Recombinant protein with CD40 activating properties

Abstract

The present disclosure relates to the field of CD40-activating proteins. More specifically, recombinant proteins comprising CD40 agonist antibodies or antigen-binding fragments thereof fused or conjugated to a CD40 ligand are disclosed herein. Also disclosed are advantageous uses of such CD40-activating proteins, particularly for inducing an immune response against delivered antigens, such as viral or cancer antigens.

Description

[Technical field]

[0001] The present disclosure relates to the field of CD40 activating proteins. More specifically, disclosed herein are recombinant proteins based on CD40 agonist antibodies having antigen-binding fragments of the CD40 agonist antibodies fused or conjugated to CD40 ligand (CD40L). Also disclosed are advantageous uses of such CD40 activating proteins, particularly for inducing an immune response against delivered antigens, such as viral or cancer antigens. [Background technology]

[0002] CD40 is a potent activating tumor necrosis factor receptor superfamily member expressed on antigen-presenting cells (APCs) and B cells. Dendritic cells (DCs) respond to infection by taking up antigens and activating pathogen-associated molecular patterns (PAMPs), thus presenting foreign antigens on their major histocompatibility complex (MHC) molecules to antigen-specific T cells, initiating a cycle of DC maturation via CD40 ligand (CD40L) expressed on activated T cells, which then induces cellular and humoral antigen-specific T and B cell responses against the pathogen [Elgueta et al., 2009]. Agonistic anti-CD40 antibodies are in clinical development based on the concept of directly activating APCs to stimulate immune responses against endogenous antigens, e.g., tumor-associated antigens (TAAs), or as vaccine adjuvants [reviewed by Dahan et al., 2016, Thompson et al., 2015, Vonderheide and Glennie, 2017]. Direct conjugation of antigens to anti-CD40 antibodies by chemical conjugation [Bar et al., 2003], non-covalent assembly [Flamar et al., 2013], or direct fusion [Flamar et al., 2014] induces strong antigen-specific cellular and humoral immunity at very low antigen doses in a variety of in vitro and in vivo settings [Flamar et al., 2013, Flamar et al., 2014, Yin et al., 2016, Yin et al., 2017]. Notably, antigen targeting to CD40 induces superior cellular T cell responses than targeting other receptors by accumulating in early endosomal compartments, unlike the rapid antigen entry into late endosomes characterized by targeting other DC receptors [Yin et al., 2016, Chattergee et al., 2012].

[0003] In addition to primary screening to identify CD40-reactive monoclonal antibodies with the most potent agonistic activity, e.g., inducing cytokine secretion or CD86 surface expression on human DCs, maximizing the agonistic efficacy and utility of clinical candidate anti-CD40 antibodies typically involves affinity maturation of the H and L chain binding regions [Mangsbo et al., 2014], as well as enhanced cross-linking of the constant regions with FcRs [Dahan et al., 2016]. Screening can also identify potent agonists without the need for Fc interactions [He et al., 2016], which may be problematic for human platelet activation if FcγRIIA interactions are maintained [Dahan et al., 2016]. Agonistic anti-CD40 antibodies can bind to sites that overlap the CD40L-interacting region or interact with sites distinct from its ligand-binding region [Gladue et al., 2011; Dahan et al., 2016; He et al., 2016], although it is unclear whether this distinction has clinical significance.

[0004] Although strong activation of CD40 is not required for effective class I and class II presentation of antigens via CD40 targeting in vitro [Chattergee et al., 2012; Flamar et al., 2014], efficacy in vivo requires co-administration of Toll-like receptor (TLR) activators such as PolyIC [Zurawski et al., 2017; Cheng et al., 2017]. However, these in vitro and in vivo studies did not utilize anti-CD40 antibody-antigen complexes or fusions with full CD40 agonistic activity, and the clear advantage of agonistic anti-CD40 antibodies combined with PolyIC for peptide-based vaccination in non-human primates [Thompson et al., 2015] suggests that CD40 targeting of antigens could be further improved by a fully agonistic anti-CD40 targeting vehicle. Furthermore, there is a need to provide a single molecule with high potency as an agonistic CD40 targeting vehicle. Disclosed herein is the nature of soluble CD40L cooperation with certain agonistic anti-CD40 vehicles to enhance the CD40 activation efficacy of both anti-CD40 antibodies and anti-CD40 antibody-antigen fusion proteins. It is further disclosed herein that methods of combining both anti-CD40 antibodies and CD40L agonist fragments into a single entity provide efficacy at least similar to, or even better than, that observed with two separate agents administered simultaneously. Such CD40 activating proteins may be of great value in treatment and adjuvant immune responses to administered antigens. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Application Publication No. 20160031988A1 [Patent Document 2] US Patent Application Publication No. 20120039916A1 [Patent Document 3] International Publication No. 2010 / 104747 [Patent Document 4] U.S. Patent No. 5,223,409 [Patent Document 5] U.S. Patent No. 5,403,484 [Patent Document 6] U.S. Patent No. 5,571,698 [Patent Document 7] U.S. Patent No. 5,427,908 [Patent Document 8] U.S. Patent No. 5,580,717 [Patent Document 9] U.S. Patent No. 5,969,108 [Patent Document 10] U.S. Patent No. 6,172,197 [Patent Document 11] U.S. Patent No. 5,885,793 [Patent Document 12] U.S. Patent No. 6,521,404 [Patent Document 13] U.S. Patent No. 6,544,731 [Patent Document 14] U.S. Patent No. 6,555,313 [Patent Document 15] U.S. Patent No. 6,582,915 [Patent Document 16] U.S. Patent No. 6,593,081 [Patent Document 17] International Application Publication No. 02 / 43478 [Patent Document 18] U.S. Patent No. 4,816,567 [Patent Document 19] U.S. Patent No. 5,225,539 [Patent Document 20] U.S. Patent No. 5,530,101 [Patent Document 21] U.S. Patent No. 5,585,089 [Patent Document 22] U.S. Patent No. 5,693,762 [Patent Document 23] U.S. Patent No. 6,180,370 [Patent Document 24] International Publication No. 2010 / 009346 [Patent Document 25] International Publication No. 2010 / 104747 [Patent Document 26] International Publication No. 2010 / 104749 [Patent Document 27] U.S. Patent No. 7,338,660 [Patent Document 28] U.S. Patent Application Publication No. 20120301488A1 [Patent Document 29] US Patent Application Publication No. 20170342159 [Patent Document 30] U.S. Patent No. 4,399,216 [Patent Document 31] U.S. Patent No. 4,634,665 [Patent Document 32] U.S. Patent No. 5,179,017 [Patent Document 33] U.S. Patent No. 3,791,932 [Patent Document 34] U.S. Patent No. 4,174,384 [Patent Document 35] U.S. Patent No. 3,949,064 [Non-patent literature]

