Agonistic CD40 antibodies as immunostimulants

Abstract

The present invention relates to a humanized monoclonal agonistic antibody, or an antigen-binding fragment thereof, which specifically binds to the human CD40 receptor, induces CD40 signaling independent of CD40 receptor crosslinking via Fcy, and can be used as an immunostimulant.

Description

[Technical field]

[0001] The present invention relates to a humanized monoclonal agonistic antibody, or an antigen-binding fragment thereof, that specifically binds to the human CD40 receptor and can induce CD40 signaling independent of Fcγ-mediated CD40 receptor crosslinking, for use as an immunostimulant. [Background technology]

[0002] Recent successes in cancer immunotherapy have revived the hypothesis that the immune system can control many, if not most, cancers, and in some cases generate durable responses in a way not seen with many small molecule drugs. Agonistic CD40 monoclonal antibodies (mAbs) offer a new therapeutic option with the potential to generate anti-cancer 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 major component of T cell "help" for immune responses. Signaling through CD40 on APCs primarily mediates 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 presented. As with several other members of the TNF receptor family, CD40 signaling is mediated by adaptor molecules rather than through the intrinsic signaling activity of the CD40 cytoplasmic tail. Receptor engagement activates downstream kinases and translocates multicomponent signaling complexes from CD40 to the cytosol, activating several well-characterized signaling pathways.

[0006] Antagonistic human CD40 antibodies are known in the art. Data show that toxicity correlates with Fcγ cross-linking, but most of the therapeutic anti-CD40 antibodies that have been clinically tested act via Fcγ cross-linking. Therefore, silent Fc mutants have been developed that reduce Fcγ-mediated CD40 receptor cross-linking. Individual mutations in the Fc region of human IgG1 are described, for example, in US Patent Publication No. 2018 / 0118843.

[0007] A recently designed immunomodulatory approach uses agonistic monoclonal antibodies (mAbs) targeting CD40 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, promote antitumor T cell responses, and cultivate cytotoxic myeloid cells capable of controlling 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] WO 2019 / 057792 describes humanized monoclonal agonistic antibodies, or antigen-binding fragments thereof, that specifically bind to the human CD40 receptor and can induce CD40 signaling independent of CD40 receptor cross-linking via Fcγ.

[0009] Thus, agonistic CD40 mAbs represent a promising strategy for novel immunotherapy, especially cancer immunotherapy. However, concerns have been raised regarding their potential cytotoxic side effects. Agonistic monoclonal CD40 antibodies may induce cytokine release syndrome, autoimmune reactions, thromboembolic syndromes (due to expression of CD40 by platelets and endothelial cells), over-immunostimulation leading to activation-induced cell death or tolerance, and tumor angiogenesis. These effects may cause untoward 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] Thus, there is a need to provide effective immunotherapies with minimal or no toxic side effects. In this application, the immunomodulatory capabilities of agonistic CD40 antibodies have been evaluated. Certain antibodies have been found to have a surprisingly wide therapeutic window that elicits immune stimulatory effects without causing excessive toxic effects. Summary of the Invention

[0011] The present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to the human CD40 receptor and induces CD40 signaling independent of Fcγ-mediated CD40 receptor cross-linking for use as an immunostimulant. According to the present invention, the antibody or antibody fragment is provided for administration to a patient in a single dose of about 0.1-1 mg per kg of patient body weight. In this range, they can elicit a strong immunostimulatory effect while causing negligible, if any, toxic side effects. The present invention also provides pharmaceutical compositions comprising the antibody suitable for use in the treatment of conditions or diseases in which it is desirable to stimulate the immune system, for example, for use in the prevention or treatment of infectious diseases, as well as for use in the treatment of patients suffering from cancer. [Brief description of the drawings]

[0012] [Figure 1] (A) Phenotype identification of CD20+ B cells, CD14+ classical monocytes (CM), CD11c+ myeloid dendritic cells (MDC) and CD123+ plasmacytoid dendritic cells (PDC) in rhesus PBMCs by flow cytometry according to the gating strategy shown. One representative animal is shown. (B) Baseline expression of CD40 on different rhesus immune cells. Mean fluorescence intensity (MFI) of CD40 is shown.

[0013] First, MAB273 was tested in vitro on rhesus macaque PBMCs to determine whether this antibody exhibited cross-reactivity and whether the rhesus macaque model could be used to study this antibody in vivo. The flow cytometric gating strategy is shown in Figure 1A.

[0014] B cells have the highest baseline expression of CD40, followed by plasmacytoid dendritic cells (PDCs) and myeloid dendritic cells (MDCs), whereas classical monocytes (CMs) have relatively low baseline expression of CD40 (Figure 1B).

[0015] [Diagram 2] Rhesus macaque PBMCs were stimulated with MAB273 (1, 10, 20 μg / ml), isotype control Ab (1, 10, 20 μg / ml), or TLR7 / 8L (5 μg / ml) for 24 h. Surface expression of cell activation markers (CD86, CD70), lymph node homing marker (CCR7), and CD40 on MDCs and B cells was assessed by flow cytometry. Data were obtained from four independent experiments. MFI values ​​are shown for n=4 mice. Statistical analysis was performed using two-tailed paired t-test.

[0016] We next analyzed the phenotypic differentiation of cells after 24 h of stimulation with MAB273 in vitro, gating on different cell subpopulations, focusing in particular on cells expressing CD40 (Figure 1). MAB273 activated MDC and B cells and upregulated the expression of CD86, CD70, and CCR7 (Figure 2A), whereas no upregulation was observed with an isotype control antibody. Regarding CD40 staining, the data indicate that CD40 staining antibodies are blocked by the binding of MAB273, so CD40 expression cannot be detected on cells after incubation with MAB273. · Activation of PDC and CM was absent or limited (Figure 2B).

