CD40 Agonistic Monoclonal Antibodies, Their Uses, and Pharmaceutical Composition

Abstract

The present invention relates to humanized monoclonal antibodies or antigen-binding fragments thereof that specifically bind to the human CD40 receptor and induce CD40 signaling independent of Fc-mediated CD40 receptor cross-linking. The antibodies of the present invention bind to a CD40 epitope that overlaps with the CD40 ligand epitope and can activate human APCs. The present invention also provides compositions comprising said antibodies and uses for the antibodies and compositions in the treatment of patients suffering from cancer.

Description

1 / 49 CD40 Agonistic Monoclonal Antibodies, Their Uses, and Pharmaceutical Composition Field of invention

[0001] The present invention relates to humanized monoclonal agonist antibodies or their antigen-binding fragments that specifically bind to the human CD40 receptor and are capable of inducing CD40 signaling independent of Fcy-mediated CD40 receptor cross-linking. The invention also relates to the uses of said antibodies and pharmaceutical compositions comprising them. Background

[0002] The recent success of immunotherapy against cancer has revived the hypothesis that the immune system can control many, if not most, cancers, in some cases producing durable responses in a way that is not seen with many small molecule drugs. CD40 agonist monoclonal antibodies (mAbs) offer a new therapeutic option that has the potential to generate anticancer immunity through several 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 in a variety of tumors.

[0004] The natural ligand for CD40 is CD154, which is primarily expressed on the surface of activated T lymphocytes and provides an important component of T cell helper immune responses: Signaling via CD40 on APCs largely mediates the ability of helper T cells to license APCs. CD40 binding to DCs, for example, induces increased surface expression of co-stimulatory molecules and MHC, production of pro-inflammatory cytokines and Petition 870260063188, dated 06 / 26 / 2026, page 5 / 118 2 / 49 increased T cell activation. CD40 binding to resting B cells enhances antigen presentation function and proliferation.

[0005] The consequences of CD40 signaling are multifaceted and depend on the type of cell expressing CD40 and the microenvironment in which the CD40 signal is delivered. Like some other members of the TNF receptor family, CD40 signaling is mediated by adapter molecules, not by the inherent signal transduction activity of the CD40 cytoplasmic tail. Downstream kinases are activated when the receptor is assembled, a multicomponent signaling complex is translocated from CD40 to the cytosol, and several well-characterized signal transduction pathways are activated.

[0006] Human anti-CD40 antibodies are known in the prior art. The respective antagonist antibodies may be silent Fc variants, showing reduced Fcy-mediated CD40 receptor cross-linking. The respective mutations of the human IgG1 FC region are described in, for example, US 2018 / 0118843.

[0007] In recently designed immunomodulatory approaches, CD40-targeted agonist monoclonal antibodies (mAbs) are used to enhance the immune system's ability to recognize and destroy cancer cells. Respective preclinical studies have shown that agonistic CD40 mAbs can activate APCs and promote antitumor T-cell responses and promote 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 blocking negative checkpoint molecules such as CTLA-4 or PD-1.

[0008] CP-870.893 is the first fully human IgG2 mAb Petition 870260063188, dated 06 / 26 / 2026, page 6 / 118 3 / 49 which operates as a potent and selective CD40 agonist. Interestingly, CP-870,893 binding does not compete with CD154 binding to CD40. In preclinical studies, CP-870,893 has been shown to mediate both immune system-dependent and immune system-independent effects on tumor cell survival. In the first human study, promising antitumor activity was observed, especially in melanoma patients. Pharmacodynamically, administration of CP-870,893 leads to a transient decrease in peripheral blood B cells and upregulation of activation markers in APCs.

[0009] Thus, agonistic CD40 mAbs represent a promising strategy for new cancer therapies. However, concerns have also been raised regarding their potential cytotoxic side effects.Monoclonal CD40 agonist antibodies are expected to trigger cytokine release syndromes, autoimmune reactions, thromboembolic syndromes (due to CD40 expression by platelets and endothelial cells), hyperimmune stimulation leading to activation-induced cell death or tolerance, and tumor angiogenesis. These effects may cause undesirable toxicity or promote tumor growth. Mechanistically, the ability of agonistic CD40 and other TNF family receptors to interact with Fcy receptors has been associated with the occurrence of toxicities in animal studies (Li & Ravetch 2012, Xu et al. 2003, Byrne et al. 2016).

[0010] For the strongest agonist tested, CP-870,893, the most common side effect reported is cytokine release syndrome, manifesting as chills, fever, stiffness, and other symptoms shortly after infusion. In addition, several cases of thromboembolic events were observed with CP-870,893. With dacetuzumab, non-infectious inflammatory eye disorders were observed.

[0011] Therefore, it is necessary to predict agonistic CD40 mABs, which Petition 870260063188, dated 06 / 26 / 2026, page 7 / 118 4 / 49 exhibit reduced cellular toxicity, leading to fewer clinical side effects, while maintaining their potency and clinical efficacy. The agonistic CD40 mAbs of the present invention can satisfy this need, allowing the exploitation of the full immunomodulatory potential of agonistic CD40 antibodies. Summary of the invention

[0012] The present invention relates to monoclonal antibodies or an antigen-binding fragment thereof that specifically bind to the human CD40 receptor and induce CD40 signaling independent of Fcy-mediated CD40 receptor cross-linking. More specifically, the antibodies of the present invention bind to a CD40 epitope that overlaps with the CD40 ligand epitope and are capable of activating human APCs. The present invention also provides compositions comprising said antibodies and uses for the antibodies and compositions in the treatment of a condition or disease in which stimulation of the immune system is desired, for example, in the treatment of patients suffering from cancer. Definitions

[0013] The term antibody encompasses various forms of antibody structures, including, but not limited to, whole antibodies and antibody fragments, provided they exhibit the properties in accordance with the invention.

[0014] “An antibody fragment refers to a molecule, with the exception of an intact antibody, comprising a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Petition 870260063188, dated 06 / 26 / 2026, p. 8 / 118 5 / 49

[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 antibodies for which heavy and light chains are humanized as a result of antibody engineering. A humanized chain is typically a chain in which the amino acid sequence of the V region has been altered so that, when analyzed as a whole, it is closer in homology to a human germline sequence than to the germline sequence of the species of origin. The assessment of humanization is based on the resulting amino acid sequence and not on the methodology itself.

[0017] The terms specific binding against the target or anti-target antibody, as used herein, refer to the binding of the antibody to the respective antigen (target) or antigen-expressing cell, measured by ELISA, wherein said ELISA preferably comprises coating the respective antigen onto a solid support, adding said antibody under conditions to permit the formation of an immune complex with the respective antigen or protein, detecting said immune complex by measuring Optical Density (OD) values ​​using a secondary antibody binding to an antibody according to the invention and using a peroxidase-mediated color development.

[0018] The term antigen according to the invention refers to the antigen used for immunization or a protein comprising said antigen as part of its protein sequence. For example, for immunization, a fragment of the extracellular domain of a protein (e.g., the first 20 amino acids) may be used, and for detection / assay and the like, the extracellular domain of the protein or the full-length protein may be used. Petition 870260063188, dated 06 / 26 / 2026, page 9 / 118 6 / 49

[0019] The term specific or specifically recognized binding in this document means that an antibody exhibits appreciable affinity for an antigen and, preferably, does not exhibit significant cross-reactivity.

[0020] An antibody that does not exhibit significant cross-reactivity is one that does not bind appreciably to another undesirable protein. Specific binding can be determined according to any method recognized in the art for determining such binding, for example, by competitive binding assays such as ELISA.

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

[0022] The variable region (or domain) of an antibody according to the invention (variable region of a light chain (VL), variable region of a heavy chain (VH)), as used herein, denotes each of the pairs of light and heavy chain regions directly involved in antibody binding to antigen. The variable regions of the light and heavy chains have the same general structure and each region comprises four structural regions (FR) whose sequences are largely conserved, connected by three complementarity-determining regions, CDRs.

[0023] The term antigen-binding portion of an antibody, when used herein, refers to the amino acid residues of an antibody that are responsible for binding to the antigen. The antigen-binding portion of an antibody preferably comprises amino acid residues from the antigen-determining regions of Petition 870260063188, dated 06 / 26 / 2026, page 10 / 118 7 / 49 complementary or CDRs. CDR sequences are defined according to 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 linear amino acid sequence may contain fewer or more additional amino acids corresponding to a shortening of or insertion in a variable region of FR or CDR. For example, a variable region of the heavy chain may include a single amino acid insertion element (residue 52a, according to Kabat) after the H2 residue 52 and inserted residues (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after the heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment in homology regions of the antibody sequence with a standard numbered Kabat sequence.

[0024] Constant domains (constant parts) are not directly involved in the binding of an antibody to an antigen, but they also exhibit, for example, effector functions. The gene fragment of the constant region of the heavy chain that corresponds to human IgG1 is called the γ1 chain. The gene fragment of the constant region of the heavy chain that corresponds to human IgG3 is called the γ3 chain. Human constant γ heavy chains are described in Kabat, EA, 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, TW, et al., Methods Enzymol. 178(1989): 515-527.

[0025] The term Fc region here is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least one portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Petition 870260063188, dated 06 / 26 / 2026, page 11 / 118 8 / 49

[0026] Unless otherwise indicated herein, the numbering of amino acid residues in an Fc region or a constant region is in accordance with the UE numbering system, also called the UE index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

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

[0028] The term Fc variant, as used herein, refers to a polypeptide comprising a modification in the Fc domain. The modification may be an addition, deletion, or substitution. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. Variants may comprise non-natural amino acids.

[0029] The term polypeptide containing the Fc region refers to a polypeptide, such as an antibody, that comprises an Fc region.