[0006] [Non-Patent Document 1] Ward et al., 1989 Nature 341:544-546 [Non-Patent Document 2] Bird et al., 1988 Science, 242:423-426 [Non-Patent Document 3] Huston et al., 1988 Proc. Natl. Acad. Sci., 85:5879-5883. [Non-Patent Document 4] Baudino et al., J. Immunol.181 (2008): pp. 6664-69 [Non-Patent Document 5] Strohl, CO, Biotechnology 20 (2009): pp. 685-91 [Non-Patent Document 6] Lim et al., Biotechnology Bioeng, 2013, 110, pp. 57-67 [Non-Patent Document 7] Nelson et al., 2010 Nature Reviews Drug discovery, “Development trends for human monoclonal antibody therapeutics” (Advance Online Publication) [Non-Patent Document 8] Hoogenboom et al., Methods in Molecular Biology, 178:1-37 [Non-Patent Document 9] O'Brien et al., eds., Human Press, Totowa, NJ, 2001 [Non-Patent Document 10] Lonberg et al., 1994 Nature 368(6474): 856-859 [Non-Patent Document 11] Kohler and Milstein, 1975 Nature 256: 495 [Non-Patent Document 12] Francisco JA et al., Cancer Res, 60: 3225-31, 2000 [Non-Patent Document 13] "Remington's Pharmaceutical Sciences" 15th edition, pages 1035-1038 and 1570-1580 [Non-Patent Document 14] Parslow et al., 2018, J Vis Exp, 2018;(138):57164 Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure provides a method for the preparation of a polypeptide comprising at least the following protein domains: (i) a CD40 agonist antibody or antigen-binding fragment thereof (αCD40); and (ii) the CD40-binding domain of CD40L (CD40L) The present invention relates to a CD40 activating protein comprising the [Means for solving the problem]

[0008] In certain embodiments, the CD40 agonist antibody specifically binds human CD40 and has at least one or more of the following properties: (i) induces B cell proliferation as measured in vitro by flow cytometric analysis or replicative dilution analysis of CFSE-labeled cells; or (ii) induces secretion of cytokines, such as IL-6, IL-12 and / or IL-15 cytokines, as measured in vitro by a dendritic cell activation assay.

[0009] In a particular embodiment, said CD40 binding domain of CD40L is a fragment of CD40L comprising SEQ ID NO:14.

[0010] In a specific embodiment, the CD40 binding domain of CD40L is fused to the C-terminus of the light or heavy chain of the CD40 agonist antibody or antigen-binding fragment thereof.

[0011] In a particular embodiment, said CD40 activating protein comprises the heavy and light chains of a CD40 agonist IgG antibody, preferably an Fc-null and disulfide stabilized IgG4 or a mutated silenced IgG antibody.

[0012] In a particular embodiment, the CD40 activating protein further comprises a peptide linker between CD40L and the light or heavy chain of the CD40 agonist antibody or antigen-binding fragment thereof, preferably the flexible linker FlexV1 of SEQ ID NO:15.

[0013] In certain embodiments, the CD40 agonist antibody is selected from the following antibodies: (i) a humanized antibody comprising an HCDR1 of SEQ ID NO: 27, an HCDR2 of SEQ ID NO: 28, an HCDR3 of SEQ ID NO: 29, an LCDR1 of SEQ ID NO: 30, an LCDR2 of SEQ ID NO: 31, and an LCDR3 of SEQ ID NO: 32; (ii) a humanized antibody comprising the VH and VL domains of SEQ ID NO: 21 and SEQ ID NO: 22, respectively; (iii) an antibody that competes with at least one of the antibodies identified in (i) or (ii) for binding to CD40-expressing cells; or (iv) an antibody that binds to the same epitope as one of the antibodies identified in (i) or (ii).