[0017] [Diagram 3] Rhesus macaque PBMCs were stimulated with anti-CD40 Ab (1, 10, 20 μg / ml), isotype control Ab (1, 10, 20 μg / ml), or TLR7 / 8L (5 μg / ml) for 24 h. TNF and IL-12 p70 levels were measured in supernatants harvested from cell cultures. n=4 animals.

[0018] This shows the following: MAB273 stimulated low levels of TNF secretion at both 1 and 10 μg / ml, but even lower levels at 20 μg / ml, likely due to toxicity. TLR7 / 8 ligands were used as positive controls. In contrast, an isotype control antibody did not induce TNF. No IL-12p70 was detected in the cultures, possibly because it was produced below the detection level of the ELISA kit. In any case, it can be concluded that the secretion of significant amounts of IL-12p70 was not induced by in vitro stimulation of rhesus macaque PBMCs with MAB273.

[0019] [Figure 4](A) Gating strategy. (B) Human PBMCs were labeled with 0.25 μM CellTrace Violet for 20 min at 37°C and then stimulated with MAB273 (1, 0.1, 0.01, 0.001 μg / ml), anti-CD40 Ab (1, 0.1, 0.01, 0.001 μg / ml), CpG B (1 μg / ml) or SEB (0.2 μg / ml) for 6 days. B cell proliferation status was assessed by flow cytometry.

[0020] We also performed a B cell proliferation assay after 6 days of culture in MAB273, which showed that: Detectable B cell proliferation was observed in PBMCs treated with different concentrations of MAB273, especially in the 0.1 μg / ml group (Figure 4). However, no proliferation was observed in the anti-CD40 Ab group. In the CpG group as a positive control, strong proliferation was observed as expected.

[0021] [Diagram 5] Fresh blood samples (350 μl) were sent to Adlego Biomedical AB (based at Karolinska Institutet Science Park) for clinical chemistry testing.

[0022] Following the in vitro studies, six rhesus monkeys were split into three groups and MAB273 was tested in vivo under three different conditions: first, they were given 1 mg / kg intravenously, the second group was given a 10-fold lower dose, and the third group was given a 10-fold lower dose intravenously. Clinical chemistry tests, including a battery of tests of liver and kidney function, were performed.

[0023] This shows the following: In the 1 mg / kg iv group, several parameters were elevated above the healthy reference range. This group also experienced side effects such as loss of appetite, vomiting, and behavioral changes. At a reduced dose of 0.1 mg / kg iv, no significant side effects were observed. No significant side effects were observed with the sc dose of 0.1 mg / kg. In the 0.1 mg / kg group (both iv and sc), almost all parameters remained within normal ranges (Figure 5).

[0024] [Figure 6] Fresh blood samples (350 μl) were sent to Adlego Biomedical AB (Karolinska Institutet Science Park) for complete blood count (CBC).

[0025] Complete blood counts showed significant changes in cell counts after administration of MAB273.

[0026] This shows the following: A rapid decrease in platelets and a rapid increase in granulocytes were observed already between 30 minutes and 4 hours after administration in all groups (Figure 6). The change in cell count was dose-dependent, with the effect being significantly increased in the 1mg / kg iv administration group.

[0027] [Figure 7] Plasma MAB273 concentrations after administration were measured by our custom ELISA assay. ULOD = upper limit of detection (67967.1 ng / mL), LLOD = lower limit of detection (0.047 ng / mL), LLOQ = lower limit of quantification (0.261 ng / mL). n = 2 animals / group.

[0028] The plasma MAB273 concentration after administration was measured using ELISA.

[0029] This shows the following: MAB273 was already well detectable 30 min after administration, especially in the intravenous administration group (Figure 7). In the high-dose group, MAB273 concentrations peaked between 30 minutes and 4 hours after administration, then began to decline gradually, becoming undetectable by day 14. In the low-dose intravenous group, peak concentrations were detected after 30 minutes and then decreased continuously until they were no longer detectable on the seventh day. In the low-dose sc group, the peak concentration of MAB273 occurred later (around day 3) and persisted for a longer period before becoming undetectable between days 7 and 14. The maximum concentration was relatively low compared to the other two groups.

[0030] [Figure 8] (A) Gating strategy. (B) Cell frequencies in rhesus PBMCs were assessed by flow cytometry. n=2 animals / group.

[0031] To further subdivide immune cell population dynamics at the subset level and analyze their activation state, PBMC samples were tracked longitudinally using a 14-parameter flow cytometry panel (Figure 8A). Analyzed cell subset frequencies were normalized to complete blood count (CBC) data (Figure 6).

[0032] This shows the following: Cellular fluctuations followed a similar trend to the CBC data, with a sharp increase in neutrophils and a transient decrease in B cells and MDCs (Figure 8B). The observed dynamics can be interpreted as a redistribution of immune cells from the circulation to tissues and regrowth of new immune cells from the bone marrow.

[0033] [Figure 9] Cell surface expression of CD40 on rhesus macaque PBMC was assessed by flow cytometry. Mean fluorescence intensity (MFI) of CD40 is shown. n=2 animals / group.

[0034] Targeting of cell surface CD40 by MAB273 was assessed by quantitating loss of competing fluorescent antibody binding by flow cytometry (as described in Fig. 2 ).

[0035] This shows the following: · MAB273 rapidly and effectively blocked CD40 on B cells and MDCs (Figure 9). The kinetics of CD40 detection followed the pharmacokinetics of MAB273 in plasma (Figure 7). Restoration of detectable CD40 expression may be due to the emergence of new cells from the bone marrow and the half-life of the antibody in vivo.

[0036] [Figure 10] Cell surface expression of CD80, a costimulatory marker, in rhesus macaque PBMCs was assessed by flow cytometry. Mean fluorescence intensity (MFI) of CD80 is shown. n=2 animals / group.

[0037] Activation by phenotyping was also analyzed by flow cytometry.

[0038] This shows the following: The expression of CD80, a costimulatory marker, increased immediately after MAB273 administration, followed by a decrease in B cells, especially in the iv administration group (Figure 10). After rapid phenotypic maturation of APCs, the cells leave the circulation and repopulation of new immature APCs occurs.