[0030] The terms Fc receptor or FcR are used to describe a receptor that binds to the Fc region of an antibody. An FcR that binds an IgG antibody (a gamma receptor) includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcyRII receptors include FcyRIIA (an activating receptor) and FcyRIIB (an inhibiting receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domain. The activating receptor FcyRIIA contains a tyrosine-based immunoreceptor activating motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcyRIIB contains a tyrosine-based immunoreceptor activating motif (ITAM) in its cytoplasmic domain (see review). Petition 870260063188, dated 06 / 26 / 2026, p. 12 / 118 9 / 49 in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcRs are analyzed 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 that should be identified in the future, are encompassed by the term FcR here. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117 (1976) 587 and Kim, et al., J. Immunol. 24 (1994) 249).

[0031] By IgG Fc ligand, as used herein, we mean a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcyRs, FcRn, Clq, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR.Fc ligands also include Fc receptor homologs (FcRHs), which are a family of Fc receptors homologous to FcyRs (Davis, et al., Immunological Reviews 190 (2002) 123-136, fully incorporated by reference). Fc ligands may include undiscovered molecules that bind to Fc. Specific IgG Fc ligands are the FcRn and Fc gamma receptors. By Fc ligand, as used herein, we mean a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex.

[0032] By Fc gamma receptor, FcyR or FcgammaR, as used herein, means any member of the protein family that binds to the Fc region of the IgG antibody and is encoded by an FcyR gene. In humans, this family includes, but is not limited to, FcyRI (CD64), including the FcyRIA, FcyRIB and FcyRIC isoforms; FcyRII (CD32), including the FcyRIIA isoforms (including H131 and R131 allotypes), FcyRIIB (including FcyRIIB-1 and FcyRIIB-2) and FcyRIIc; and FcyRIII (CD16), including Petition 870260063188, dated 06 / 26 / 2026, p. 13 / 118 10 / 49 as the FcyRIIIA isoforms (including allotypes V158 and F158) and FcyRIIIb isoforms (including allotypes FcyRIIB-NA1 and FcyRIIB-NA2) (Jefferis et al., Immunol Lett 82 (2002)), as well as any other undiscovered isoforms or allotypes of human FcyRs or FcyRs. An FcyR can be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FCYRIII-2 (CD16-2), as well as any undiscovered isoforms or allotypes of mouse FcyRs or FcyRs.

[0033] By FcRn or neonatal Fc receptor, as used herein, refers a protein that binds to the Fc region of the IgG antibody and is encoded at least in part by an FcRn gene. The FcRn can be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. As is known in the art, the functional FcRn protein comprises two polypeptides, often referred to as the heavy chain and the light chain. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated, FcRn or an FcRn protein refers to the FcRn heavy chain complex with beta-2-microglobulin.

[0034] The “percentage (%) of amino acid sequence identity” with respect to a reference peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that is identical with the amino acid residues in the specific peptide or polypeptide sequence, after sequence alignment and introducing gaps, if necessary, to obtain the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining the percentage of identity of. Petition 870260063188, dated 06 / 26 / 2026, page 14 / 118 The 11 / 49 amino acid sequence can be achieved in several ways that are within the specialty of the technique, for example, BLAST, BLAST2, ALIGN or Megalign (DNASTAR).

[0035] Antibody-dependent cell-mediated cytotoxicity and ADCC refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcRs (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize antibody bound to a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, while monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression in hematopoietic cells is summarized in Table 3, on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.

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

[0037] The term antibody effector function(s) or effector function, as used herein, refers to a function contributed by an Fc effector domain(s) of an IgG (e.g., the Fc region of an immunoglobulin). This function may be effected, for example, by the binding of an Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity or by the binding of an Fc effector domain(s) to components of the complement system. Typical effector functions are ADCC, ADCP, and CDC.

[0038] “C1q is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q, along with two serine proteases, C1r and C1s, forms the C1 complex, the first component of the complement-dependent cytotoxicity (CDC) pathway. Petition 870260063188, dated 06 / 26 / 2026, page 15 / 118 12 / 49

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

[0040] An effective amount of an agent, for example, a pharmaceutical formulation, refers to an effective quantity, in doses and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0041] The term cancer, as used herein, may include, for example, lung cancer, non-small cell lung cancer (NSCL), bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer with advanced pancreatic carcinoma, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma,Hepatocellular carcinoma, biliary cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymal tumors, medulloblastomas, meningiomas, squamous cell carcinomas, Petition 870260063188, dated 06 / 26 / 2026, page 16 / 118 13 / 49 pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Detailed Description of the Invention

[0042] The present invention addresses the need to provide agonistic CD40 mAbs that exhibit reduced cellular toxicity, leading to fewer clinical side effects, while their signaling potency and clinical efficacy are at least maintained, if not increased, compared to prior art agonistic CD40 antibodies.

[0043] The antibodies or antigen-binding fragment of the present invention provide these advantageous properties because they are able to bind specifically to the human CD40 receptor and induce CD40 signaling independent of Fcy-mediated CD40 receptor cross-linking.

[0044] In preferred embodiments, the antibodies according to the invention are humanized IgG1LALA antibodies, humanized IgG1-type antibodies possessing at least two alanine amino acids at positions 234 and 235 of the human Fc1 region. Thus, according to a preferred embodiment, an IgG1LALA comprises the L234A and L235A mutation of the human Fc1 region.

[0045] It is still preferred that the antibodies according to the invention be recombinant molecules.

[0046] The present invention provides that an agonistic monoclonal antibody, or an antigen-binding fragment thereof, is capable of binding to the human CD40 receptor and inducing CD40 signaling independent of Fcy-mediated CD40 receptor crosslinking (see also Example 5, Figure 6 and text below). Furthermore, an antibody according to the present invention may exhibit reduced or depleted signaling capacity through the receptor. Petition 870260063188, dated 06 / 26 / 2026, p. 17 / 118 14 / 49 Human FcY signaling when compared to wild-type IgG Fcy receptor signaling or Fcy antibody signaling from the prior art technique.

[0047] In certain embodiments, the CD40 agonist monoclonal antibodies of the invention, or their antigen-binding fragments, may exhibit reduced or depleted affinity for human Fcy receptors compared to wild-type Fcy IgG. According to a preferred embodiment, the antibodies of the invention do not bind to Fcy receptors – correspondingly, the antibodies of the invention do not trigger Fcy-mediated CD40 receptor crosslinking.

[0048] In a preferred embodiment, the antibodies of the present invention comprise at least amino acid substitutions at L234A and L235A of the Fc region of human IgG1 or S228P and L235E of the Fc region of human IgG4.

[0049] It is further preferred for the present invention that the antibodies 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 antibodies tested. It is also preferred that the CD40 antibodies compete with CD40L for binding to the CD40 receptor. In epitope competition assays, the antibodies of the invention therefore compete with CD40L. Such assays are described in Examples 3 and 11, and the results of experiments with antibodies of the invention are shown in Figures 3 and 16. Thus, according to another preferred embodiment, the antibodies of the invention inhibit the binding of CD40L to the CD40 receptor. Figure 19 demonstrates that the antibodies of the invention do not compete with CP-870,893 for binding to CD40.

[0050] The antibodies according to the invention have very high binding activity to the CD40 receptor. Therefore, in a Petition 870260063188, dated 06 / 26 / 2026, page 18 / 118 In a 15 / 49 cell binding assay, as described in Example 1, the antibodies according to the 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, less than 9 ng / ml, 7 ng / ml, 6 ng / ml, 5 ng / ml, 4 ng / ml. Most preferably, the EC50 is 3 ng / ml in a cell binding assay, as described in Example 1 and as shown in Figure 1.

[0051] The humanized anti-CD40 agonist antibodies according to the invention can be characterized by biochemical affinities for the soluble trimeric CD40 protein of humans or cynomolgus monkeys (cf. Example 13; Figure 18). The antibodies of the invention can show KD values ​​equal to or less than 15.7 nM for human CD40. The antibodies of the invention can be cross-reactive with the cynomolgus monkey CD40 protein with a KD value equal to or less than 10.3 nM.

[0052] Furthermore, the antibodies of the present invention are capable of inducing NF-κB cell signaling with high potency. A summary of the experiments (cf. Example 2) is shown 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.

[0053] It will also be understood that the antibodies of the invention can bind to macaque cynomolgus-CD40. The binding activity of humanized anti-CD40 IgG1-LALA monoclonal antibodies to macaque cynomolgus (Macaca fascicularis) is shown in ELISA experiments using recombinant macaque cynomolgus CD40 protein (cf. Example 4). The EC50 values ​​shown in Figure 4 indicate potent antibody binding.

[0054] Another feature of the antibodies of the present invention is that they can activate human APCs. For example, the antibodies Petition 870260063188, dated 06 / 26 / 2026, page 19 / 118 16 / 49 can activate selected cells from the group comprising dendritic cells (DCs), B cells, monocytes, and myeloid cells. Preferably, the antibodies activate DCs.

[0055] This potent agonist activity of CD40 in the activation of APCs are not due to Fc1 receptor-mediated cross-linking of CD40 proteins (cf. experimental results shown in Figures 5, 6 and 7 and described in Example 5).

[0056] As such, in one embodiment, the antibodies of the present invention induce the release of IL-12p40 in a dendritic cell maturation assay, as described in Example 5. The results of experiments conducted with the antibodies of the present invention in such an assay are shown in Figures 5 to 7.

