / ml in RPMI-1640 containing 10% FCS, 1x Pen / Strep (penicillin / streptomycin), 1x L-glutamine, 50 ng / ml recombinant human GM-CSF (R&D Systems), and 10 ng / ml recombinant human IL-4 (R&D Systems). Every 48 hours, 90% of the medium was replaced with fresh cytokine-containing medium. In vitro differentiated immature dendritic cells (iDCs) were harvested on day 5 and cultured at 100 μl in the same medium. 6 cells / m Cells were seeded into cell culture 96-well plates at a cell density of 1.

[0121] b. In one experiment, iDCs were stimulated by adding anti-CD40 antibody at a concentration of 5 μg / ml. Forty-eight hours after stimulation, secreted IL12p40 cytokine was quantified in the culture supernatant using a commercially available ELISA kit (R&D Systems) according to the manufacturer's instructions. Figure 5 shows that the humanized anti-CD40 IgG1-LALA monoclonal antibody stimulated IL12p40 release by monocyte-derived dendritic cells to varying degrees, ranging from less than 1 ng / ml to more than 24 ng / ml, whereas the control IgG1-LALA antibody, which does not bind CD40, did not stimulate detectable levels of IL12p40.

[0122] c. The humanized anti-CD40 IgG1-LALA monoclonal antibody of the present invention was unable to bind to the Fcγ receptors expressed on monocyte-derived dendritic cells. To compare these antibodies with antibodies having various Fcγ receptor binding activities, we constructed reference CP-870,893 anti-CD40 antibodies containing human IgG1, IgG1-LALA, IgG2, and IgG-V11 Fc portions, and stimulated monocyte-derived dendritic cells with various concentrations of these antibodies. The IgG1-V11 form has four mutations (G237D, H268D, P271G, A330R) in the heavy chain Fc portion. These mutations are said to selectively increase the affinity for the Fc receptor RIIB (Mimoto et al. 2013). Figure 6 demonstrates that the CP-870,893 variant stimulates the release of IL12p40 in an Fc-dependent manner (IgG1-LALA < IgG2 < IgG1 < IgG1-V11). Notably, stimulation with the humanized anti-CD40 IgG1-LALA monoclonal antibody of the present invention resulted in Fc-independent IL12p40 secretion at levels that covered and even exceeded the range brought about by the CP-870,893 variant. Thereby, the anti-CD40 antibody of the present invention provides potent agonist activity to primary, monocyte-derived dendritic cells without cross-linking of the CD40 protein mediated by Fcγ receptors.

[0123] d. In other experiments, the potency of the humanized anti-CD40 IgG1-LALA monoclonal antibody to induce IL12p40 secretion by monocyte-derived dendritic cells was measured by stimulating with antibodies at concentrations in the range of 0.005 - 10 μg / ml. Figure 7 shows that the EC50 values of the various antibodies are in the range between 380 - 743 ng / ml.

[0124] Example 6: TNF-alpha Release in a High-Density PBMC Assay To determine whether the humanized anti-CD40 IgG1-LALA monoclonal antibody induces a general release of cytokines in blood cells, PBMCs were stimulated with the antibody according to the protocol of Romer et al. 2011. PBMCs were isolated as described above and placed in a T175 cell culture flask at 1 × 10 7The cells were cultured in RPMI-1640 containing 10% human AB serum and 1x non-essential amino acids (NEAA) at a cell density of 1x10 cells / ml. Cells were harvested two days later and plated in triplicate in 96-well cell culture plates at a density of 1x10 cells / ml. 6 Cells were seeded at a density of 1000 cells / ml. Antibodies were added at a concentration of 10 μg / ml and incubated with PBMCs for 3 days at 37°C, 5% CO2, and 95% humidity. OKT-3 antibody (Abcam) was included as a positive control in the experiment. TNF-alpha released into the cell culture supernatant was quantified using a commercially available human TNF-alpha ELISA kit (R&D Systems) according to the manufacturer's instructions. Figure 8 shows the results of the quantification of TNF-alpha in contrast to the OKT-3 antibody. 1 shows that the anti-CD40 antibodies of the present invention and the IgG1-LALA control antibody, which does not bind to CD40, did not stimulate significant release of TNF-alpha by PBMCs.