[0039] [Figure 11] CCR7, a lymph node homing marker, was assessed by flow cytometry on rhesus PBMCs. Mean fluorescence intensity (MFI) of CCR7 is shown. n=2 animals / group.

[0040] Similar to CD80 expression, we found the following. Expression of the lymph node homing marker CCR7 was also rapidly increased in B cells and subsequently decreased (Figure 11). The reason is probably the same as for CD80.

[0041] [Figure 12] Plasma cytokine (IFN-γ and IL-6) concentrations were measured using ELISA kits according to the manufacturer's protocol. n=2 animals / group.

[0042] Systemic levels of inflammatory cytokines in plasma were assessed by ELISA. We detected: Both IFN-γ and IL-6 were significantly increased only on days 1 or 2 of high-dose administration (Figure 12). TNF-α and IL-12 p70 were out of the detection range even when high-sensitivity ELISA kits (TNF ELISA kits: BMS223HS, BMS654, 3512M-1H-6, IL-12 p70 ELISA kit: BMS646) were added.

[0043] This shows the following: The absence of systemic cytokine release in the lower dose group is consistent with the more favorable safety profile observed.

[0044] [Figure 13] Time course of Ag-specific T cells in PBMC (A) and BAL (B). Data summarized are background subtracted. Prime and booster immunizations were performed at weeks 0 and 7, respectively (three animals received 0.1 mg / kg sc HIV envelope peptide alone, and another three animals received 0.1 mg / kg sc HIV envelope peptide in combination with 0.1 mg / kg sc MAB273 for both prime and boost).

[0045] A second booster immunization was also performed, this time with 1 mg / kg HIV envelope peptide sc in all six animals, followed by 0.1 mg / kg MAB273 sc on week 11. Individual data are shown as dots. n=3 animals / group.

[0046] This shows the following: Envelope-specific CD4 and CD8 T cell responses were induced in both groups, but at low levels, possibly because the dose of 0.1 mg / kg HIV1-envelope peptide was not optimal. · Nevertheless, the frequencies of envelope-specific CD4 and CD8 T cells were overall higher in the MAB273 group compared to the group receiving peptide alone. A second booster, in which all animals received MAB273 plus a higher dose of peptide, boosted T cell responses in both groups of animals. In addition to responses in PBMC, T cell responses were also detected in BAL.

[0047] [Figure 14] Histograms of Alexa Fluor 680 signal from different cell populations in different tissues. Control = peripheral blood B cells from the same animal, but just before the chase immunization. n=3 animals.

[0048] Finally, to assess the biodistribution of MAB273 following treatment, three animals were administered 0.1 mg / kg sc Alexa Fluor 680-labeled MAB273. Animals were withdrawn from treatment after 24 or 48 hours and necropsied to assess the presence of MAB273 in tissues.

[0049] This shows the following: ·Alexa Fluor 680 signal was only detected at the injection site (L. skin) and in specific LNs draining the injection site (L. inguinal, L. com. iliac and paraaortic LNs). ·Alexa Fluor 680 signal gradually decreased the further away the LN was from the injection site. More MAB273 reached more distant LNs after 48 hours compared with 24 hours. The main cell populations targeted by MAB273 in vivo were monocytes, MDCs, and neutrophils. This is likely because anti-MAB273 IgG prevents systemic dissemination in these non-naive animals.

[0050] [Figure 15] Sequences (amino acids in one-letter code) Complete sequences of the variable regions (VR): Heavy chain: VH full length: SEQ ID NO: 1 Light chain: VL full length: SEQ ID NO: 2

[0051] Complementarity Determining Regions (CDRs): CDR-H1: SEQ ID NO:3 Heavy chain: CDR-H2: SEQ ID NO:4 CDR-H3: SEQ ID NO:5

[0052] CDR-L1: SEQ ID NO:6 Light chain: CDR-L2: SEQ ID NO:7 CDR-L3: SEQ ID NO:8 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] definition The term "antibody" includes, but is not limited to, whole antibodies and antibody fragments, and encompasses various forms of antibody structures as long as they exhibit the properties of the present invention.

[0054] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an 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.

[0055] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0056] 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. 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 the human germline sequence than the germline sequence of the original species. The assessment of humanization is based on the resulting amino acid sequence, and not on the methodology per se.

[0057] The term "specifically binds to a target or anti-target antibody" as used herein 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 allowing the formation of an immune complex with the respective antigen or protein, detecting the immune complex by measuring the optical density value (OD) using a secondary antibody that binds to the antibody according to the invention and using peroxidase-mediated color development.

[0058] The term "antigen" in the present invention refers to an antigen used for immunization or a protein that contains said 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 of the protein can be used for detection / assay etc.

[0059] By "specifically binds" or "specifically recognized" herein is meant an antibody that exhibits adequate affinity for an antigen, preferably one that does not exhibit significant cross-reactivity.

[0060] 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, for example, competitive binding assays such as ELISA.

[0061] 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.

[0062] As used herein, the term "variable region (or domain) of an antibody according to the invention" (variable region of the light chain (VL), variable region of the heavy chain (VH)) refers to each of the pair of light and heavy chain regions that are directly involved in binding the antigen to the antibody. The variable light and heavy chain regions have the same general structure, each region comprising four framework (FR) regions, the sequences of which are largely conserved and which are linked by three complementarity determining regions, CDRs.

[0063] The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Antigen-binding portions of an antibody preferably comprise amino acid residues from the "complementarity determining regions" or "CDRs". 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 linear amino acid sequence 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, etc. according to Kabat). The Kabat numbering of residues for a given antibody may be determined by alignment of the homologous regions of that antibody's sequence with the "standard" Kabat numbered sequences.

[0064] The "constant region (constant part)" is not directly involved in binding of the 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, 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.

[0065] 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.

[0066] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to 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).