[0057] According to another preferred embodiment, the antibodies of the invention induce the maturation of antigen-presenting cells, as determined by the release of IL12p70, which is at least equal to the release induced by stimulation with the antibody CP-870,893-IgG2 and with an EC50 value equal to or less than 208 ng / ml (Figures 12 and 14). Furthermore, the antibodies of the invention induce the maturation of antigen-presenting cells, as determined by the induction of CD86 at least 7.5 times and with an EC50 equal to or less than 148 ng / ml (Figures 11 and 13).

[0058] Preferably, antibodies induce a release of IL12p40 release from monocyte-derived DCs is at least equal to the release induced by stimulation with the antibody CP-870.893-IgG2 (see Figure 6). As previously stated, this potential to induce DC maturation is not due to signaling via Fc receptors. The humanized anti-CD40 IgG1-LALA monoclonal antibodies of the invention potently induce activation of monocyte-derived dendritic cells in an Fc receptor-independent manner (see Figure 6). Petition 870260063188, dated 06 / 26 / 2026, p. 20 / 118 17 / 49 Example 5 and Figure 6). Figure 6 demonstrates that the IL12p40 release levels induced by the CP-870,893 variants correlate with the variants' ability to bind to Fc receptors (IgG1LALA). <IgG2 <IgG1 <IgG1-V11). Surpreendentemente, a estimulação por anticorpos monoclonais anti-CD40 IgG1-LALA humanizados da invenção resultou em níveis de secreção de IL12p40 independentes de Fc que cobriram e até excederam o intervalo gerado com as variantes CP-870,893. Assim, os anticorpos anti-CD40 da invenção proporcionam atividade agonística potente em células dendríticas primárias derivadas de monócitos, sem reticulação mediada pelo receptor Fc receptor de proteínas CD40.

[0059] Furthermore, the antibodies of the present invention are very specific in their activation. They do not induce a general release of inflammatory cytokines, such as TNF-alpha (cf. Example 6 and Figure 8).

[0060] Another characteristic of the antibodies of the present invention is the reduced clearance of antibodies from the cell surface. CP-870,893 is known to internalize after binding to the CD40 receptor on cells. Clinical studies have shown that CP-870,893 is rapidly cleared from circulation in patients, with an estimated half-life of less than 6 hours, reflecting a large CD40 receptor scavenger in patients that may be caused by cellular internalization (Rüter et al 2010).

[0061] The antibodies of the invention are retained on the cell surface under conditions that permit endocytosis and internalization (cf. Example 7 and Figure 9). On the other hand, the CP-870,893 variants are not retained on the cell surface under conditions that permit endocytosis and internalization (Figure 9).

[0062] It will be appreciated that the antibodies according to the invention have an indirect (immunity-mediated) effect on the death of tumor cells. Thus, the antibodies exhibit a cytotoxic effect mediated by Petition 870260063188, dated 06 / 26 / 2026, p. 21 / 118 18 / 49 indirect immune cells in tumor cells.

[0063] In a specific embodiment, the antibodies according to the invention do not result in the depletion of immune cells expressing CD40 by ADCC, ADCP or CDC mechanisms.

[0064] Thus, in summary, the antibodies of the invention can be further characterized by

[0065] (a) no binding to Fcy receptor;

[0066] (b) have binding affinity to CD40 cells with an EC50 value equal to or less than approximately 49.5 ng / ml;

[0067] c) have KD values ​​equal to or less than approximately 15.7 nM;

[0068] (d) be cross-reactive to monkey cynomolgus CD40 with a KD value equal to or less than about 10.3 nM;

[0069] (e) inhibiting the binding of CD40L to CD40;

[0070] (f) avoid synergistic and additive effects of functions mediated by CD40L;

[0071] (g) inducing maturation of antigen-presenting cells, as determined by the release of IL12p70, which is at least equal to the release induced by stimulation with the antibody CP870,893-IgG2 and with an EC50 value equal to or less than 208 ng / ml

[0072] and / or as determined by the induction of CD86 in dendritic cells at least 7.5 times and with an EC50 equal to or less than 148 ng / ml; and / or

[0073] (h) reduce the level of CD40 on the cell surface to a lesser degree than CP-870,893.

[0074] According to a preferred embodiment, the antibodies of the invention are characterized by having at least one, two, three, four, five, six, seven or eight or all of the above properties (a - h).

[0075] Due to the favorable properties of the antibodies of the invention, they are able to inhibit tumor growth. Petition 870260063188, dated 06 / 26 / 2026, p. 22 / 118 19 / 49 humans.

[0076] In certain embodiments, an antibody according to the invention may comprise a VH region selected from the group of VH regions comprising CDR regions selected from the group consisting of a CDR1H region of SEQ ID NO: 29 + n, a CDR2H region of SEQ ID NO: 43 + n and a CDR3H region of SEQ ID NO: 57 + n, wherein 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 a CDR1L region of SEQ ID NO: 71 + m, a CDR2L region of SEQ ID NO: 85 + m and a CDR3L region of SEQ ID NO: 99 + m, wherein m 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 diminish their activity according to the invention.

[0077] Preferably, an antibody comprises a VH region selected from the group of VH regions comprising CDR regions selected from the group consisting of a CDR1H region of SEQ ID NO: 29 + n, a CDR2H region of SEQ ID NO: 43 + n and a CDR3H region of SEQ ID NO: 57 + n, wherein 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 a CDR1L region of SEQ ID NO: 71 + m, a CDR2L region of SEQ ID NO: 85 + m and a CDR3L region of SEQ ID NO: 99 + m, wherein m is a number selected from the group consisting of 0 to 13.

[0078] In certain embodiments, an antibody according to the invention may comprise a VH region selected from the group of VH regions comprising the CDR regions selected from the group consisting of a CDR1H region of SEQ ID NO: 29 + n, a region Petition 870260063188, dated 06 / 26 / 2026, page 23 / 118 20 / 49 CDR2H of SEQ ID NO: 43 + ne a CDR3H region of SEQ ID NO: 57 + m, and a VL region selected from the group of VL regions comprising the CDR regions selected from the group consisting of a CDR1L region of SEQ ID NO: 71 + n, a CDR2L region of SEQ ID NO: 85 + n and a 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 diminish their activity according to the invention.

[0079] Preferably, CDRs have a sequence identity for their respective SEQ ID NOs of at least 91%, preferably 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0080] In another embodiment, the antibodies or antigen-binding fragments according to the invention comprise a variable region of the heavy chain (HC) that is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, most preferably at least 85% identical to an HC region selected from the group consisting of HC regions of SEQ ID NO: 1 to 14.

[0081] Preferably, said antibodies comprise a variable heavy chain (VH) region sequence with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence selected from the group of VH sequences of SEQ ID NO: 1 to 14.

[0082] In certain embodiments, a VH sequence with at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, whereby the antibody Petition 870260063188, dated 06 / 26 / 2026, p. 24 / 118 21 / 49 retains the ability to bind specifically to the respective antigen according to the invention.

[0083] The present invention also encompasses an antibody comprising a variable heavy chain (VH) region comprising an amino acid sequence selected from the SEQ ID NO: 1 to 14 group.

[0084] Preferably, the sequence of the variable region of the heavy chain (VH) is SEQ ID NO:1, alternatively 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.

[0085] The present invention also relates to an antibody comprising a variable heavy chain (VL) region that is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, most preferably at least 85% identical to a VL region selected from the group consisting of VL regions of SEQ ID NO: 15 to 28.

[0086] Preferably, said antibodies comprise a VL sequence with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence selected from the group of VL sequences of SEQ ID NO: 15 to 28.

[0087] In certain embodiments, a VL sequence with at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, whereby the antibody retains the ability to bind specifically according to the invention to the respective antigen.

[0088] The present invention also encompasses an antibody that Petition 870260063188, dated 06 / 26 / 2026, p. 25 / 118 22 / 49 comprises a variable region of the light chain (VL) comprising an amino acid sequence selected from the SEQ ID NO: 15 to 28 group.

[0089] Preferably, the sequence of the variable region of the light chain (VL) is SEQ ID NO:15, alternatively 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.

[0090] In certain embodiments, a total of 1 to 10 amino acids were substituted, inserted and / or deleted in the so-called VL sequences. In other embodiments, a total of 1 to 10 amino acids were substituted, inserted and / or deleted in the so-called VH sequences. In certain embodiments, a total of 1 to 10 amino acids were substituted, inserted and / or deleted in each of the so-called VH or VL sequences. The said substitutions, insertions or deletions may occur in regions outside the CDRs (i.e., in the FRs).

[0091] The invention also comprises affinity-matured antibodies that can be produced according to methods known in the art. Marks et al. Bio / Technology, 10:779-783 (1992) describe affinity maturation by shuffling the VH and VL domains. Random mutagenesis of CDR and / or structural residues is described by Barbas et al., Proc Nat. Acad. Sci, USA 91: 3809-3813 (1994); Schier et al., Gene 169: 147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 1 54(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992) and WO2010 / 108127.

[0092] The present invention also encompasses an antibody comprising a VH region and a VL region comprising the respective CDR1, CDR2 and CDR3 regions of an antibody selected from the group comprising the antibodies listed in Figure 10, that is, Petition 870260063188, dated 06 / 26 / 2026, page 26 / 118 23 / 49 comprising antibodies MAB-16-0283, MAB-16-0377, MAB-160267, 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-160484, MAB-16-0400, MAB-16-0489.

[0093] The present invention also encompasses an antibody comprising SEQ ID NO.: 1 and 15, or SEQ ID NO.: 2 and 16. An antibody according to the invention may also comprise SEQ ID NO.: 3 and 17, or SEQ ID NO.: 4 and 18, or SEQ ID NO.: 5 and 19, or SEQ ID NO.: 6 and 20, or SEQ ID NO.: 7 and 21, or SEQ ID NO.: 8 and 22, or SEQ ID NO.: 9 and 23, or SEQ ID NO.: 10 and 24, or SEQ ID NO.: 11 and 25, or SEQ ID NO.: and 26. Alternatively, an antibody according to the invention comprises SEQ ID NO.: 13 and 27, or SEQ ID NO.: 14 and 28.