[0125] Example 7: Cellular pulse-chase assay Pulse-chain immunoprecipitation using HEK-Blue-CD40L™ cells (Invivogen) The kinetics of cell binding and internalization of anti-CD40 antibodies were analyzed in a ELISA. Two black 384-well plates with clear bottoms were seeded at 2000 cells / well in DMEM medium containing 10% FCS. After overnight culture, the anti-CD40 antibodies of the present invention and the CP-870,893 Fc variant antibody described above were added to one plate at a concentration of 0.8 μg / ml and incubated for 15 minutes at 37°C and 5% CO2. Both plates were then washed three times with cell wash buffer (PBS, 0.05% Tween) and incubated in culture medium for 1 hour. During the final 15 minutes, antibodies were added to the second plate at a concentration of 0.8 μg / ml. Both plates were then washed three times with cell wash buffer, placed on ice, and incubated with 0.8 μg / ml of a secondary anti-human Alexa Fluor-488-conjugated antibody (Jackson Laboratories) and 5 μg / ml of Hoechst stain (Invitrogen) on ice for 30 minutes. Cell surface fluorescent signals were quantified using a CellInsight high-content imager (Thermo Fisher Scientific). Figure 9 shows that the CP-870,893 mutant antibody strongly reduced the surface signal after 1 hour of incubation under conditions permissive for internalization. In contrast, many of the anti-CD40 antibodies of the present invention only slightly reduced the cell surface signal when incubated under the same conditions, suggesting a limited rate of internalization.

[0126] Example 8: Correlation between gene reporter induction and dendritic cell maturation activity of humanized anti-CD antibodies a. Cellular gene reporter (HEK-Blue-CD40L™) and dendritic cell (DC) maturation assays were performed as described in Examples 2 and 5, respectively. Antibodies were used at 5 μl / ml in the DC assay and at concentrations ranging from 13 to 20,000 ng / ml in the HEK-Blue-CD40L™ gene reporter assay. Figure 10 compares the maximal induction observed in the gene reporter assay with IL12p40 cytokine release by DCs after stimulation with 5 μl / ml of antibody. While all 88 humanized anti-CD40 IgG1-LALA antibodies induce gene reporter expression to a similar extent, several antibodies show significantly greater stimulation of IL12p40 release by DCs. HEK-Blue-CD40L™ cells do not express Fcγ receptors. The assay captures basal, Fcγ receptor-independent agonistic activity of CD40 antibodies. DCs express Fcγ receptors, and the agonistic activity of anti-CD40 antibodies such as CP-870 and CP-893 depends on Fcγ receptor binding (Example 5). Nevertheless, although many of the 88 IgG1-LALA antibodies lack strong DC activation, a small subset can induce very strong DC activation without Fcγ receptor-mediated cross-linking. Thus, highly agonistic anti-CD40 antibodies whose activity in primary dendritic cells is independent of Fcγ receptor cross-linking are rare, and their identification requires the screening of numerous candidate antibodies with basal agonistic activity.

[0127] Example 9: Stimulation of costimulatory receptors and cytokine release in dendritic cells by agonistic anti-CD40 antibodies a. To test the activity of the humanized agonistic anti-CD40 IgG1-LALA antibody in stimulating costimulatory receptor expression and inflammatory cytokine release by DCs, immature monocyte-derived DCs (iDCs) were generated from three separate donors as described in Example 5. iDCs were treated for 48 hours with either 2 μg / ml of antibody or 20 μg / ml of CD40L (R&D Systems 6245-CL-050). Stimulated, mature DCs were harvested and stained with fluorescently labeled antibodies against HLA-DR, CD86, CD80, CD83, CD54, and CD95 (all from Miltenyi Biotec) and analyzed by flow cytometry on a BD FACSVerse instrument. Figure 11 shows receptor stimulation expressed as fold induction relative to isotype control antibody treatment. The data demonstrate strong induction of costimulatory receptors, particularly CD86. CP-870,893 is a compound similar to MAB-16-0262, MAB-16-0451, MAB-16-0464 and M Compared to the AB-16-0406 antibody, it exhibits overall lower activity. A further decrease in activity was observed when the CP-870,893 variant containing the IgG1-LALA constant region was used, confirming the Fcγ receptor binding dependency of this antibody.