[0014] In certain embodiments, one or more antigens are fused to the heavy or light chain of said CD40 agonist antibody or antigen-binding fragment thereof.

[0015] Typically, the one or more antigens are viral, bacterial, or cancer antigens fused (directly via a peptide bond or non-conjugated, e.g., via dockerin-cohesin technology) to the heavy or light chain of the CD40 agonist antibody.

[0016] In a particular embodiment, the CD40 activating protein comprises a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy-(PL-Ag)x, αCD40Light is the light chain of the CD40 agonist antibody described above; αCD40Heavy is the heavy chain of the CD40 agonist antibody described above; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; ·Ag is a viral or cancer antigen, same or different; x is an integer from 1 to 20, for example 3, 4, or 5; CD40L is a binding domain of a ligand of CD40, e.g., a CD40 binding domain of CD40L comprising SEQ ID NO: 14; - is a bond.

[0017] In a particular embodiment, the viral antigen is selected from an HIV peptide antigen, preferably an HIV-1 antigen, such as GNG of SEQ ID NO:48 or HIV5pep of SEQ ID NO:57.

[0018] The present disclosure also relates to a pharmaceutical composition comprising a CD40 activating protein as defined above and one or more pharma- ceutically acceptable excipients.

[0019] The present disclosure further relates to a CD40 activating protein for use as a vaccine, in particular, said CD40 activating protein can be used to enhance T cell specific responses, in particular CD8+ T cell specific responses against viral antigens in a subject.