[0094] In another aspect, the antibodies of the invention are for use in the treatment of patients suffering from cancer.

[0095] The cancer in question may be one or more of the cancer types selected from the group comprising pancreatic cancer, advanced pancreatic carcinoma lung cancer, non-small cell lung cancer (NSCL), bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, rectal cancer, anal region cancer, 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.

[0096] In certain forms, cancer is a solid tumor.

[0097] It is also possible in some forms that the cancer is a CD40-expressing cancer. However, this is not necessary for the effective functioning of the antibodies of the invention.

[0098] It will also be appreciated that the antibody of the invention can be Petition 870260063188, dated 06 / 26 / 2026, page 27 / 118 24 / 49 is used as a single treatment for cancer in a patient or as part of a combination treatment (the additional treatment may be a cytotoxic or cytostatic pharmaceutical agent, radiotherapy, targeted therapy and / or surgery).

[0099] Thus, the patient may also receive one or more additional treatments for cancer, for example, pharmaceutical agents (such as cytotoxic or cytostatic agents, targeted therapy), radiotherapy and / or surgery.

[0100] Thus, in some embodiments, the antibody according to the invention is used in cancer treatment in combination with cytotoxic or cytostatic agents, radiotherapy, targeted therapy and / or immunotherapy.

[0101] The antibodies of the invention can also be used in the treatment of patients who respond insufficiently to and / or resist cytotoxic or cytostatic agents, radiotherapy, targeted therapy and / or immunotherapy.

[0102] The aforementioned radiotherapy may be selected from the group comprising external beam radiotherapy, contact X-ray brachytherapy, brachytherapy, systemic radioisotope or intraoperative radiotherapy.

[0103] Cytotoxic or cytostatic anticancer agents according to the invention may be from the group comprising taxanes, anthracyclines, alkylating agents, histone deacetylase inhibitors, topoisomerase inhibitors, kinase inhibitors, kinase inhibitors, nucleotide analogs, peptide antibiotics and platinum-based agents.

[0104] Preferably, targeted anticancer agents are used in targeted therapy and selected from one of the following, or combinations thereof: anti-EGFR compounds, such as cetuximab, gefitinib, erlotinib, lapatinib, panitumumab, compounds Petition 870260063188, dated 06 / 26 / 2026, page 28 / 118 25 / 49 anti-HER2 inhibitors, such as trastuzumab, ado-trastuzumab emtansine, pertuzumab, VEGF-targeting compounds such as bevacizumab, aflibercept and pegaptanib, and tyrosine kinase inhibitors such as sunitinib, pazopanib, axitinib, vandetanib, cabozantinib and regorafinib.

[0105] If patients are receiving immunotherapy, this may involve immune checkpoint inhibition and one or more immune checkpoint inhibitors may be used. One or more immune checkpoint inhibitors may be selected from the group comprising anti-PD-L1, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40 and / or anti-GITR.

[0106] The antibody according to the invention can also be used in combination with an antibody that specifically binds to PD-L1, CTLA-4, LAG-3, TIM-3, CD137, OX40, GITR and / or in combination with the drug Nivolumab, Pembrolizumab, Urelumab, Utomilumab, Atezolizumab, Avelumab, Durvalumab, Tremelimumab, Ipilimumab.

[0107] In some embodiments according to the invention, the antibody is used in the treatment of cancer in a weekly to monthly dosing regimen.

[0108] One of the special advantages of this invention is that, due to their mutations in the Fc region, the antibodies exhibit less dose- or treatment-limiting toxicity compared with prior art antibodies. The antibodies cause the typical side effects of CD40 antibodies, if any, only to a very limited extent. Such side effects are selected conditions from the group comprising cytokine release syndrome, thrombosis, cerebral embolism, transaminase elevations, lymphopenia, fatigue, peripheral neuropathy, alopecia, constipation, nausea, and neutropenia.

[0109] In another aspect, the present invention relates to a Petition 870260063188, dated 06 / 26 / 2026, page 29 / 118 26 / 49 pharmaceutical composition comprising a pharmaceutically acceptable vehicle and a therapeutically effective amount of the antibody according to the invention.

[0110] The pharmaceutical composition according to the invention can be used in the treatment of patients suffering from cancer. This type of cancer can be a solid tumor. The cancer can be further selected from the group comprising pancreatic cancer, advanced pancreatic carcinoma lung cancer, non-small cell lung cancer (NSCL), bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, rectal cancer, anal region cancer, 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.

[0111] The composition can also be used in cancer treatment in combination with chemotherapy, radiotherapy, targeted therapy and / or immunotherapy. The aforementioned immunotherapy may be an immune checkpoint inhibition.

[0112] Patients treated with the aforementioned composition may respond insufficiently to and / or be resistant to chemotherapy, radiotherapy, targeted therapy and / or immunotherapy.

[0113] The pharmaceutical composition according to the invention can also be used in the treatment of cancer in combination with one or more cytotoxic, cytostatic or targeted anticancer compounds.

[0114] It can be used in cancer treatment in combination with one or more immune checkpoint inhibitors, wherein such immune checkpoint inhibitors may be selected from Petition 870260063188, dated 06 / 26 / 2026, page 30 / 118 27 / 49 group comprising anti-PD-LI, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40 and / or anti-GITR.

[0115] The composition can also be used in combination with an antibody that binds specifically to PD-L1, CTLA-4, LAG-3, TIM-3, CD137, OX40, GITR and / or in combination with the drug Nivolumab, Pembrolizumab, Urelumab, Utomilumab, Atezolizumab, Avelumab, Durvalumab, Tremelimumab, Ipilimumab.

[0116] It can also be used in cancer treatment on a weekly to monthly dosing regimen.

[0117] Patients will particularly appreciate that the antibody and composition according to the invention exhibit any dose- or treatment-limiting toxicity, if any, only to a very limited extent and, in particular, to a lesser extent than prior art antibodies and compositions.

[0118] In another aspect, the present invention also relates to methods of treatment, comprising administering an effective amount of the antibody according to the invention to individuals in need. Such individuals may be patients suffering from cancer. Thus, the present invention also relates to methods of cancer treatment, wherein the cancer may be a solid tumor.

[0119] The administration step to an individual in need of the same may comprise local administration, for example, local administration to a tumor in a patient (e.g., intratumoral or peri-tumoral).

[0120] As the antibody-based agents of the invention are suitable for use in the treatment of any type of cancer for which CD40 activation may provide a therapeutic benefit, the methods comprising the administration of said antibodies are also suitable for the treatment of any type of cancer. Petition 870260063188, dated 06 / 26 / 2026, page 31 / 118 28 / 49 for which CD40 activation may provide a therapeutic benefit.

[0121] For example, cancer may be selected from the group consisting of: prostate cancer; breast cancer; colorectal cancer; pancreatic cancer; ovarian cancer; lung cancer; cervical cancer; rhabdomyosarcoma; neuroblastoma; multiple myeloma; leukemia, acute lymphoblastic leukemia, melanoma, bladder cancer and glioblastoma.

[0122] It will also be appreciated that the treatment methods according to the invention may comprise a single administration of antibody-based agents of the invention to a patient or as part of a combination treatment (the additional treatment may be a pharmaceutical cytotoxic or cytostatic agent, radiotherapy, targeted therapy and / or surgery).

[0123] In fact, all the favorable characteristics and properties of the antibodies of the invention, as detailed above, are also reflected and understood in the treatment methods and uses of the antibodies according to the invention. Examples

[0124] The following examples are used in conjunction with the figures and tables to illustrate the invention. Example 1: Binding of anti-CD40 antibodies to the cell

[0125] To determine the potency of humanized anti-CD40 IgG1-LALA monoclonal antibodies in binding to CD40 expressed on cells, HEK-Blue-CD40L™ (InvivoGen) cells were seeded in 25 µl of DMEM containing 10% FBS at a cell density of 1000 cells / well in a 384-well, clear-bottom plate treated with cell culture. Antibodies were added at final concentrations ranging from 1.25 pg / ml to 0.01 ng / ml in 5 µl of medium. After 24 h, cells were washed three times with 25 µl of washing buffer (PBS, Tween 0.05%) before the addition of IgG. Petition 870260063188, dated 06 / 26 / 2026, page 32 / 118 Human 29 / 49 anti-goat conjugate with Alexa-Fluor-488 (Jackson Laboratories) was injected at a concentration of 0.8 μg / ml in 20 μl of medium. Four hours later, 5 μl of Hoechst dye was added to the medium until a final concentration of 5 μg / ml was reached. Binding signals to fluorescent cells were measured using an automated high-content imager, CellInsight (Thermo Fisher Scientific). Fitting curves and EC50 calculations were obtained using Excel (Microsoft) and XLfit (IDBS). Figure 1 summarizes the EC50 binding values ​​ranging from 3 to 49.5 ng / ml. Example 2: Induction of NF-kB cell signaling by anti-CD40 agonist antibodies.