[0128] b. Figure 12 shows cytokine measurements in DC culture supernatants using the BD Human Inflammatory Cytometric Bead Array Kit (BD #551811) according to the manufacturer's instructions. The MAB-16-0262, MAB-16-0451, MAB-16-0464, and MAB-16-0406 antibodies of the invention released very high levels of IL-12p40 and IL-12p70, while producing and secreting other cytokines (e.g., TNF-α, IL-1β, IL-10, and IL-6) to a much lower extent. The release of IL-12p40 and IL-12p70 from DCs treated with the CP-870,893 IgG2 and IgG1 variants was significantly lower compared to the release observed with the antibodies of the invention. In a similar experiment, in vitro-differentiated iDCs from three separate donors were treated for 48 hours with agonistic anti-CD40 antibodies at concentrations ranging from 10,000 to 5 ng / ml. Receptor expression and cytokine release were analyzed as described above. Excel (Microsoft) and Xfit (IDBS) were used to generate fitted curves and calculate EC50 values. Data shown in Figures 13 and 14 demonstrate exemplary dose-dependent effects observed in one donor. Results from two additional donors demonstrate quantitatively similar effects. In summary, the potency of the humanized anti-CD40 antibodies of the present invention, as measured by EC50 values, is similar to that of CP-870,893, while the maximal induction effects, particularly with regard to IL-12 cytokine release and coreceptor expression, are significantly greater than those of CP-870,893.

[0129] Example 10: Stimulation of costimulatory receptors on B cells by agonistic anti-CD40 antibodies Stimulation of costimulatory receptors by humanized anti-CD40 IgG1-LALA antibody was also tested on B cells. PBMCs from three different donors were isolated from human buffy coats by Ficoll density gradient centrifugation, and untouched B cells were purified by negative magnetic enrichment using the B Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer's instructions. 2×105 B cells were stimulated for 48 hours with antibody concentrations ranging from 500 to 0.2 ng / ml in 100 μl of RPMI-1640 + 10% human AB serum. Stimulated B cells were harvested, stained with fluorophore-conjugated antibodies against HLA-DR, CD86, and CD80 (all from Miltenyi Biotec), and analyzed by flow cytometry on a BD FACSVerse instrument. Figure 15 shows the dose-dependent stimulation of receptor expression in single donor B cells as fold induction relative to isotype control antibody treatment. Fit curves and EC50 calculations were obtained using Excel (Microsoft) and Xfit (IDBS). The results indicate that the antibodies of the present invention also stimulate costimulatory receptors on B cells, although the level of upregulation was lower than that observed in DCs.

[0130] Example 11: Competition between CD40L and anti-CD40 antibodies for binding to CD40 on cells a. Binding of CD40 antibodies of the present invention to HEK-Blue-CD40L™ cells in the presence of CD40L was tested to verify whether the antibodies bind to the CD40L-binding site on cell-surface CD40. HEK-Blue-CD40L™ cells were preincubated with antibodies at their EC90 binding concentrations at 40°C for 30 minutes. CD40L containing a mouse IgG2a Fc tag (AB Biosciences) was added at concentrations ranging from 10,000 to 9.8 ng / ml, and the cells were incubated at 4°C for 60 minutes. Anti-CD40 antibodies and CD40L bound to cell-expressed CD40 were detected using secondary DyLight405-conjugated anti-mouse IgG and Alexa-Fluor-488-conjugated anti-human IgG (Jackson Laboratories) and analyzed using a FACSVerse instrument (BD). Figure 16A shows that, with the exception of CP-870,893, the anti-CD40 antibodies showed stable binding across multiple concentrations, with C The antibody binding signal of P-870,893 was slightly reduced at higher CD40L concentrations. CD40L bound to cells in a dose-dependent manner, and P-870,893 did not significantly interfere with CD40L binding (Fig. 16B). In contrast, the antibodies of the present invention strongly prevented CD40L binding to CD40 expressed on cells, suggesting that these antibodies bind to the CD40L-binding portion of CD40 and inhibit CD40L binding to CD40 (Fig. 16B).

[0131] Example 12: Induction of FasR (CD95) death receptor expression by agonistic CD40 antibodies in combination with CD40L To test for the interference of CD40L with anti-CD40 antibody-mediated effects on cells, Ramos cells were treated with CD40L alone or in combination with an agonistic anti-CD40 antibody. 1.25 x 10 cells were plated in RPMI containing 10% FCS in a 96-well plate. 6 Ramos cells were seeded at a cell density of 1000 cells / ml. Antibodies were added to wells at a concentration of 10 μg / ml, and the plates were incubated for 10 minutes at 37°C, 5% CO2, and 95% humidity. CD40L (R&D Systems) was then added to some wells to a final concentration of 10 μg / ml, and the plates were incubated overnight at 37°C, 5% CO2, and 95% humidity. Cells were washed with DPBS and stained with an FITC-labeled antibody against CD95 (Miltenyi Biotec). Figure 17 shows that CD95 induction by CP-870,893 is strongly enhanced by the addition of CD40L, while the effect of CD40L is reduced by co-treatment with all of the anti-CD40 antibodies of the present invention tested. The data indicate that agonistic anti-CD40 antibodies of the present invention that bind to the CD40L binding site on CD40 prevent the synergistic and additive effects of CD40L, thereby enabling controlled and safe pharmacology.