[0020] The CD40 activating proteins may also be used to induce B cell proliferation and / or induce cytokine proliferation of dendritic cells in a subject. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram showing the anti-receptor antibody-ligand fusion concept exemplified by anti-CD40 11B6-human CD40 IgG4. Relevant domains are indicated: VL is the light chain variable region; VH is the heavy chain variable region; CH is the heavy chain constant region 1-3; CL is the light chain constant region; linker sequences and important disulfide bonds are indicated by grey lines. The present invention envisages alternative fusion configurations, including the ligand fused to the C-terminus of the heavy chain, and various antibody forms and isotypes. [Diagram 2]Figure 1 shows the proliferative response of human B cells to a dose range of anti-CD40 antibodies incubated with and without a constant low dose of soluble human CD40L. Human PBMCs were incubated with human IL-4, human IL-21, and a dose range of different human IgG4 isotype-matched antibodies (shown from left to right: 10, 1, 0.1, 0.01 nM) determined after 6 days by flow cytometric analysis of CFSE dilution. The curves marked with filled circles are the responses to antibody dose, and the curves marked with filled grey boxes are the responses to antibody dose in the presence of 100 ng / ml (6 nM) soluble human CD40L. Data represent the average of 7 (11B6, 12B4, CP, IgG4) or 3 (12E12, 24A3) independent experiments for different donors normalized for maximum proliferation (80±22%) versus baseline replication without antibody or sCD40L (range 6±4%) or without antibody but with sCD40L (11±7%). [Diagram 3] Figure 1 shows the cytokine secretion response of human MDDCs to a dose range of anti-CD40 IgG4 antibodies incubated with and without a constant low dose of soluble CD40L. Human MDCs were cultured with a dose range of each anti-CD40 mAb (shown from left to right: 10, 1, 0.1, 0.01, 0.001 nM) and the extent of cytokine secretion was determined after 2 days. The maximum response was set to 100%. The curves marked with black circles are the responses to antibody alone, and the curves marked with grey squares are the responses to antibody in the presence of 1 μg / ml soluble human CD40L (60 nM). Data represent a single experiment normalized to the maximum secretion of each cytokine tested (panel A, IL-12 p40, 561 ng / ml; panel B, IL-15, 62 ng / ml). A similar dose-response trend was observed for IL-6 secretion (maximum 446 pg / ml, 30 pg / ml response to sCD40L). [Figure 4]Figure 1. Anti-CD40 antibody inhibition of CD40L binding. A titration series of anti-human CD40 mouse (left panel) or human IgG4 (right panel) mAbs was added to a constant amount (2 μg / ml) of human CD40 ectodomain human Fc (left panel) or mouse Fc (right panel) fusion protein at 2 μg / ml, the mixture was incubated on ice for 1 h, then added to L cells stably transfected with an equal amount of 200K human CD40L, incubated on ice for 1 h, washed, incubated with goat anti-mouse (top panel) or anti-human (bottom panel) IgG-PE reagent, washed again, and analyzed on a FACS array instrument. Loss of binding indicates that the test mAb binds soluble CD40 in a manner that prevents binding to cell surface CD40L. [Diagram 5] Figure 1 shows the proliferative response of human B cells to a range of doses of anti-CD40 HIV5pep antigen fusion protein incubated with and without a constant low dose of soluble CD40L. Human PBMCs were incubated with human IL-4, human IL-21, and a range of doses (shown from left to right: 10, 1, 0.1 nM) of mAb or fusion protein, and the extent of proliferation was determined after 6 days by flow cytometric analysis of CFSE dilution. The curve marked with solid grey squares is the response to parental hIgG4 antibody dose in the presence of 100 ng / ml (6 nM) soluble human CD40L, the curve marked with open circles is the response to antibody-HIV5pep fusion protein dose, and the curve marked with filled grey circles is the response to antibody-HIV5pep fusion protein dose in the presence of 100 ng / ml (6 nM) soluble human CD40L. Data represent a single experiment normalized for maximum proliferation (58%) versus baseline replication without antibody or sCD40L (range 6%) or without antibody but with sCD40L (12.5%). For reference, the proliferative B cell responses elicited by these antibodies without fusion antigen and with and without the addition of sCD40L are shown in replicates in FIG. 2. [Figure 6]Figure 1 shows cell surface activation marker responses of human MDDCs to a dose range of anti-CD40 IgG4 antibodies incubated with and without fusion HIV5pep antigen and with and without a fixed low dose of soluble CD40L. Human MDDCs with a dose range (shown from left to right: 10, 1, 0.1 nM) of each anti-CD40 mAb and anti-CD40.HIV5pep fusion protein antibody in the presence or absence of 100 ng / ml (6 nM) soluble human CD40L, and the percentage of cells with high expression of CD86 and DR as determined by flow cytometry. The curves marked with filled circles are the responses to antibody alone, the curves marked with filled grey squares are the responses to antibody in the presence of soluble human CD40L, the curves marked with open circles are the responses to antibody to HIV5pep alone, and the curves marked with filled grey circles are the responses to antibody fused to HIV5pep in the presence of soluble human CD40L. Data are from a single representative experiment. [Figure 7] Figure 1. CD40-targeted HIV5pep expansion of HIV-1 specific T cells in HIV-1 infected donor PBMC cultures incubated with and without sCD40. HIV-1+ donor PBMCs were cultured for 9 days with a range of doses of anti-CD40.HIV5pep fusion protein (1, 0.1, 0.01 nM from left to right) with and without low doses of sCD40L (100 ng / ml) and IL-2, followed by stimulation with a pool of five HIV-1 Gag, Nef, and Pol long peptides with BFA for 6 hours and then analyzed by ICS. Data show the percentage of antigen-specific CD8+ and CD4+ T cells producing IFNγ at the end of culture in response to peptide stimulation. Y-axis shows the percentage of IFNγ+ CD3+ CD4+ or CD3+ CD8+ cells. [Figure 8]Figure 1 shows the proliferative response of human B cells to a dose range of anti-CD40hIgG4 mAbs incubated with and without directly fused CD40L or with a fixed low dose of soluble CD40L. Human PBMCs were incubated with human IL-4, human IL-21, and a dose range of mAbs or fusion proteins (shown from left to right: 10, 1, 0.1, 0.01 nM), and the extent of proliferation was determined after 6 days by flow cytometric analysis of CFSE dilution. The curves marked with filled circles are the responses to hIgG4 antibody doses, the curves marked with filled