[0126] The agonistic activity of humanized anti-CD40 IgG1-LALA monoclonal antibodies was tested by stimulating HEK-Blue-CD40L™ (InvivoGen) cells harboring an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) gene construct. 25,000 cells / well in 20 μl of DMEM containing 10% FBS were seeded in a 384-well, clear-bottomed cell culture plate 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 were transferred to a white, transparent-bottomed 384-well plate containing 20 μl of 2x QUANTI-Blue™ reagent (InvivoGen). After incubation for one hour at 37 °C and 5% CO2, the optical density with a wavelength of 620 was measured, reflecting the NF-kB-dependent activation of phosphatase secretion.The fitting curves and EC50 calculation were obtained using Excel (Microsoft) and XLfit (IDBS). The EC50 values ​​in Figure 2 indicate the potency of anti-CD40 antibodies to induce NF-kB signaling in the HEK-Blue-CD40L™ cell line. Petition 870260063188, dated 06 / 26 / 2026, page 33 / 118 30 / 49 Example 3: Competition with CD40L binding

[0127] The competition of humanized anti-CD40 IgG1LALA monoclonal antibodies with CD40L binding to CD40 was tested using an ELISA assay. CD40L was coated onto the surface of a 384-well Nunc™ MaxiSorp™ plate in a volume of 25 μL of PBS and at a concentration of 1 μg / ml for one hour at room temperature. The antibodies were pre-incubated at a concentration of 5 μg / ml with recombinant CD40 protein at a concentration of 1.7 μg / ml in a total volume of 40 μL for 1.5 hours at room temperature in ELISA buffer (PBS, 0.5% BSA, 0.05% Tween). The Nunc™ MaxiSorp™ plate was washed three times with wash buffer (PBS, 0.1% Tween) and blocked for one hour at room temperature with PBS, 2% BSA, 0.05% Tween. After three washes in wash buffer, 25 μl of the antibody-CD40 complex were added to the wells of the Nunc™ MaxiSorp™ plate and incubated for one hour at room temperature.After 3 washes in washing buffer, the wells were incubated with 25 μl of a 1:2000 dilution of species-specific F(ab)2 fragment bound to goat anti-human peroxidase (AbD Serotec) in ELISA buffer for one hour at room temperature. The wells were washed six times with washing buffer and 30 μl / well of TMB substrate solution (Invitrogen) was added. After 10 minutes at room temperature, 30 μl of blocking solution (1M HCl) was added per well and the absorbance at wavelengths of 450 and 620 nm was measured using a Tecan M1000 microplate reader. The ELISA signal for samples incubated with CP-870,893 indicates a lack of competition with CD40L, whereas the humanized anti-CD40 IgG1-LALA monoclonal antibodies according to the invention compete with the binding of CD40L to CD40 (see Figure 3). Example 4: CD40 binding activity of cynomolgus monkeys

[0128] The binding of anti-CD40 IgG1-LALA monoclonal antibodies Petition 870260063188, dated 06 / 26 / 2026, p. 34 / 118 31 / 49 humanized cynomolgus monkey CD40 protein was tested in a biochemical ELISA. Recombinant cyno-CD40 protein (Acro Biosystems) was incubated in a 384-well Nunc™ MaxiSorp™ plate at a concentration of 0.5 pg / ml in PBS for one hour at room temperature. After washing three times with washing buffer (PBS, 0.1% Tween), the plates were blocked with PBS, 2% BSA, 0.05% Tween for one hour at room temperature. The plates were washed again three times with washing buffer and antibodies at concentrations ranging from 500 to 0.03 ng / ml in PBS, 0.5% BSA, 0.05% Tween were incubated for one hour at room temperature. After 3 washes in washing buffer, the wells were incubated with 12.5 µl of a 1:3000 dilution of species-specific F(ab)2 fragment bound to goat anti-human peroxidase (AbD Serotec) in ELISA buffer for one hour at room temperature.The wells were washed six times with washing buffer, and 15 µl / well of TMB substrate solution (Invitrogen) was added. After 10 minutes at room temperature, 15 µl of blocking solution (1M HCl) was added per well, and absorbance at wavelengths of 450 and 620 nm was measured using a Tecan M1000 microplate reader. Fitting curves and EC50 calculations were obtained using Excel (Microsoft) and XLfit (IDBS). As shown in Figure 4, most antibodies bind to cyno-CD40 with EC50 values ​​between 8 and 31.8 ng / ml. Example 5: Induction of dendritic cell maturation

[0129] Monocyte-derived dendritic cells were generated to test the ability of humanized anti-CD40 IgG1LALA monoclonal antibodies to stimulate dendritic cell maturation, as measured by IL12p40 cytokine secretion. Human buffy coat preparations from different donors were used to differentiate dendritic cells from monocytes in vitro. The Petition 870260063188, dated 06 / 26 / 2026, page 35 / 118 32 / 49 buffy coating received from the Bavarian Red Cross was diluted 1:4 with DPBS and layered onto FicollPaque density gradients (GE Healthcare). After centrifugation, 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. Monocytes were cultured in RPMI-1640 containing 10% FCS, 1x Pen / Strep, 1x L-Glutamine, 50 ng / ml recombinant human GM-CSF (R&D Systems), and 10 ng / ml recombinant human IL-4 (R&D Systems) at a cell density of 1.2x10⁶ cells / ml in T-175 cell culture flasks. Every 48 hours, 90% of the medium was replaced with fresh medium containing cytokine. On day five, immature dendritic cells (iDCs) differentiated in vitro were harvested and distributed to a 96-well cell culture plate at a cell density of 10⁶ cells / ml in 100 µl of the same medium.

[0130] In an experiment, iDCs were stimulated by the addition of anti-CD40 antibodies at a concentration of 5 pg / ml. 48 hours after stimulation, the secreted IL12p40 cytokine was quantified in the medium supernatant using a commercially available ELISA kit (R&D Systems) according to the manufacturer's instructions. Figure 5 shows that humanized anti-CD40 IgG1-LALA monoclonal antibodies stimulated IL12p40 release by monocyte-derived dendritic cells to varying extents, ranging from less than 1 ng / ml to more than 24 ng / ml, while a control IgG1-LALA antibody not bound to CD40 did not lead to stimulation of detectable levels of IL12p40.

[0131] The humanized anti-CD40 IgG1-LALA monoclonal antibodies of the invention are unable to bind to Fcy receptors that are expressed on monocyte-derived dendritic cells. To compare these antibodies with antibodies harboring different Petition 870260063188, dated 06 / 26 / 2026, p. 36 / 118 33 / 49 Fcy receptor binding activities, we constructed CP870.893 reference anti-CD40 antibodies containing Fc portions of human IgG1, IgG1LALA, IgG2, and IgG1-V11, and monocyte-derived dendritic cells stimulated with different concentrations of these antibodies. The IgG1-V11 form harbors four mutations (G237D, H268D, P271G, A330R) in the Fc portion of the heavy chain. These mutations have been described to selectively increase affinity for the Fc y RIIB receptor (Mimoto et al. 2013). Figure 6 demonstrates that the CP870.893 variants stimulate IL12p40 release in an Fc-dependent manner (IgG1-LALA). <IgG2 <IgG1 <IgG1-V11). Surpreendentemente, a estimulação por anticorpos monoclonais anti-CD40 IgG1-LALA humanizados da invenção resultou em níveis de secreção de IL12p40 independentes de Fc que cobriram e até excederam o intervalo gerado com as variantes CP-870,893.The anti-CD40 antibodies of the invention provide potent agonist activity in primary dendritic cells derived from monocytes, without Fcy receptor-mediated cross-linking of CD40 proteins.

[0132] In another experiment, the potency of humanized anti-CD40 IgG1-LALA monoclonal antibodies to induce IL12p40 secretion by monocyte-derived dendritic cells was determined by stimulation with antibody concentrations ranging from 0.005 to 10 pg / ml. Figure 7 shows that the EC50 values ​​for different antibodies ranged from 380 to 743 ng / ml. Example 6: Release of TNF alpha in a high-density CMSP assay

[0133] To determine if anti-CD40 monoclonal antibodies Humanized IgG1-LALA antibodies induce a general release of cytokines in blood cells. CMSPs were stimulated with these antibodies according to the protocol of Romer et al. 2011. CMSPs were isolated as previously described and cultured in RPMI-1640 containing serum. Petition 870260063188, dated 06 / 26 / 2026, page 37 / 118 34 / 49 Human antibody (AB) at 10% and 1x non-essential amino acids (NEAA) were added to a cell density of 1x10⁷ cells / ml in a T175 cell culture flask. After two days, cells were harvested and seeded at a density of 1x10⁶ cells / ml in triplicate in a 96-well cell culture plate. Antibodies were added at a concentration of 10 pg / ml and incubated with CMSPs for three days at 37 °C, 5% CO₂, and 95% humidity. An OKT-3 antibody (Abcam) was included in the experiments as a positive control. TNF-alpha release was quantified in the cell culture supernatant using a commercially available human TNF-alpha ELISA kit (R&D Systems) according to the manufacturer's instructions. Figure 8 shows that the anti-CD40 antibodies of the invention and a CD40-unbound IgG1LALA control antibody did not stimulate significant TNF-alpha release by CMSPs, in contrast to the OKT-3 antibody. Example 7: Cellular pulse-chase assay

[0134] Cell binding and internalization dynamics of anti-CD40 antibodies were analyzed in a pulse-chase assay using HEK-Blue-CD40L™ cells (InvivoGen). 2000 cells / well were seeded onto two 384-well black plates with a clear background in DMEM medium containing 10% FCS. After overnight culture, the anti-CD40 antibodies of the invention and the Fc variant antibodies CP870,893, as described above, were added to one plate at a concentration of 0.8 pg / ml and incubated for 15 min at 37 °C and 5% CO2. Subsequently, both plates were washed three times with cell wash buffer (PBS 0.05% Tween) and incubated for one hour in culture medium. In the last 15 minutes, antibodies were added to plate 2 at a concentration of 0.8 pg / ml. Both plates were washed three times with cell wash buffer, placed on ice, and incubated with 0.8 pg / ml of Alexa-Fluor-488 anti-human coupled secondary antibody (Jackson Laboratories) and 5 Petition 870260063188, dated 06 / 26 / 2026, page 38 / 118 35 / 49 pg / ml of Hoechst dye (Invitrogen) for 30 minutes on ice. Cell surface fluorescent signals were quantified using a CellInsight high-content imager (Thermo Fisher Scientific). Figure 9 shows that the CP-870,893 variant antibodies exhibit strongly reduced cell surface signals after one hour of incubation under conditions that allow internalization. In contrast, incubation of many of the anti-CD40 antibodies according to the invention under the same conditions results in only a small reduction in cell surface signals, indicating limited internalization rates. Example 8: Correlation of reporter gene induction and dendritic cell maturation activity of humanized anti-CD40 antibodies.