[0067] A "variant Fc region" comprises an amino acid sequence, which differs from that of a "native" or "wild-type" Fc region sequence by virtue of at least one "amino acid modification" as defined herein.

[0068] As used herein, the term "Fc variant" refers to a polypeptide that contains 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.

[0069] The term "Fc region-containing polypeptide" refers to a polypeptide that includes an Fc region, such as an antibody.

[0070] 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. FcγRIIA, an activating receptor, contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. FcγRIIB, an inhibitory receptor, contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcRs 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 delivery of maternal IgG to the fetus (Guyer et al., J. Immunol. 117 (1976) 587 and Kim et al., J. Immunol. 24 (1994) 249).

[0071] As used herein, "IgG Fc ligand" refers to 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, FcγR, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that share homology with FcγR (Davis et al., Immunological Reviews 190 (2002) 123-136, see generally). (Incorporated by reference herein). Fc ligands may 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, from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0072] 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, but is not limited to, FcγRI (CD64), which includes isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32), which includes isoforms FcγRIIA (including allotypes H131 and R131), FcγRIIB (including FcγRIIB-1 and FcγRIIB-2), and FcγRIIc; and FcγRIII (CD16), which includes 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 undiscovered human FcγR or FcγR isoforms or allotypes. FcγR may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0073] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the IgG antibody Fc region and is encoded, at least in part, by the FcRn gene. FcRn may be from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As is 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 FcRn heavy chain and beta-2-microglobulin.

[0074] "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 alignment of the sequences and introduction of 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, for example, using publicly available computer software BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

[0075] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing 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, 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.

[0076] 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 immunoglobulin Fc region.

[0077] As used herein, the term "antibody effector function(s)" or "effector function" refers to a function contributed by the Fc effector domain(s) of IgG (e.g., the Fc region of an immunoglobulin). Such a function is brought about, 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.

[0078] "C1q" is a polypeptide that contains a binding site for the Fc region of immunoglobulins. Together with two serine proteases, C1r and C1s, C1q forms the complex C1, which is the first component of the complement-dependent cytotoxicity (CDC) pathway.

[0079] 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.

[0080] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount that is effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0081] The term "infectious disease" as used herein refers to any disease caused by infectious organisms.Infectious organisms can include viruses (e.g., RNA viruses, DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B and C viruses, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus (HPV)), parasites (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria, especially M.tuberculosis, Salmonella, Streptococci, E.coli, Staphylococci), fungi (e.g., Candida species, Aspergillus species), Pneumocystis carinii, and prions.

[0082] 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. The cancer may be 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, brain stem gliomas, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, lymphomas, lymphocytic leukemias (including refractory versions of any of the above cancers), or a combination of one or more of the above cancers.

[0083] As used herein, the term "immunotherapy" refers to the treatment of a disease or condition, e.g., therapeutic or prophylactic treatment, that aims at and / or results in an immune response (e.g., an active or passive immune response).

[0084] The term "immunotherapeutic agent" refers to an agent that includes or consists of one or more immunogens, immunoglobulins, antibodies, antibody fragments, or combinations thereof for use in immunotherapy. Thus, the term "immunotherapeutic agent" as used herein also includes nucleic acids that code for immunogens, immunoglobulins, antibodies, or antibody fragments. Such nucleic acids can be DNA or RNA. The nucleic acid segment that codes for the immunogen is typically linked to regulatory elements such as promoters and enhancers that allow expression of the DNA segment in the intended target cells of the subject or patient.

[0085] An "immunogenic agent" or "immunogen" is capable of inducing an immunological response against itself upon administration to a patient, optionally in combination with an adjuvant. An "immunogenic composition" is a composition that includes an immunogenic agent.

[0086] "Adjuvant" refers to a compound that enhances the immune response to an immunogen when administered together with the immunogen, but does not produce an immune response when administered alone. Adjuvants can enhance the immune response through several mechanisms, including recruitment of lymphocytes, stimulation of B cells and / or T cells, and stimulation of macrophages.

[0087] Detailed Description of the Invention The present invention addresses the need to provide agonistic CD40 antibodies or antigen-binding fragments thereof as immunostimulants in medical treatment. According to the present invention, antibodies or antibody fragments are provided for administration to patients at a dose of about 0.1-1 mg / kg of patient body weight. In this range, the specific antibodies described herein are capable of providing the desired signaling capabilities and clinical effects without eliciting significant cytotoxicity.

[0088] Of WO2019 / 057792, one of the agonistic CD40 antibodies was found to have a particularly large therapeutic window. It is already effective at very low doses of 0.1 mg per patient body weight, with major toxic effects occurring only at doses more than 10 times higher. An antibody or antigen-binding fragment thereof for use according to the present invention comprises a VH region comprising the CDR1H region of SEQ ID NO: 3, the CDR2H region of SEQ ID NO: 4, and the CDR3H region of SEQ ID NO: 5, and a VL region comprising the CDR1L region of SEQ ID NO: 6, the CDR2L region of SEQ ID NO: 7, and the CDR3L region of SEQ ID NO: 8, where the CDRs may comprise any one or more amino acid mutations that do not reduce their activity according to the present invention.

[0089] Preferably, the CDRs have at least 91%, preferably 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to their respective SEQ ID NOs.

[0090] Preferably, the antibody comprises a VH region comprising the CDR1H region of SEQ ID NO:3, the CDR2H region of SEQ ID NO:4 and the CDR3H region of SEQ ID NO:5, and a VL region comprising the CDR1L region of SEQ ID NO:6, the CDR2L region of SEQ ID NO:7 and the CDR3L region of SEQ ID NO:8.

[0091] In other embodiments, the antibody or antigen-binding fragment thereof for use according to the invention comprises a heavy chain variable (VH) region having 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 of SEQ ID NO:1.

[0092] In certain embodiments, the VH sequence having 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, such that the antibodies retain the ability to specifically bind to their respective antigens. Preferably, the heavy chain variable region (VH) sequence is SEQ ID NO:1.