squares are the responses to hIgG4 antibody doses in the presence of 100 ng / ml (6 nM) soluble human CD40L, and the curves marked with filled squares are the responses to hIgG4 antibody doses with directly fused CD40L. Data represent a single experiment normalized for maximum proliferation (95%) to baseline replication without antibody and sCD40L (10%) or without antibody but with sCD40L (44.5%). Figures 2 and 5 contain replication data for Ab and Ab+sCD40L. [Figure 9]Figure 1 shows the cytokine secretion response of human MDDCs to a dose range of anti-CD40 IgG4 antibodies incubated with and without directly fused human CD40L and with and without a fixed low dose of soluble CD40L. Human MDCs were cultured with a dose range of each anti-CD40 mAb or control mAb (shown from left to right: 100, 10, 1, 0.1, 0.01, 0.001 nM) and the extent of cytokine secretion was determined after 2 days. The curves marked with filled circles are the responses to antibody alone, the curves marked with grey squares are the responses to antibody in the presence of 1 μg / ml (6 nM) soluble human CD40L, and the curves marked with filled squares are the responses to antibody directly fused to human CD40L. Data represent a single experiment normalized to the maximum secretion of each cytokine tested. In the left panel, the maximum values ​​were IL-6, 469ng / ml; IL-12p40, 561ng / ml; IL-15, 62ng / ml). In the right panel, the maximum values ​​were IL-6, 6192ng / ml; IL-12p40, 5776ng / ml; IL-15, 194ng / ml). Note that the Ab and Ab+sCD40L data are identical to those shown in Figure 3. [Figure 10A] Figure 1 shows human B cell and MDDC responses to a dose range of anti-CD4011B6 mAb fused to HIV5 pep antigen at the C-terminus of the H chain and / or to human CD40L fused to the C-terminus of the L chain. Human PBMC or MDDC were incubated with a dose range of each IgG4 mAb (shown from left to right: 10, 1, 0.1 nM) and the extent of B cell proliferation (left panel) or the extent of MDDC cytokine secretion (right panel) was determined. These are the results of a single experiment in which the maximal range of B cell proliferation was 31% with a 4% baseline, while cytokine production data are within-experiment averages of the maximal production percentages for IL-6 (610 pg / ml), IL-15 (2,250 pg / ml), TNFα (27,500 pg / ml), IL-12 p40 (9,900 pg / ml) and IL-12 p70 (3,300 pg / ml). [Figure 10B]Figure 1 shows human B cell proliferation response to a range of antibody or antibody fusion protein doses. Human PBMCs were incubated with human IL-4, human IL-21 at a range of doses (10, 1, 0.1, 0.01 nM from left to right) and the extent of proliferation was determined after 6 days by flow cytometric analysis of CFSE dilution. Curves are response to antibody dose. Data represent the average of two donors, each normalized for maximum proliferation (31% and 28% of B cells replicated in response to maximum signal, whereas baseline cell only values ​​were 3.5% and 3.6%). 11B6-HIV5pep+sCD40L titration series was 11B6-HIV5pep supplemented with a fixed dose of 1 μg / ml (60 nM) soluble CD40L. [Figure 10C] Figure 1 shows the response of human MDDCs to a range of doses of Mega sCD40L and anti-CD40 11B6-CD40L mAb, with and without HIV5pep antigen fused to the H chain C-terminus. Human MDDCs were incubated with a range of doses (indicated from left to right: 10, 1, 0.1, 0.01 nM) of each IgG4 mAb or Mega sCD40L, and the extent of cytokine secretion was determined at 24 hours. Results presented are averaged data across three experiments with two different donors, where the maximum range of cytokine production was normalized to the maximum signal and then averaged for IL-12 p40, TNFα, and IL-15 secretion. The mean values ​​of maximum production were IL-12 p40 (14,236 pg / ml), TNFα (37,145 pg / ml), and IL-15 (295 pg / ml). Error bars are standard deviation of the mean. 11B6-CD40L-HIV5pep carries the five linked HIV-1 antigenic regions on the IgG4 heavy chain termini as previously described (Flamar et al., 2013), while 11B6-CD40L-HIV5pepk has two of the five antigenic regions on one heavy chain and three on the other heavy chain using knobs-in-holes technology (Flamar et al., 2018; Ridgway et al., 1996). [Figure 11]Figure 1 shows the proliferative response of human B cells to a dose range of anti-CD40 or control antibodies fused to HIV-1 Gag p24, Nef, and Gag p17, or HPV16 E6 / E7 antigens fused at the C-terminus of the H chain, with and without human CD40L fused to the C-terminus of the L chain, or co-cultured with a low dose of soluble CD40L. Human PBMCs were incubated with human IL-4, human IL-21, a dose range indicated (shown from left to right: 10, 1, 0.1, 0.01 nM), and the extent of proliferation was determined after 6 days by flow cytometric analysis of CFSE dilution. The curves marked with filled circles are responses to the indicated antibody or antibody-antigen fusion protein doses, the curves marked with filled grey squares are responses to the indicated antibody or antibody-antigen fusion protein doses in the presence of 1 μg / ml (60 nM) soluble human CD40L, and the curves marked with green triangles are responses to antibody-CD40L or antibody-CD40L-antigen fusion protein doses. Data represent a single experiment of normalized maximum proliferation (95%) versus baseline proliferation without antibody (10%). [Figure 12] Figure 1 shows the cytokine secretion response of human MDDCs to a range of doses of anti-CD40 or anti-CD40-GNG fusion antibodies, with and without directly fused human CD40L. Human MDCs were cultured with a range of doses of each mAb (shown from left to right: 10, 1, 0.1 nM) and the extent of cytokine secretion was determined after 2 days. Curves marked with open circles are dose responses to antibody or antibody-antigen fusion protein, curves marked with filled squares are dose responses to antibody or antibody-antigen fusion protein directly fused to human CD40L. Data represent a single experiment normalized to maximum secretion of each cytokine tested. Maximum values ​​were IL-10, 30 ng / ml; IL-12p40, 359 ng / ml; IL-15, 36 ng / ml, TNFα, 6134 ng / ml). [Figure 13]Figure 1. Expansion of HIV-1 specific T cells in HIV-1 infected donor PBMC cultures with CD40 targeted Gag p17-Nef-Gag p24 or fusion CD40L with and without soluble CD40. HIV-1+ donor PBMC were cultured with IL-2 and anti-CD40-GNG and control IgG4-GNG fusion proteins (0.1 nM) with and without soluble CD40L (0.1 nM) or directly linked CD40L for 9 days, followed by stimulation with Gag and Nef overlapping peptides for 6 hours with BFA and analyzed by ICS. The top panel shows data for donor A17 and the bottom panel shows data for donor A15. [Figure 14] CD40-targeted HIV5pep antigen with fused CD40L