[0135] Cellular reporter gene maturation assays (HEK-BlueCD40LTM) and dendritic cell (DC) assays were performed as described in Examples 2 and 5, respectively. Antibodies were used at 5 pg / ml for the DC assay and at concentrations ranging from 13 to 20000 ng / ml in the HEK-Blue-CD40L™ reporter gene assay. Figure 10 compares the maximum induction observed in the reporter gene assay with IL12p40 cytokine release by DCs after stimulation with 5 pg / ml of antibodies. While all 88 humanized anti-CD40IgG1-LALA antibodies induce reporter gene expression to similar extents, some antibodies show very high stimulation of IL12p40 release by DCs. HEK-Blue-CD40L™ cells do not express Fc receptors. The assay is capturing basic agonist activities of CD40 antibodies, independent of Fc receptor binding. DCs express Fc receptors and agonist activities of anti-CD40 antibodies, such as CP-870.893, are dependent on binding to the Fc receptor (Example 5). However, while most of the 88 IgG1-LALA antibodies do not have strong binding. Petition 870260063188, dated 06 / 26 / 2026, page 39 / 118 36 / 49 activation of DCs, a smaller fraction can induce very strong DC activation without Fc receptor-mediated cross-linking. Therefore, a highly agonistic anti-CD40 antibody, whose activity on primary dendritic cells is independent of Fc receptor cross-linking, is a rare case, and identification requires screening a large number of candidate antibodies with basic agonist activities. Example 9: Stimulation of co-stimulatory receptors and cytokine release in dendritic cells by anti-CD40 agonist antibodies.

[0136] a. To test the activity of humanized and agonistic anti-CD40 lgG1-LALA antibodies in stimulating co-stimulatory receptor expression and inflammatory cytokine release by DCs, monocyte-derived immature DCs (iDCs) were generated from three independent donors, as described in Examples 5. The iDCs were treated with the antibodies at a concentration of 2 pg / ml or CD40L (R&D Systems 6245-CL-050) at 20 pg / ml for 48 h. The stimulated mature DCs were harvested, stained using fluorophore-labeled antibodies against HLA-DR, CD86, CD80, CD83, CD54, and CD95 (all from Miltenyi Biotech), and analyzed by flow cytometry on a BD FACSVerse device. Figure 11 shows the stimulation of receptor expression as a fold of induction upon isotypic antibody control treatment. The data demonstrate strong induction of costimulatory receptors, particularly CD86. CP-870.893 shows lower overall activity compared to antibodies MAB-16-0262, MAB-16-0451, MAB-16-0464, and MAB-16-0406 of the invention. A further reduction in activity is observed when a variant CP-870.893 containing a constant moiety of IgG1-LALA is used, confirming the Fc receptor binding dependence of this antibody.

[0137] b. Figure 12 shows the results of the measurements of Petition 870260063188, dated 06 / 26 / 2026, p. 40 / 118 37 / 49 cytokines in DC culture supernatants, using a 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 show very high levels of IL-12-p40 and IL-12p70 release, while other cytokines, e.g., TNF-α, IL-1, IL-10 and IL-6 are produced and secreted to a much lesser extent. The release of IL-12p40 and IL-12p70 from DCs treated with the lgG2 and lgG1 variants of CP-870,893 is significantly lower compared to the release observed with the antibodies of the invention. In similar in vitro experiments, differentiated iDCs from three independent donors were treated with agonistic anti-CD40 antibodies for 48 hours at concentrations ranging from 10,000 to 5 ng / ml. Receptor expression and cytokine release were analyzed as described above.The fitting curves and EC50 calculation were obtained using Excel (Microsoft) and XLfit (IDBS). The data presented in Figures 13 and 14 exemplify the dose-dependent effects observed in one donor. Results from two other donors showed qualitatively similar effects. In summary, the potency of the humanized anti-CD40 antibodies of the invention, as determined by the EC50 values, is similar to that of CP-870,893, while the maximum induced effect, particularly on IL-12 cytokine release and coreceptor expression, is significantly greater than that of CP-870,893. Example 10: Stimulation of costimulatory receptors in B cells by anti-CD40 agonist antibodies.

[0138] a. Stimulation of co-stimulatory receptors by anti-CD40 IgG1-LALA antibodies was also tested in B cells. CMSPs from three different donors were isolated from the human buffy coat by Ficoll density gradient centrifugation, and the untouched B cells were purified by magnetic enrichment. Petition 870260063188, dated 06 / 26 / 2026, page 41 / 118 38 / 49 negative using a B cell isolation kit II (Miltenyi Biotech) according to the manufacturer's instructions. 2x10⁵ B cells in 100 μL of RPMI-1640 + 10% human AB serum were stimulated with antibody concentrations ranging from 500 to 0.2 ng / ml for 48 h. The stimulated B cells were harvested, stained using fluorophore-labeled antibodies against HLA-DR, CD86, and CD80 (all from Miltenyi Biotech), and analyzed by flow cytometry on a FACSVerse BD device. Figure 15 shows the dose-dependent stimulation of receptor expression in donor B cells as a fold of induction over the isotype antibody control treatment. Fitting curves and EC50 calculation were obtained using Excel (Microsoft) and XLfit (IDBS). The results demonstrate that the antibodies of the invention also stimulate co-stimulatory receptors on B cells; however, the level of upregulation is lower than that observed on DCs. Example 11: Competition of CD40L with anti-CD40 antibody bound to CD40 on cells

[0139] a. The binding of anti-CD40 antibodies of the invention to cells HEK-Blue-CD40L™ in the presence of CD40L was tested to verify if antibodies bind to the CD40L binding site on the CD40 cell surface. HEK-Blue-CD40L™ cells were pre-incubated with antibodies at their EC90 binding concentration for 30 minutes at 40°C. CD40L containing a mouse IgG2a Fc tag (AB Biosciences) at a concentration ranging from 10000 to 9.8 ng / ml were incubated for 60 minutes at 4°C. Anti-CD40 and CD40L antibodies bound to cell-expressed CD40 were detected using DyLight 405-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-human IgG (Jackson Laboratories) and analyzed using the FACSVerse instrument (BD). Figure 16A shows the stable binding of anti-CD40 concentrations. Petition 870260063188, dated 06 / 26 / 2026, page 42 / 118 39 / 49 except for CP-870,893, whose antibody binding signal decreases slightly at higher CD40L concentrations. CD40L binds to cells in a dose-dependent manner, and CP-870,893 does not significantly interfere with CD40L binding (Figure 16B). In contrast, the antibodies of the invention strongly prevent CD40L binding to cell-expressed CD40, indicating that these antibodies bind to the CD40L binding site of CD40 and block CD40L from binding to CD40 (Figure 16B). Example 12: Induction of FasR (CD95) death receptor expression by CD40 agonist antibodies in combination with CD40L

[0140] a. To test the interference of CD40L with anti-CD40 antibody-mediated effects on cells, Ramos cells were treated with CD40L alone or in combination with agonistic anti-CD40 antibodies. Ramos cells were seeded in 96-well RPMI plates containing 10% FCS at a cell density of 1.25 x 10⁶ cells / ml. Antibodies were added to the wells at a concentration of 10 pg / ml and incubated for 10 minutes at 37 °C, 5% CO₂, 95% humidity. CD40L (R&D Systems) was then added to the wells at a final concentration of 10 pg / ml and incubated overnight at 37 °C, 5% CO₂, 95% humidity. Cells were washed with DPBS and stained with a FITC-labeled antibody against CD95 (Miltenyi Biotech). Figure 17 shows that the induction of CD95 by CP-870,893 is strongly increased by the addition of CD40L, while co-treatment with all tested anti-CD40 antibodies of the invention reduces the effect of CD40L.The data indicate that the agonistic anti-CD40 antibodies of the invention, which bind CD40L to CD40, prevent synergistic and additive effects by CD40L and therefore allow for controlled and safe pharmacology. Example 13: Affinities of humanized and agonistic antibodies Petition 870260063188, dated 06 / 26 / 2026, p. 43 / 118 40 / 49 anti-CD40 lgG1-LALA

[0141] a. The biochemical affinities of the antibodies of the invention were determined by surface plasmon resonance measurements. The antibodies were reversibly immobilized on a CM5 sensor chip surface using an anti-human Fc antibody. The kinetics of the interaction of immobilized antibodies with soluble human or monkey cynomolgus monomeric protein CD40 (Acro Biosystems) were analyzed on a Biacore T200 SPR instrument. Kinetic data were determined using a 1:1 Langmuir binding model. Figure 18 shows that antibodies MAB-16-0451 and MAB-16-0464 have a KD of 1.2 and 2.6, respectively, while MAB-16-0262 and CP870,893 show KD values ​​of 15.7 or 8.9, respectively. KD values ​​generated using monkey cynomolgus CD40 protein demonstrate similar affinities. Example 14: Competitive binding of anti-CD40 antibodies to CD40