[0132] Example 13: Affinity of humanized, agonistic anti-CD40 IgG1-LALA antibodies a. The biochemical affinity of the antibodies of the present invention was measured by surface plasmon resonance measurements. The antibodies were reversibly immobilized on the surface of a CM5 sensor chip via an anti-human Fc antibody. The interaction kinetics between the immobilized antibodies and soluble human or cynomolgus monkey CD40 monomer protein (Acro Biosystems) was analyzed using a Biacore T200 SPR instrument. The kinetic data were measured using a Langmuir 1:1 binding model. Figure 18 shows that the MAB-16-0451 and MAB-16-0464 antibodies have K values ​​of 1.2 and 2.6. D whereas MAB-16-0262 and CP-870,893 have K values ​​of 15.7 and 8.9, respectively. D This demonstrates that the K value is obtained using cynomolgus monkey CD40 protein. D The values ​​demonstrated similar affinities.

[0133] Example 14: Competitive binding of anti-CD40 antibodies to CD40 a. A competitive binding ELISA was performed to test whether the humanized, agonistic anti-CD40 antibodies of the present invention bind to overlapping regions on the CD40 molecule. A 384-well Maxisorp plate was coated with antibody at a concentration of 625 ng / ml in PBS for 60 minutes, followed by a 70-minute blocking step with PBS, 2% BSA, and 0.05% Tween. All antibodies were incubated individually in tubes at a concentration of 10 μg / ml in ELISA buffer (PBS, 0.5% BSA, and 0.05% Tween) with 330 ng / ml HIS-tagged CD40 recombinant protein (Acro Biosystems) and 4 μg / ml peroxidase-conjugated anti-HIS detection antibody (Sigma-Aldrich) for 60 minutes. The plate was washed three times with PBS and 0.1% Tween before the antibody / HIS-CD40 / anti-HIS peroxidase mixture was added to the plate wells. The plate was incubated for 60 minutes. The wells were washed six times with PBS, 0.1% Tween, and 15 μl / well of TMB substrate solution (Invitrogen) was added. The reaction was stopped with 15 μl / well of stop solution (1 M HCl), and absorbance at wavelengths of 450 and 620 nm was measured using a Tecan M1000 microplate reader. Figure 19 shows the results of the present invention. These results demonstrate that none of the antibodies compete with CP-870,903 for binding to CD40. However, each antibody competes with any other antibody of the invention, demonstrating that these antibodies bind to the same region of CD40. Importantly, the agonistic activity of the antibodies of the invention covers a broad range (Examples 9 and 10), indicating that the paratope of an agonistic anti-CD40 antibody primarily determines the agonistic activity of the anti-CD40 antibody.

[0134] Example 15: Antibody-mediated effector functions of IgG1-LALA anti-CD40 antibodies To test the efficacy of the LALA mutation in the constant portion of an IgG1 antibody in reducing antibody-mediated effector function (e.g., ADCC), a Jurkat effector cell reporter cell line-based assay (Promega ADCC Bioassay, #G701A) was applied according to the manufacturer's instructions using HEK-Blue-CD40L™ cells as target cells. Five thousand HEK-Blue-CD40L™ cells were seeded per well in 25 μl of DMEM + 10% FCS in a white, flat-bottomed 384-well assay plate and incubated at 37°C, 5% CO2 for 20 hours. The medium was replaced with 8 μl of RPMI medium containing 4% low IgG FCS before adding 4000 effector cells per well in 8 μl of the same medium. Finally, CP-870,903 anti-CD40 antibody containing either the IgG1 or IgG1-LALA Fc portion was added in 8 μl of medium at concentrations ranging from 10,000 to 0.002 ng / ml. The plates were incubated at 37°C and 5% CO2 for 6 hours. Luciferase activity in effector cells was measured using BioGlo Luciferase Assay Reagent (Promega) according to the manufacturer's instructions. Luminescence intensity was read using a Tecan M1000 microplate reader. Fold induction was calculated using the formula RLU (antibody treatment minus background) / RLU (vehicle minus background). Fitting curves were obtained using Excel (Microsoft) and Xfit (IDBS). Figure 20 demonstrates that the LALA mutation in IgG1 abolishes Fc receptor-mediated signaling in effector cells.