preferentially expands HIV-1-specific CD8+ T cell responses in HIV-1-infected donor PBMC cultures. HIV-1+ donor PBMCs were cultured with IL-2 and anti-CD40 HIV5 peptide fusion proteins (1 nM; 2 nM for KIH proteins) for 9 days, followed by stimulation for 6 hours with long peptides specific for each of the five HIV-1 gag, nef, and pol regions, and then analyzed by ICS. This is a pooled data set from experiments with each of the four indicated proteins tested in four donors. A. Each point represents the value for % of IFNγ+TNFα+ HIV5pep-specific CD8+ (squares) or CD4+ (diamonds) T cells for each response specific for each long peptide elicited in each donor comparing αCD40 11B6-CD40L.HIV5pep KIH Gen 2 and αCD40 11B6-CD40L.HIV5pep Gen 1 (X-axis) with αCD40 12E12.HIV5pep KIH Gen 2 (X-axis) and αCD40 E1212.HIV5pep Gen 2 (Y-axis). High values ​​of nef66 responses in one donor are not shown (58, 44 vs. 35, 44). B. Comparison of HIV5pep KIH constructs vs. non-KIH constructs showed no significant differences in HIV5pep-specific CD4+ or CD8+ T cell responses. [Figure 15]CD40-targeted HIV5pep antigen was tested with and without fusion CD40L via in vitro expansion of HIV-1 specific T cells in HIV-1 infected donor PBMC cultures. PBMCs from HIV-1+ donor Patient 1 were cultured with IL-2 and anti-CD40 HIV5pep fusion protein (1 nM) for 9 days, followed by BFA stimulation for 6 hours with long peptides specific for each of the five HIV-1 gag, nef, and pol regions, and then analyzed by intracellular cytokine staining (ICS). This is ICS data from an experiment with two of the four proteins tested in one donor shown in FIG. 14. PSApep and sol represent non-relevant peptide and solvent negative control, respectively, and PMA represents polyclonal stimulation with phorbol 12-myristate 13-acetate and ionomycin. For each ICS panel, the Y-axis represents INFγ staining intensity and the X-axis represents TNFα staining intensity. [Figure 16]CD40-targeted HIV-1 gp140 antigen induces gp140-specific serum IgG responses in human CD40 transgenic mice with and without fused CD40L. Taconic CD40 homozygous transgenic mice (strain 12692) were vaccinated by intraperitoneal injection with (group 1, G1) anti-CD40 11B6-CD40L directly fused to HIV-1 gp140, (group 2, G2) anti-CD40 11B6-CD40L-dockerin complexed to cohesin gp140, (group 3, G3) anti-CD40 11B6-dockerin complexed to cohesin gp140, or (group 4, G4) cohesin gp140. Doses were normalized to be the molar equivalent of 1 μg of group 2 vaccine, except for the non-targeted control group 3 (2 μg or approximately 3 molar equivalents). Vaccinations were administered on days 1, 14, and 21, and blood was collected on days 13 (D13), 27 (D27), and 34 (D34). Graphs show serial dilutions of sera analyzed for anti-gp140 IgG reactivity by ELISA as described in Zurawski et al., 2016, except anti-mouse IG-HRP reagent was the detection reagent. Group sizes were G1, n=3 and G2-4, n=4. Directional error bars are SE of the mean. [Figure 17]Figure 1 shows the effect of anti-CD40 CP and anti-CD40 11B6-CD40L on B cells in human CD40 transgenic mice. Mice were administered CP-870,893hIgG4 (CP) and anti-CD40 11B6-CD40L (11B6-CD40L). Human CD40 BAC transgenic mice on either wild type (WT, white bars), wild type (hCD40 Taconic strain, black bars) or CD40 KO (ImmuRx hCD40 strain, grey bars) C57BL / 6 background were injected (intraperitoneally) with a molar equivalent of CP-870,893hIgG4 (CP, 10 μg≈0.5 mg / Kg) or anti-CD40 11B6-CD40L (11B6-CD40L) and sacrificed 24 hours later. Blood was collected for PBMC preparation, and cells were prepared from the skin-draining lymph nodes and spleen, then analyzed by flow cytometry. B cell activation was characterized by analysis of the activation marker CD69. Data are the average of two animals from each of the six groups. [Figure 18] Fusing CD40L to anti-CD40 11B6 increases both the efficacy and potency of cell killing induced by CD40 ectodomain fused to FAS transmembrane and intracellular domains. CHO cells stably transfected with human CD40-FAS fusion constructs were incubated with a dilution series of anti-CD40 IgG4 antibodies for 48 hours. Cells were then incubated with MTT for colorimetric detection of mitochondrial reduction activity, an indicator of cell viability. Untransfected CHO cells were not affected by any of the tested agents (not shown). [Figure 19]Analysis of anti-CD40 mAb binding to CHO cells expressing CD40 ectodomain fusion proteins. CHO cells (250K) stably transfected with expression plasmids expressing human CD40 or CD40 ectodomain fused to FAS transmembrane and intracellular residues were incubated with a dilution series of anti-CD40 antibodies starting at 0.1 μM final (3-fold dilutions across 11 wells) at 250K per point (with specific cell type). Binding was monitored for 1 hour on ice followed by 2× washing with 1× PBS, 2% FCS, 2 mM EDTA (also binding buffer) and then probed with goat anti-human IgG PE (Prozyme: Phycolink anti-hIgG-RPE) at a dilution of 1:500 followed by two washes with the above buffer. Samples were analyzed on a FACS array (BD Biosciences): P1% calculated using gate set above background cells with PE (parent) histogram only. All events from a sample / particular event within the P1 gate give the specified P1% value. WT CD40-FAS CHO are cells expressing wild type (non-mutated) CD40 ectodomain fused to FAS. CD40 R27A and E28A FAS are cells with mutated CD40 ectodomain. [Figure 20] Figure 1 shows the cytokine secretion response of human bone marrow-derived dendritic cells (MDDCs) to a range of doses of anti-CD40 11B6 IgG4 antibody with direct fusion human CD40L fused to the C-terminus of the H or L chain, with and without a flexible linker region. Human MDDCs were cultured with a range of doses of each anti-CD40 11B6-CD40L isoform (indicated from left to right: 10, 1, 0.1, 0.01 nM) and the extent of cytokine secretion was determined after 24 hours. Data represent the sum of values ​​for each cytokine tested normalized to the highest value within the experiment and averaged over repeated responses in two different donors. Error bars are standard error of the mean. Schematics shown below each titration series show the L or H chain CD40L fusion isoforms, with F indicating the presence of the Flex V1 linker. [Figure 21]The superagonistic activity of CD40L fused to anti-CD40 11B6 is independent of CD40L positioning, as defined by the efficacy and potency of cell killing directed by the CD40 ectodomain fused to the FAS transmembrane and intracellular domains. CHO cells stably transfected with a construct for expressing human CD40 ectodomain fused to FAS transmembrane and intracellular residues (Mam-cetHS-puro [hCD40--Ecto hFas-TM-IC], SEQ ID NO: 151) were incubated with