[0142] To test whether the humanized and agonistic anti-CD40 antibodies of the invention bind to overlapping regions on the CD40 molecule, a competitive binding ELISA was performed. Antibodies were coated onto 384-well Maxisorp plates at a concentration of 625 ng / ml in PBS for 60 minutes, followed by a blocking step with PBS, 2% BSA, 0.05% Tween for 70 minutes. All antibodies were incubated separately at a concentration of 10 pg / ml in tubes, along with 330 ng / ml of HIS-labeled recombinant CD40 protein (Acro Biosystems) and 4 pg / ml of peroxidase-coupled anti-HIS detection antibody (Sigma-Aldrich) for 60 minutes in ELISA buffer (PBS, 0.5% BSA, 0.05% Tween). The plate was washed three times with PBS, 0.1% Tween before adding the antibody / HIS-CD40 / anti-HIS-peroxidase mixtures to the wells of the plate. The plate was incubated for 60 minutes. The wells were washed six times with Petition 870260063188, dated 06 / 26 / 2026, page 44 / 118 41 / 49 PBS, 0.1% Tween, and 15 μl / well of TMB substrate solution (Invitrogen) were added. The reaction was blocked with 15 μl / well of blocking solution (1M HCl), and the absorbance at wavelengths of 450 and 620 nm was measured using a Tecan M1000 microplate reader. Figure 19 demonstrates that the antibodies of the invention do not compete with CP-870,893 for binding to CD40. Each antibody, however, competes with any other antibody of the invention, demonstrating that these antibodies bind to the same region on CD40. Importantly, since the agonistic activities of the antibodies of the invention cover a wide range (Examples 9 and 10), this shows that the paratopes of the anti-CD40 agonistic antibodies primarily determine the agonistic activity of the anti-CD40 antibodies. Example 15: Antibody-mediated effector function of anti-CD40 IgG1-LALA antibodies

[0143] a. To test the potency of an LALA mutation in the constant moiety of an IgG1 antibody in decreasing antibody-mediated effector function, e.g., ADCC, a Jurkat reporter effector cell line-based assay (Promega ADCC Bioassay, # G701A) was applied according to the manufacturer's instructions and using HEK-Blue-CD40L™ cells as target cells. 5000 HEK-Blue-CD40L™ cells were seeded per well in a 384-well white flat-bottom assay plate in 25 μL DMEM + 10% FCS and incubated 20 ha at 37 °C, 5% CO2. The medium was replaced with 8 μL of RPMI medium containing 4% FCS with low IgG levels, before 4000 effector cells per well were added in 8 μL of the same medium. Finally, CP-870.893 anti-CD40 antibodies, containing an Fc IgG1 or IgG1-LALA moiety, were added to 8 μl of medium at concentrations ranging from 10000 to 0.002 ng / ml. The plate was incubated for 6 hours at 37 °C, 5% CO2.The luciferase activity of effector cells was measured using luciferase assay reagents. Petition 870260063188, dated 06 / 26 / 2026, page 45 / 118 42 / 49 BioGlo (Promega) was administered according to the manufacturer's instructions. Luminescence was read using a Tecan M1000 microplate reader. The fold induction was calculated using the formula RLU (antibody treatment - background) / RLU (vehicle - background). Fitting curves were obtained using Excel (Microsoft) and XLfit (IDBS). Figure 20 demonstrates that the LALA mutation in IgG1 abolishes Fc receptor-mediated signaling in effector cells. Example 16: Safety of agonistic anti-CD40 antibody therapy in a humanized mouse model

[0144] a. To assess safety, a humanized mouse model with stem cells was applied. Nod / Scid / gamma(c)(null) FcRg- / - mice lack mouse activating Fc receptors. Therefore, the binding of therapeutic antibodies to the Fc receptor by human immune cells is not compromised by binding to the mouse Fc receptor in this model. Mice were sublethally irradiated with 1.4 Gy within the first 24 hours after birth. After 4–6 hours, mice were iv-grafted with 20,000–50,000 human hematopoietic stem cells isolated from umbilical cord blood. 12 weeks post-graft, the presence of human immune cells was validated by flow cytometry of peripheral blood cells. Successfully humanized mice were injected once intravenously with 3 pg / g of CP-870.893, MAB-16-0451, or isotype control antibody.Body weight and temperature were measured before treatment and at different times after antibody injection (Figure 21). The data show a significant reduction in body temperature in 3 of 6 mice treated with CP-870.893, and these mice had to be sacrificed due to severe impairment of their organism conditions. On the other hand, mice treated with MAB-16-0451 showed no effects. Petition 870260063188, dated 06 / 26 / 2026, page 46 / 118 43 / 49 significant changes in body temperature, nor were there other evident signs of impaired body condition. This indicates that highly agonistic anti-CD40-IgG1-LALA antibodies, without Fc receptor binding activity, can be applied therapeutically without obvious signs of toxicity, while the toxic effects of the less active agonistic CP-870,893-IgG2 antibody can be demonstrated in vivo in this model. Figure captions Figural: Cellular connection

[0145] Anti-CD40 IgG1-LALA monoclonal antibodies were tested for binding to CD40 antigen expressed on HEK-Blue-CD40L™ cells (InvivoGen). EC50 values ​​demonstrate potent binding of the tested antibodies. Figure 2: Determination of EC50 of HEK-Blue in 8-point analysis

[0146] Agonistic activity of anti-CD40 monoclonal antibodies Humanized IgG1-LALA was tested in a cell-based NFkB gene reporter assay. HEK-Blue-CD40L™ (InvivoGen) cells were incubated for 24 hours with different concentrations of the antibodies. EC50 values ​​demonstrate the potency of the antibodies to induce NF-kB signaling. Figure 3: Competition between epitope and CD40 binding

[0147] To test whether humanized anti-CD40 IgG1LALA monoclonal antibodies bind to an epitope that overlaps with the CD40L binding site, a CD40L competition ELISA was performed. Different concentrations of anti-CD40 antibodies were pre-incubated with recombinant CD40 protein to form a binding complex. Subsequently, the complex was added to microtiter plates coated with recombinant CD40L. After washing, the bound CD40-anti-CD40 complexes were detected using a Petition 870260063188, dated 06 / 26 / 2026, page 47 / 118 44 / 49 anti-human F(ad)2 peroxidase-linked antibody. ELISA signals such as for the reference antibody CP-870,893 indicate no competition with CD40L and therefore bind to an epitope distinct from the CD40L binding site. The data demonstrate that the tested humanized anti-CD40 IgG1-LALA monoclonal antibodies bind to an epitope that overlaps with the CD40L binding site. Figure 4: CD40 binding activity of cynomolgus monkeys

[0148] The binding activity of anti-CD40 monoclonal antibodies Humanized IgG1-LALA antibodies to cynomolgus macaques (Macaca fascicularis) were tested in an ELISA using recombinant cynomolgus macaque CD40 protein (Acro Biosystems). EC50 values ​​indicate potent antibody binding. nd = not detectable in the tested concentration range. Figure 5 a) b): Induction of dendritic cell maturation

[0149] To test the agonistic activity of humanized anti-CD40 IgG1-LALA monoclonal antibodies in primary target cells, maturation of immature dendritic cells derived from monocytes was analyzed. Immature dendritic cells, which were differentiated in vitro from monocytes, were incubated for 48 h with agonistic anti-C40 antibodies at a concentration of 5 pg / ml. Secreted IL12p40 derived from dendritic cells was subsequently quantified in the supernatant medium by biochemical ELISA. Figure 6: Dendritic cell maturation

[0150] The activity of humanized anti-CD40 IgG1-LALA monoclonal antibodies to stimulate IL12p40 secretion by dendritic cells was determined at different antibody concentrations and the activity was compared to CP-870,893 antibodies carrying different Fc portions (IgG1, IgG1-LALA, IgG2, and IgG1-V11). The antibodies were incubated for 48 h with differentiated immature dendritic cells in vitro. IL12p40 release was measured by ELISA. Petition 870260063188, dated 06 / 26 / 2026, page 48 / 118 45 / 49 Figure 7: Determination of EC50 of anti-CD40 antibodies in dendritic cell maturation.

[0151] The EC50 values ​​of humanized anti-CD40 IgG1-LALA monoclonal antibodies on dendritic cell-mediated IL12p40 secretion were determined by testing antibody concentrations ranging from 10 - 0.005 pg / ml. The antibodies were incubated for 48 h with immature dendritic cells differentiated in vitro. IL12p40 release was measured by ELISA. Figure 8: Cytokine release assay

[0152] Humanized anti-CD40 IgG1-LALA monoclonal antibodies were tested in the high-density CMSP cytokine release assay at 10 pg / ml to determine the overall induction of inflammatory cytokines such as TNF-alpha. The data indicate that, unlike an anti-CD3 antibody (OKT3), anti-CD40 antibodies do not induce significant TNF-alpha secretion. Figure 9: Antibody pulse-chase cell assay

[0153] Antibody binding dynamics and internalization were tested in a pulse-chase cell assay. Antibodies were incubated with HEK-Blue-CD40L™ cell cultures for 15 min at a concentration of 0.8 pg / ml. After washing, antibodies were allowed to internalize for 60 min before cells were washed again and the remaining cell surface anti-CD0 antibody was detected by a secondary antibody labeled with Alexa488. Under conditions that do not allow internalization, cells were treated similarly, but antibodies were incubated for only 15 min, followed by washing and secondary antibody incubation. Data show that under conditions that allow internalization, the signals of surface-located antibodies are reduced to different extents for the humanized anti-CD40 IgG1-LALA monoclonal antibodies tested. A strong signal reduction is Petition 870260063188, dated 06 / 26 / 2026, page 49 / 118 46 / 49 observed for all CP-870,893 antibody isoforms. Figure 10: Correlation of reporter gene induction and dendritic cell maturation activity of humanized anti-CD40 antibodies.