[0135] Example 16: Safety of agonistic anti-CD40 antibody therapy in a humanized mouse model a. To evaluate safety, we used a stem cell-humanized mouse model. Nod / Scid / gamma(c)(null)FcRg- / - mice lack mouse Fc-activating receptors. Therefore, in this model, mouse Fc receptor binding does not impair Fc receptor binding of therapeutic antibodies by human immune cells. Mice were sublethally irradiated with 1.4 Gy within the first 24 hours after birth. Four to six hours later, mice were intravenously transplanted with 20,000 to 50,000 human hematopoietic stem cells isolated from umbilical cord blood. Twelve weeks after transplantation, the presence of human immune cells was verified by flow cytometry of peripheral blood cells. Successfully humanized mice were intravenously injected with 3 μg / g of CP-870,893, MAB-16-0451, or an isotype control antibody. Body weight and temperature were measured before treatment and at various time points after antibody injection (Figure 21). The data showed that body temperature was significantly reduced in three of six mice treated with CP-870,893, and these mice had to be sacrificed due to severe impairment of body condition. In contrast, mice treated with MAB-16-0451 showed no significant effect on body temperature or any other obvious signs of impaired body condition. This suggests that the highly agonistic anti-CD40-IgG1-LALA antibody, which lacks Fc receptor binding activity, can be applied therapeutically without obvious signs of toxicity, whereas the toxic effects of the less potent agonistic CP-870,893-IgG2 antibody were demonstrated in vivo in this model.

Claims

1. An agonistic monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to the human CD40 receptor and is capable of inducing CD40 signaling independent of Fcγ-mediated CD40 receptor cross-linking, a) the antibody comprises a heavy chain variable (VH) region represented by an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1, and comprises CDR regions (CDR1H region of SEQ ID NO: 29, CDR2H region of SEQ ID NO: 43, and CDR3H region of SEQ ID NO: 57); and b) The antibody comprises a light chain variable (VL) region represented by an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 15, and comprises CDR regions (CDR1L region of SEQ ID NO: 71, CDR2L region of SEQ ID NO: 85, and CDR3L region of SEQ ID NO: 99).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized IgG1LALA antibody or antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof according to claim 2, comprising amino acid substitutions at least at L234A and L235A in the human IgG1 Fc region.

4. An antibody or antigen-binding fragment thereof described in claim 1, comprising amino acid substitutions at least at S228P and L235E in the human IgG4 Fc region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof binds to an epitope that overlaps with the binding site of CD40L.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof activates human APCs.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof activates cells selected from the group including dendritic cells (DCs), B cells, monocytes, myeloid cells, and tumor cells.

8. The antibody of claim 1 , wherein the antibody or antigen-binding fragment thereof has an indirect immune cell-mediated cytotoxic effect on tumor cells.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, (a) does not bind to Fcγ receptors; (b) has a CD40 cell-binding affinity with an EC50 value of 49.5 ng / ml or less; (c) has a K value of 15.7 nM or less; (d) cross-reactive with cynomolgus CD40 with a K value of 10.3 nM or less; (e) inhibiting CD40L by binding to CD40; (f) preventing the synergistic and additive effects of CD40L-mediated functions; (g) IL12p70 release, as measured by IL12p70 release, with an EC50 value of 208 ng / ml or less; and / or as measured by at least a 7.5-fold induction of CD86 in dendritic cells with an EC50 value of 148 ng / ml or less. Inducing maturation of antigen-presenting cells; and / or (h) reducing the level of CD40 on the cell surface to less than 50%; An antibody or antigen-binding fragment thereof further having at least one property of

10. 10. The antibody or antigen-binding fragment thereof of claim 1 for use in treating a patient suffering from cancer.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the cancer is a solid tumor.

12. 12. The antibody or antigen-binding fragment thereof of claim 10 or 11, wherein the cancer is selected from the group including pancreas cancer, advanced pancreatic carcinoma lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, kidney cancer, Hodgkin's lymphoma, liver cancer, gallbladder cancer, bladder cancer, prostate cancer, thyroid cancer, salivary gland cancer, or uterine cancer.

13. 13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is used in the treatment of cancer in combination with cytotoxic or cytostatic agents, radiation therapy, targeted therapy, immunotherapy, or surgery.

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 13.

Images