a dilution series of anti-CD40 IgG4 antibody for 48 hours. The cells were then incubated with MTT for colorimetric detection of mitochondrial reduction activity. In the graph, h indicates humanized 11B6 mAb and m indicates the original mouse V region. [Figure 22] Schematic diagram showing the structure of each fusion protein, with antigen either at the C-terminus of the light or heavy chain, and / or the presence of a flexible linker. [Diagram 23]Anti-CD40 11B6-CD40L enhances CD40 cluster formation. Human CD40-mCherrry CHO cells were incubated alone (top panel), with 10 nM anti-CD40 12E12 (middle panel) or with 10 nM anti-CD40 11B6-CD40L (bottom panel) on cover slides at 37°C in culture medium for 1 hour. Cells were then fixed with 1% PFA, washed, and mounted on ultra-frozen slides with ProLong Gold antifade reagent with DAPI. Images were taken at UTSW and analyzed using FIJI software (open source from ImageJ) for quantification of clusters per beam area. An average of 5 cells per group were analyzed using spatial image correlation spectroscopy, with three 32 × 32 squares analyzed per cell. The protocol used utilizes autocorrelation and image mathematical functions of FIJI software to quantify the fluorescence intensity of the labeled receptor as a function of the beam area of ​​the confocal microscope, providing a quantitative measurement of the state of target molecule aggregation (clustering) on ​​the cell surface (Parslow et al., 2018, J Vis Exp 2018;(138):57164). MDDCs were incubated in culture medium with 100 nM anti-CD40 11B6-CD40L-Dock_Coh-eGFP or 100 nM anti-CD40 23E12-Dock_Coh-eGFP at 37 °C on cover slides for 6 h. Cells were then washed, fixed, permeabilized, stained for anti-EEA1 or anti-Lamp1, washed, and mounted on ultra-frozen slides with ProLong Gold antifade reagent with DAPI. Images were taken and analyzed with FIJI software for quantification of clusters per beam area as previously described. Eight images for anti-CD40 11B6-CD40L treatment with four 32x32 squares per image and seven images for anti-CD40 12E12 treatment with four or five 32x32 squares per image were used for analysis (right). The difference in scales on the left and right is due to the difference in fluorescence intensity between the pictures, taken at different moments, and different cell types (not comparable). However, for the different cell types used, the statistical significance between the treatments is the same (p-value < 0.0001). [Figure 24] Anti-CD40 11B6-CD40L enhances CD40-mediated internalization. Human CD40-CHO cells were incubated with 100 nM mCherry-labeled anti-CD40 12E12 (A), anti-CD40 11B6-CD40L (B), or anti-CD40 11B6 (C) in medium at either 37°C or 0°C for various times, after which the cells were washed with PBS or treated with isotonic acid removal buffer (pH 2.5) for 1 min, neutralized, and then washed with PBS. Total label or acid-resistant (i.e., internalized) label was then measured by fluorescence. The total binding signal from the 30-270 min time points was averaged and set to 100% to normalize data between three independent replicate experiments. Background fluorescence with cells alone or 100 nM mCherry not conjugated to anti-CD40 mAb was 2 ± 1%. Detectable binding at time zero reflects binding that occurs during the first approximately 6 minute centrifugation and washing steps. [Diagram 25] Figure 1 shows data from an Ala mutagenesis study. Underlined are the four CDR structural homology regions of CD40. Residual numbers of some mutations are indicated. Grey highlighting indicates altered residues. P is Pfizer CP mAb; E is 12E12; B is 11B6; L is 11B6-CD40L; - means no effect on any mAb tested; lower case letters below the sequence indicate reduced binding; CAPS letters mean no binding. [Figure 26] Figure 1. CD40-targeted GNG antigen-induced expansion of HIV-1-specific T cells in HIV-1-infected donor PBMC cultures. HIV-1+ donor PBMCs were cultured for 9 days with a dose range of anti-CD40-GNG fusion protein (from left to right: 1, 0.1, 0.01 nM) in the presence and absence of low doses of sCD40L (100 ng / ml; 6 nM) and IL-2, followed by stimulation with pooled HIV-1 Gag p17, Nef, and Gag p24 peptides for 6 hours and then analyzed by ISC. Data show the percentage of antigen-specific (A) CD4+ and (B) CD8+ T cells producing IFNγ+TNFα in response to peptide stimulation at the end of culture. [Figure 27] Figure 1. CD40-targeted GNG antigen-induced expansion of HIV-1-specific T cells in HIV-1-infected donor PBMC cultures. HIV-1+ donor PBMCs were cultured for 9 days with a range of doses of anti-CD40-GNG fusion protein (from left to right: 1, 0.1, 0.01 nM) in the presence and absence of low doses of sCD40L (100 ng / ml; 6 nM) and IL-2, followed by stimulation with pooled HIV-1 Gag p17, Nef, and Gag p24 peptides for 6 hours and then analyzed by ICS. Data show the percentage of IFNγ+TNFα production in response to peptide stimulation at the end of culture of antigen-specific (A) CD4+ and (B) CD8+ T cells. Panels C and D show matched responses of antigen-specific CD4+ and CD8+ T cells, respectively, up to the three highest doses from three patients, spiked across all three peptide regions tested, normalized to the highest response for each patient, and then averaged. Significant differences indicated were based on unpaired tests with Welch's correction. [Figure 28]Anti-CD40-CD40L targeting HIV5pep antigen preferentially expands numerous HIV-1 specific CD8+ T cell responses in HIV-1 infected donor PBMC cultures. HIV-1+ donor PBMC were cultured with IL-2 and anti-CD40 HIV5 peptide fusion protein (1 nM; 2 nM for KIH protein) for 9 days, followed by stimulation with long peptides specific for each of the five HIV-1 gag, nef, and pol regions with BFA for 6 hours, then analyzed by ICS. This is collated with data from experiments with each indicated protein pair tested in four donors. Each point represents the % values ​​for IFNγ++TNFα+HIV5pep-specific CD8+ (panel A, black squares) or CD4 (panel B, open circles) for each long peptide-specific response elicited in each donor comparing anti-CD40 11B6-CD40L-HIV5pep KIH Gen 2 and anti-CD40 11B6-CD40L-HIV5pep Gen 1 (Y-axis) with anti-CD40 12E12-HIV5pep KIH Gen 2 and anti-CD40 12E12-HIV5pep Gen 2 (X-axis). Panel A highlights responses below 20% of expansion cultures and excludes the strong patient 2 response to the nef66 peptide (38±7% elicited by anti-CD40 11B6-CD40L-HIV5pep vaccine vs. 48±10% elicited by anti-CD40-HIV5pep vaccine). Panel B shows the entire data set. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition In order that this disclosure may be more readily understood, certain terms are first defined. Further definitions are provided throughout the detailed description.