[0093] The present invention also relates to antibodies comprising a light chain variable (VL) region having 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 of SEQ ID NO:2.

[0094] In certain embodiments, the VL sequence having 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 antibodies retain the ability to specifically bind to their respective antigens according to the present invention. Preferably, the light chain variable region (VL) sequence is SEQ ID NO:2.

[0095] In a particular embodiment, 1 to 10 amino acids are substituted, inserted and / or deleted in the VL sequence. In another embodiment, 1 to 10 amino acids are substituted, inserted and / or deleted in the VH sequence. In a particular embodiment, 1 to 10 amino acids are substituted, inserted and / or deleted in each of the VH or VL sequences. The substitution, insertion or deletion may occur in a region outside the CDR (i.e., within the FR).

[0096] In a preferred embodiment, the antibody is a humanized IgG1LALA antibody, in particular a humanized IgG1 type antibody, with at least two alanine amino acids at positions 234 and 235 of the human Fc1 region. Thus, according to a preferred embodiment, the IgG1LALA comprises the mutations L234A and L235A in the human Fc1 region. In another embodiment, the antibody comprises at least the amino acid substitutions S228P and L235E in the human IgG4 Fc region.

[0097] More preferably, the antibody is a recombinant molecule.

[0098] Agonistic monoclonal antibodies, or antigen-binding fragments thereof, for use according to the invention can bind to the human CD40 receptor and induce CD40 signaling independent of Fcγ-mediated CD40 receptor cross-linking, and may further exhibit reduced or depleted ability to signal through human Fcγ receptors when compared to wild-type IgG Fcγ receptor signaling, or Fcγ signaling of prior art antibodies.

[0099] In certain embodiments, the agonistic monoclonal CD40 antibody, or antigen-binding fragment thereof, for use according to the invention exhibits reduced or depleted affinity for human Fcγ receptors compared to wild-type IgG Fcγ. According to a preferred embodiment, the antibody does not bind to Fcγ receptors and correspondingly, the antibody does not induce Fcγ-mediated cross-linking of the CD40 receptor.

[0100] The antibodies for use according to the invention rapidly bind to rhesus CD40-expressing immune cells after administration and show upregulation of costimulatory and homing markers (e.g., CD80, CCR7). Pharmacokinetic analysis shows that the antibodies or antibody fragments of the invention persist in the circulation for about one week. Systemic cytokine secretion (e.g., TNF, IL-12) is low. Only at high doses is a transient increase in IFNγ and IL-6 observed. The antibodies induce a transient decrease in platelets and lymphocytes and an increase in granulocytes in the periphery, indicating immune activation. In addition, an adjuvant effect is observed that enhances antigen-specific immunity. Administration in the above range of 0.1-1 mg / kg (intravenous or subcutaneous) induces a comparable and long-lasting robust cellular and cytokine systemic immune response. This indicates that classical signaling by the antibody induces very strong CD40 activation.

[0101] In summary, classical activation of CD40 by the above defined antibodies or fragments thereof induces an appropriate systemic immune response in NHPs without toxic effects. The agonistic anti-CD40 antibody of the present invention (MAB273) shows a strong activation profile of antigen-presenting cells and enhances antigen-specific T cell responses, making it a unique potential adjuvant for inducing cytotoxic T cell immunity.

[0102] Further characteristics of antibodies for use according to the invention are described in WO2019 / 057792, the disclosure of which is incorporated herein by reference.

[0103] According to the invention, the antibody or fragment thereof is provided for administration to a patient at a dose of about 0.1-1 mg / kg of patient body weight. Preferably, the dose is less than 1 mg, e.g., less than 0.9 mg / kg, or less than 0.8 mg / kg. In preferred embodiments, the dose is in the range of about 0.2-0.7 mg / kg, 0.3-0.6 mg / kg, or 0.4-0.5 mg / kg.

[0104] The advantageous properties of said antibodies enable them to be used as immunomodulatory agents, in particular for the treatment or prevention of infectious diseases and / or cancer.

[0105] In one embodiment, the antibody or fragment thereof is used to prevent or treat infectious diseases. Infectious diseases include viral infections (e.g., RNA viruses, DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B and C viruses, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus (HPV)), parasitic infections (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacterial infections (e.g., Mycobacteria, especially M. tuberculosis, Salmonella, Streptococci, E. coli, Staphylococci), fungal infections (e.g., Candida species, Aspergillus species, Pneumocystis carinii), and prions.

[0106] The antibody or fragment thereof as defined above can support the immune response of a patient suffering from an infectious disease. Moreover, the antibody or fragment thereof can enhance the immunological response to an immunogen administered to the patient. For example, the antibody or fragment thereof can therefore be administered in or in addition to a vaccine that contains an immunogen intended to induce an immune response in a patient to protect against an infectious disease. Optionally, the antibody or fragment thereof can be combined with further adjuvants and excipients for this purpose.

[0107] It will be further understood that the patient may also receive one or more additional treatments, such as medications, such as immunotherapeutic agents. Immunotherapy may include, for example, immune checkpoint inhibition, using one or more immune checkpoint inhibitors, such as anti-TIGIT, anti-PD-L1, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40, and / or anti-GITR.

[0108] In another embodiment, the antibody or fragment thereof is used to treat a patient suffering from cancer.

[0109] The cancer may be one or more types of cancer selected from the group including pancreatic cancer, advanced pancreatic carcinoma, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolarcell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and 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.

[0110] In certain aspects, the cancer is a solid tumor.

[0111] In some embodiments, the cancer may be a CD40-expressing cancer, however this is not required for the effective functioning of the antibodies of the invention.

[0112] It will further be appreciated that the antibodies can be used as the sole treatment for a patient or as part of a combination treatment, which may include pharmaceutical cytotoxic or cytostatic agents, radiation therapy, targeted therapy, and / or surgery.