[0154] 88 anti-CD40 IgG1-LALA antibodies were tested for their activity in a HEK-Blue gene reporter assay and a dendritic cell maturation assay. HEKBlue reporter gene activity is quantified by OD@ 655 correlated with SEAP-induced secretion, dendritic cell maturation is quantified by IL12p40 release (ELISA). Figure 11: Stimulation of costimulatory receptors in dendritic cells by anti-CD40 agonist antibodies.

[0155] Immature iDCs differentiated in vitro were stimulated with agonistic CD40 antibodies, isotype control antibodies, or CD40L for 48 hours. The expression of co-stimulatory receptor molecules was measured by flow cytometry. Mean fluorescence intensities were normalized to isotype control treatments or, in the case of CD40L, to untreated samples. The induction of expression is expressed as the induction fold (FOI) over the control treatment. Figure 12: Cytokine release by dendritic cells treated with anti-CD40

[0156] Immature iDCs differentiated in vitro were stimulated with agonistic CD40 antibodies, isotype control antibodies, or CD40L for 48 hours. Cytokine release was measured by ELISA (IL12p40) or flow cytometry using a BD cytometry microsphere array. Figure 13: Dose-dependent stimulation of costimulatory receptors in dendritic cells by anti-CD40 agonist antibodies. Petition 870260063188, dated 06 / 26 / 2026, page 50 / 118 47 / 49

[0157] Immature iDCs differentiated in vitro were stimulated with agonistic CD40 antibodies or isotype control antibodies for 48 hours at concentrations ranging from 10000 to 5 ng / ml. The expression of co-stimulatory receptor molecules was measured by flow cytometry. Mean fluorescence intensities were normalized to isotype control treatments. The induction of expression is expressed as the induction fold (FOI) over the control treatment. Calculated EC50 values ​​are presented in the table. Figure 14: Dose-dependent cytokine release by dendritic cells treated with anti-CD40

[0158] Immature iDCs differentiated in vitro were stimulated with agonistic CD40 antibodies and isotype control antibodies for 48 hours. Cytokine release was measured by flow cytometry using a BD cytometry microsphere array. Figure 15: Dose-dependent stimulation of costimulatory receptors in B cells by anti-CD40 agonist antibodies.

[0159] iB cells were stimulated with CD40 agonist antibodies or isotype control antibodies for 48 hours at concentrations ranging from 500 to 0.2 ng / ml. The expression of co-stimulatory receptor molecules was measured by flow cytometry. Mean fluorescence intensities were normalized to isotype control treatments. Induction of expression is expressed as induction fold (FOI) over the control treatment. Calculated EC50 values ​​are presented in the table. Figure 16: Competition of anti-CD40 antibodies with CD40L-binding CD40 expressed in cells

[0160] HEK-Blue-CD40L™ cells were pre-incubated with anti-CD40-IgG1-LALA antibodies at EC90 concentrations before the addition of CD40L at concentrations ranging from 10000 to 9.8 ng / ml. The Petition 870260063188, dated 06 / 26 / 2026, page 51 / 118 48 / 49 anti-CD40 antibodies and the binding of CD40L to CD40 expressed on the cell surface were detected using different fluorophore-coupled secondary antibodies. Figure 17: Induction of FasR (CD95) death receptor expression by CD40 agonist antibodies in combination with CD40L

[0161] B-cell lymphoma Ramos cells were stimulated overnight with CD40L alone or in combination with anti-CD40 agonist antibodies or isotype control antibodies. CD95 expression was quantified by flow cytometry, the upregulation of expression is expressed as fold induction (FOI) over the control treatment. Figure 18: Affinities of humanized and agonistic anti-CD40 IgG1-LALA antibodies

[0162] Biochemical affinities were measured by surface plasmon resonance on a Biacore T200 SPR instrument. Kinetic data were determined using a 1:1 Langmuir binding model. Figure 19: Antibody competition for CD40 binding.

[0163] Binding of a pre-incubated mixture of HIS-labeled CD40 protein and different anti-CD40 antibodies to plates coated with different anti-CD40 antibodies. Consecutive binding of both anti-CD40 antibodies is detected by anti-HIS-labeled POD antibodies. Figure 20: Antibody-mediated effector function of anti-CD40 IgG1-LALA antibodies

[0164] Anti-CD40 antibody-mediated effector function was analyzed using a Jurkat effector luciferase reporter gene cell line and HEK-Blue-CD40L™ as target cells. IgG1-LALA or IgG1 anti-CD40 antibodies were incubated at doses ranging from 10000 to 0.002 ng / ml with target and effector cells for 6 hours. The fold Petition 870260063188, dated 06 / 26 / 2026, page 52 / 118 A 49 / 49 induction of measured luciferase activity indicates activation of effector cells mediated by anti-CD40 antibodies. Figure 21: Safety of agonistic anti-CD40 antibody therapy in a humanized mouse model.

[0165] Nod / Scid / gamma (c) (null) FcRg - / - mice were injected with 3 pg / g of MAB-16-0451 or CP-870.893 antiCD40 antibodies on day 0. Temperature was measured before and at different times after injection. Three mice treated with CP870.893, which showed a striking reduction in body temperature, had to be sacrificed after 3 days. Figure 22: Sequences (amino acids in one-letter code)

[0166] Complete sequences of variable regions (VR):

[0167] Heavy chain: VH complete: SEQ ID NO: 1-14

[0168] Light chain: Full VL: SEQ ID NO: 15-28

[0169] Complementar Determining Region (CDR):

[0170] Heavy chain:

[0171] CDR-H1: SEQ ID NO: 29-42

[0172] CDR-H2: SEQ ID NO: 43-56

[0173] CDR-H3: SEQ ID NO: 57-70

[0174] Light chain:

[0175] CDR-L1: SEQ ID NO: 71-84

[0176] CDR-L2: SEQ ID NO: 85-98

[0177] CDR-L3: SEQ ID NO: 99-112 Petition 870260063188, dated 06 / 26 / 2026, page 53 / 118

Claims

1 / 3 CLAIMS 1. Monoclonal agonist antibody or an antigen-binding fragment thereof, characterized in that it binds specifically to the human CD40 receptor and is capable of inducing CD40 signaling independent of Fcy-mediated CD40 receptor cross-linking, wherein the antibody comprises a CDR1H region of SEQ ID NO: 33, a CDR2H region of SEQ ID NO: 47 and a CDR3H region of SEQ ID NO: 61; and a CDR1L region of SEQ ID NO: 75, a CDR2L region of SEQ ID NO: 89 and a CDR3L region of SEQ ID NO:

103.

2. Antibody, according to claim 1, characterized in that the antibody is a humanized IgG1LALA comprising amino acid substitutions at L234A and L235A of the Fc region of human IgG1, wherein the numbering follows the EU numbering system.

3. Antibody, according to claim 1, characterized in that the antibody comprises 4 amino acid substitutions at S228P and L235E of the Fc region of human IgG4, wherein the numbering follows the EU numbering system.

4. Antibody, according to any one of claims 1 to 3, characterized in that the antibody binds to an epitope that overlaps with the CD40L binding site.

5. Antibody, according to any one of claims 1 to 4, characterized in that it has at least one of the following features: (a) no binding to the Fcy receptor; (b) has binding affinity to CD40 cells with an EC50 value equal to or less than 49.5 ng / ml; (c) has KD values ​​equal to or less than 15.7 nM; (d) is cross-reactive to cynomolgus monkey CD40 with a KD value equal to or less than 10.3 nM; (e) inhibits CD40L by binding to CD40; (f) avoids synergistic and additive effects of CD40L-mediated functions; (g) induces maturation of antigen-presenting cells, as determined by the release of IL12p70 with an EC50 value equal to or less than 208 ng / ml and / or as determined by the induction of CD86 in dendritic cells by at least 7.5 times and with an EC50 equal to or less than 148 ng / ml; and / or (h) reduces the level of CD40 on the cell surface by less than 50%.

6. Antibody or antigen-binding fragment, according to any one of claims 1 to 5, characterized in that the antibody comprises a variable region of the heavy chain (VH) of SEQ ID NO: 5; and a variable region of the light chain (VL) of SEQ ID NO:

19.

7. Antibody, according to any one of claims 1 to 6, characterized in that it is for use in the treatment of patients suffering from cancer.

8. Antibody, according to claim 7, characterized in that cancer is a solid tumor.

9. Antibody, according to claim 7 or 8, characterized in that the cancer is selected from the group comprising pancreatic cancer, advanced pancreatic carcinoma, lung cancer, non-small cell lung cancer (NSCLC), bronchioloalveolar cell lung cancer, bone cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, Petition 870260063188, dated 06 / 26 / 2026, page 55 / 118 3 / 3 kidney cancer, Hodgkin's lymphoma, liver cancer, gallbladder cancer, bladder cancer, prostate cancer, thyroid cancer, salivary gland cancer or uterine cancer.

10. Antibody, according to any one of claims 7 to 9, characterized in that the antibody is used in the treatment of cancer in combination with cytotoxic or cytostatic agents, radiotherapy, targeted therapy, immunotherapy or surgery.

11. Pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable vehicle and a therapeutically effective amount of the antibody, as defined in any one of claims 1 to 6.

12. Use of an antibody, as defined in any one of claims 1 to 6, characterized in that it is for the manufacture of a medicament or composition for the treatment of cancer in patients suffering from the same.

13. Use of an antibody, as defined in any one of claims 1 to 6, in combination with cytotoxic or cytostatic agents, characterized in that it is for the manufacture of a medicament or composition for the treatment of cancer. Petition 870260063188, dated 06 / 26 / 2026, p. 56 / 118

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