[0113] Thus, the patient may also be receiving one or more further treatments (especially for infections and cancers), such as medicines (such as cytotoxic or cytostatic agents, radiation therapy, targeted therapy, and / or surgery).

[0114] Thus, in some embodiments, the antibodies of the invention are used to treat cancer in combination with cytotoxic or cytostatic agents, radiation therapy, targeted therapy and / or immunotherapy.

[0115] 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, radiation therapy, targeted therapy, and / or immunotherapy.

[0116] The radiation therapy may be selected from the group comprising external beam radiation therapy, contact x-ray brachytherapy, brachytherapy, total body radiation therapy, or intraoperative radiation therapy.

[0117] The cytotoxic or cytostatic anti-cancer agents of the present invention may be from the groups including taxanes, anthracyclines, alkylating agents, histone deacetylase inhibitors, topoisomerase inhibitors, kinase inhibitors, nucleotide analogues, peptide antibiotics, and platinum-based agents.

[0118] Preferably, targeted anti-cancer agents are used in targeted therapy and are 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 regorafinibe.

[0119] If the patient is undergoing immunotherapy, this may be an immune checkpoint inhibitor, and one or more immune checkpoint inhibitors may be used. The one or more 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.

[0120] 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, GITR, and / or in combination with the following drugs: nivolumab, pembrolizumab, urelumab, utomilumab, atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab.

[0121] In some embodiments of the present invention, the antibody or fragment thereof is used in a weekly to monthly dosing regimen. The dosage is such that the patient dose does not exceed 1 mg / kg body weight at any time. Preferably, the patient dose is less than 1 mg / kg body weight at any time, for example, less than 0.9 mg / kg. To obtain substantial effect, the dosage can be kept at 0.1 mg / kg body weight or more for a long period of time.

[0122] In another aspect, the invention relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of an antibody or antibody fragment, the pharmaceutical composition adapted for administration of the antibody or fragment at a dose of 0.1-1 mg / kg body weight.

[0123] The pharmaceutical compositions of the invention can be adapted for controlled release of the antibody or fragment over an extended period of time, and are preferably a depot formulation for sustained release of the antibody or fragment.

[0124] In a particular embodiment, the pharmaceutical composition is a vaccine comprising the above-mentioned antibody or a fragment thereof in combination with an immunogen. Such a vaccine can be used in particular for the prevention of infectious diseases. In this case, the immunogen is a disease-associated antigen capable of inducing an immunological response.

[0125] In another embodiment, the pharmaceutical composition is for use in treating patients suffering from cancer. Such cancer can be solid tumor. Cancer can also be selected from the group including pancreatic cancer, advanced pancreatic carcinoma, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin 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.

[0126] The compositions may also be used in the treatment of cancer in combination with chemotherapy, radiotherapy, targeted therapy and / or immunotherapy, which may be immune checkpoint inhibition.

[0127] The patient treated with the composition may be one that exhibits an inadequate response and / or resistance to chemotherapy, radiation therapy, targeted therapy and / or immunotherapy.

[0128] The pharmaceutical compositions of the invention may also be used in the treatment of cancer in combination with one or more cytotoxic, cytostatic or targeted anti-cancer compounds.

[0129] It may be used in combination with one or more immune checkpoint inhibitors, which may be selected from the group including anti-TIGIT, anti-PD-L1, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40, and / or anti-GITR.

[0130] The compositions may also be used in combination with antibodies that specifically bind to human PD-L1, TIGIT, CTLA-4, LAG-3, TIM-3, CD137, OX40, GITR, and / or in combination with the agents nivolumab, pembrolizumab, urelumab, utomilumab, atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab.

[0131] It can also be administered once a week to once a month.

[0132] In another aspect, the present invention also relates to a method of treatment comprising administering to an individual in need of treatment an effective amount of an antibody, the dosage being in the range of 0.1-1 mg / kg body weight. Such an individual may be a patient suffering from an infectious disease and / or cancer. Thus, the present invention also relates to a method of treatment of infectious diseases and cancer (e.g. solid cancer).

[0133] The step of administering to an individual in need thereof may include local administration, e.g., local administration to a tumor of the patient (e.g., intratumoral or peritumoral). Preferably, administration is subcutaneous or intravenous.

[0134] For example, the 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.

[0135] It will further be appreciated that the therapeutic methods of the invention may consist of the administration of the antibody-based agents of the invention to a patient alone, or as part of a combination treatment (which further treatment may be immunotherapy, pharmaceutical cytotoxic or cytostatic agents, radiation therapy, targeted therapy and / or surgery).

[0136] Indeed, all of the features and advantageous properties of the antibodies of the invention detailed above are also reflected and encompassed in the methods of treatment and use of the antibodies according to the invention. EXAMPLES

[0137] The following examples together with the figures and tables are used to explain the invention.

[0138] Materials and Methods animal The study was approved by the local ethical committee for animal experiments. Six male (toxicity studies) / three male and three female (immunogenicity / biodistribution studies) Indian native rhesus macaques were housed at the Astrid Fagraeus Laboratory, Karolinska Institutet. All procedures were performed in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care.

[0139] Vaccination and sample collection[1-4] Toxicity studies were performed at staggered times, with animals divided into three groups. The first two received 1 mg / kg of anti-CD40 mAb (MAB273, Icano MAB GmbH) intravenously (iv), the next two received 0.1 mg / kg MAB273 iv, and the last two received 0.1 mg / kg MAB273 subcutaneously (sc). For iv administration, the antibody was infused into the saphenous vein in a total volume of 25 ml, administered stepwise at approximately 4 ml per minute every 5 min, for a total of 25 ml administered over 30 min. For sc administration, the antibody was administered as a single 0.5 ml subcutaneous injection into the skin over the left quadriceps. Blood samples were taken in 4 mL heparin tubes before, 30 min, 4 h, 24 h, 48 h, 72 h, 1 week, 2 weeks, 3 weeks, and 4 weeks after MAB273 administration. For immunogenicity studies, priming immunization was performed on day 0. Six rhesus macaques were divided into two groups (n=3). Group 1 was administered 0.1 mg / kg HIV envelope peptide (GenScript, peptide synthesis service) alone, and group 2 was administered 0.1 mg / kg HIV envelope peptide in combination with 0.1 mg / kg MAB273, sc. The second booster immunization was performed on week 11, and all six rhesus macaques were administered 1 mg / kg HIV envelope peptide in combination with 0.1 mg / kg MAB273, sc. Blood samples were collected on day 0, 48 hours after priming, and at weeks 1, 2, 3, 7, 8, 9, and 11, 48 hours after booster, and at weeks 12 and 13. Bronchoalveolar lavage (BAL) samples were collected at weeks 9 and 13. For biodistribution studies, Alexa Fluor 680-labeled MAB273 was administered at 0.1 mg / kg sc, and animals were necropsied 24 or 48 hours later, after which tissues were harvested and analyzed.

[0140] Safety Clinical Chemistry and Hematology Testing [5,6] Haematological analysis of heparinized blood was performed within 8 h of collection using an Exigo Vet instrument (Model H400, Boule Diagnostics AB, Spanga, Sweden) after QC with Boule Vet Con control blood. Parameters analysed were RBC, HCT, MCV, RDW%, RDWa, HGB, MCH, MCHC, PLT, MPV, WBC, LYM, GRAN, and MONO.

[0141] Heparinized plasma samples were analyzed using an ABAXIS Vetscan VS2 3.1.35 Chemistry analyzer (Triolab, Solna, Sweden). Parameters analyzed were TBIL, BUN, BA, ALP, ALB, ALT, CHOL, and GGT on a Mammalian Liver Profile rotor (Triolab) with individual QC controls.

[0142] Blood sample processing [2-4,7] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Fairfield, CT) density gradient centrifugation of blood samples at 2200 rpm for 25 min without braking or acceleration. PBMCs were washed and maintained in phosphate-buffered saline (PBS). Samples were either stained immediately or frozen in 90% heat-inactivated fetal bovine serum (FBS) and 10% DMSO (Sigma-Aldrich) and stored at -170°C.

[0143] Flow cytometry for natural cell profiling [2,3,7,8] Fresh LIVE / DEAD PBMCs TM After staining with the Fixable Blue Dead Cell Stain Kit (Invitrogen, L23105), cells were blocked with FcR Blocking Reagent (Miltenyi Biotec, 130-059-901) according to the manufacturer's protocol, followed by a surface staining panel as follows:

[0144] [Table 1]

[0145] After staining and washing, PBMCs were resuspended in 1% paraformaldehyde (PFA) and acquired on an LSRFortessa flow cytometer (BD). Data analysis was performed using FlowJo v10.

[0146] B cell proliferation assay [2,4] Human PBMCs at a cell concentration of 1 million / ml were labeled with 0.25 μM CellTrace Violet (Invitrogen) for 20 min at 37°C. Labeled human PBMCs were stimulated with 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, or 0.001 μg / ml MAB273 or anti-CD40 Ab (clone 5C3, BioLegend, 334306). As controls, cells were stimulated with 1 μg / ml CpG B (Invivogen), 0.2 μg / ml SEB (Sigma), or left unstimulated and cultured for 6 days in complete medium (RPMI1640, 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin). After incubation, cells were washed with PBS and cultured for 6 days in LIVE / DEAD medium. TM After staining with the Fixable Blue Dead Cell Stain Kit (Invitrogen, L23105), cells were blocked with FcR Blocking Reagent (Miltenyi Biotec, 130-059-901) according to the manufacturer's protocol, followed by a surface staining panel as follows:

[0147] [Table 2]

[0148] After staining and washing, PBMCs were resuspended in 1% paraformaldehyde (PFA) and acquired on an LSRFortessa flow cytometer (BD). Data analysis was performed using FlowJo v10.

[0149] T cell stimulation [2-4,7] Fresh PBMCs or cells from BAL samples at 1 million cells / mL were cultured overnight at 37°C in 0.2% DMSO + CD107a staining antibody + R10 (unstim), 2ug / mL HIV Envelope peptides + CD107a staining antibody + R10, or 1ug / mL SEB + CD107a staining antibody + R10. Six hours before staining, 10ug / ml Brefeldin A (BFA; Invitrogen) and 10ug / ml monensin (Invitrogen) were added to the culture medium. After incubation, cells were washed with PBS and LIVE / DEAD TM The cells were stained with the Fixable Blue Dead Cell Stain Kit (Invitrogen, L23105), followed by a surface staining panel as follows:

[0150] [Table 3]

[0151] After staining and washing, the cells were permeabilized using Cytofix / Cytoperm kit (BD Biosciences) and intracellular staining was performed as shown in the lower panel.

[0152] [Table 4]

[0153] After staining and washing, cells were resuspended in 1% paraformaldehyde (PFA) and acquired on an LSRFortessa flow cytometer (BD). Data analysis was performed using FlowJo v10.

[0154] Flow cytometry follow-up experiments [2,4] Fresh cells from different tissues were analyzed using LIVE / DEAD TMAfter staining with the Fixable Blue Dead Cell Stain Kit (Invitrogen, L23105), cells were blocked with FcR Blocking Reagent (Miltenyi Biotec, 130-059-901) according to the manufacturer's protocol, followed by a surface staining panel as follows:

[0155] [Table 5]

[0156] After staining and washing, cells were resuspended in 1% paraformaldehyde (PFA) and acquired on an LSRFortessa flow cytometer (BD). Data analysis was performed using FlowJo v10.

[0157] ELISA assay for pharmacokinetic analysis of MAB273 [5,9] MAB273 concentrations in plasma were measured by an in-house custom ELISA assay. Greiner-Bio One 96 well half-area ELISA plates (VWR, 738-0032) were coated with CD40 protein (ThermoFisher, A42565) in fresh PBS overnight at 4°C. Plates were blocked with 5% milk in PBS for 1 h at room temperature (RT). Diluted plasma was added to the plates and incubated for 2 h at room temperature. MAB273 was detected by adding a 1:5,000 dilution of monkey cross-adsorbed polyclonal goat anti-human IgG HRP antibody (Southern Biotech, 2049-05), and TMB substrate (BioLegend) was added to develop the signal. The reaction was stopped by adding an equal volume of 1 M H2SO4, and the optical density (OD) was read at 450 nm and background at 550 nm. Between each incubation step, plates were washed three times with PBS supplemented with 0.05% Tween 20.

[0158] ELISA assays for detecting cytokines in plasma [2,3,7] Rhesus plasma samples were assessed for IFN-γ, IL-6, TNF and IL-12p70 levels by ELISA kits (EP8RB, BMS223HS, BMS654 and BMS646 from ThermoFisher, and 3460-1H-6 and 3512M-1H-6 from Mabtech). The assays were performed according to the manufacturer's protocols.

[0159] References 1. F, L., et al., Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques. Molecular therapy: the journal of the American Society of Gene Therapy, 2017. 25(12). 2. EA, T., et al., Human Anti-CD40 Antibody and Poly IC:LC Adjuvant Combination Induces Potent T Cell Responses in the Lung of Nonhuman Primates. Journal of immunology (Baltimore, Md. : 1950), 2015. 195(3). 3. EA, T., et al., Monocytes Acquire the Ability to Prime Tissue-Resident T Cells via IL-10-Mediated TGF-βRelease. Cell reports, 2019. 28(5). 4. S, O., et al., Route of Vaccine Administration Alters Antigen Trafficking but Not Innate or Adaptive Immunity. Cell reports, 2020. 30(12). 5. J, R., et al., Immune modulation with weekly dosing of an agonist CD40 antibody in a phase I study of patients with advanced solid tumors. Cancer biology & therapy, 2010. 10(10). 6. DA, K., D. R, and R. JV, Toxicity of an Fc-engineered anti-CD40 antibody is abrogated by intratumoral injection and results in durable antitumor immunity. Proceedings of the National Academy of Sciences of the United States of America, 2018. 115(43). 7. EA, T., et al., TLR-adjuvanted nanoparticle vaccines differentially influence the quality and longevity of responses to malaria antigen Pfs25. JCI insight, 2018. 3(10). 8. F, L., et al., Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques. Journal of immunological methods, 2015. 425. 9. NH, S., et al., Results from an Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody. Clinical cancer research : an official journal of the American Association for Cancer Research, 2017. 23(8).

Claims

1. An immunostimulant comprising an agonistic monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the human CD40 receptor and can induce CD40 signaling independently of CD40 receptor crosslinking via Fcγ, The antibody, a) a VH region comprising a CDR1H region of SEQ ID NO: 3, a CDR2H region of SEQ ID NO: 4, and a CDR3H region of SEQ ID NO: 5; b) a VL region comprising a CDR1L region of SEQ ID NO: 6, a CDR2L region of SEQ ID NO: 7, and a CDR3L region of SEQ ID NO: 8; Including, The CDRs may contain any one or more amino acid mutations that do not impair their activity according to the present invention; An immunostimulant wherein the antibody or fragment thereof is administered at a dose of 0.1 to 1 mg / kg body weight.

2. The immunostimulant of claim 1, wherein the antibody or fragment thereof is administered at a dose of 0.2 to 0.9 mg / kg body weight, preferably 0.3 to 0.7 mg / kg, or 0.4 to 0.5 mg / kg.

3. The immunostimulatory agent of claim 1 , wherein the antibody is a humanized IgG1 LALA antibody.

4. 4. The immunostimulatory agent of claim 3, wherein the antibody comprises amino acid substitutions at least at L234A and L235A in the human IgG1 Fc region or S228P and L235E in the human IgG4 Fc region.

5. 2. The immunostimulatory agent of claim 1, wherein the antibody comprises a heavy chain variable (VH) region that is at least 85% identical to the VH region of SEQ ID NO:

1.

6. 2. The immunostimulatory agent of claim 1, wherein the antibody comprises a light chain variable (VL) region that is at least 85% identical to the VL region of SEQ ID NO:

2.

7. The immunostimulant according to claim 1 for the prevention or treatment of infectious diseases and / or cancer.

8. The immunostimulatory agent of claim 7 , wherein the cancer is a solid tumor.

9. 8. The immunostimulant of claim 7, wherein the cancer is selected from the group including pancreatic cancer, advanced pancreatic cancer, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic 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, kidney cancer, Hodgkin's lymphoma, liver cancer, gallbladder cancer, bladder cancer, prostate cancer, thyroid cancer, salivary gland cancer, or uterine cancer.

10. The immunostimulant of claim 1, wherein the antibody or fragment thereof is used in combination with a cytotoxic or cytostatic agent, radiation therapy, targeted therapy, immunotherapy or surgery.

11. 2. The immunostimulant of claim 1, wherein the antibody or fragment thereof is used in combination with at least one immune checkpoint inhibitor, preferably at least one immune checkpoint inhibitor selected from the group consisting of anti-TIGIT, anti-PD-L1, anti-PD-1, anti-CTLA-4, anti-CD137, anti-LAG-3, anti-TIM-3, anti-OX40, and / or anti-GITR.

12. The immunostimulatory agent of claim 1 , wherein the antibody or fragment thereof is administered as a single dose or multiple doses.

13. 13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody or fragment of any one of claims 1 to 12, wherein the pharmaceutical composition is adapted for administration of the antibody or fragment at a dose of 0.1 to 1 mg / kg body weight.

14. 14. The pharmaceutical composition according to claim 13, which is a depot formulation for controlled release of the antibody or fragment over a longer period of time, preferably for sustained release of the antibody or fragment.

15. 14. The pharmaceutical composition of claim 13 adapted for intravenous or subcutaneous administration.

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