[0023] As used herein, the term "CD40" has its general meaning in the art and refers to the human CD40 polypeptide receptor, including CD40 of SEQ ID NO: 13. In certain embodiments, CD40 is an isoform of the human standard sequence reported by UniProtKB-P25942 (also called human TNR5). The ectodomain of CD40 recognized by certain anti-CD40 antibodies may typically be comprised between residues 21 and 193 of SEQ ID NO: 13.

[0024] As used herein, the term "CD40L" has its general meaning in the art and refers to the human CD40L polypeptide as reported by UniProtKB-P25942, e.g., comprising the CD40 binding domain of SEQ ID NO: 14. CD40L can be expressed as a soluble polypeptide and is the natural ligand of the CD40 receptor.

[0025] As used herein, the term "protein" refers to any organic compound consisting of amino acids arranged in one or more linear chains (also called "polypeptide chains") and folded into a globular form. The amino acids in such polypeptide chains are linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term "protein" further includes, but is not limited to, peptides, single-chain polypeptides, or any composite protein consisting primarily of two or more chains of amino acids. It further includes, but is not limited to, glycoproteins or other known post-translational modifications. It further includes known natural or artificial chemical modifications of natural proteins, such as glycoengineering, pegylation, hesylation, etc., incorporation of non-natural amino acids, chemical conjugation, or modification of amino acids for other molecules, etc.

[0026] As used herein, a "composite protein" more specifically refers to a protein consisting of at least two polypeptide chains, which are bound together under appropriate conditions, either non-covalently or covalently, e.g., via disulfide bridges or peptide bonds.

[0027] A "heterodimeric protein" refers to a protein that is composed of at least two polypeptide chains that form a complex protein, and the two polypeptide chains have different amino acid sequences.

[0028] The terms "polypeptide," "peptide," and "protein" explicitly include glycoproteins, as well as non-glycoproteins. In certain embodiments, the terms "polypeptide" and "protein" refer to any polypeptide or protein that is genetically encoded and can be translated using cellular expression systems and DNA recombinant means, e.g., a mammalian host cell expression system.

[0029] The term "recombinant protein," as used herein, includes proteins prepared by recombinant means, such as, for example, (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom (described further below), (b) fusion proteins isolated from host cells transformed to express the corresponding protein, e.g., transfectomas, etc.

[0030] As used herein, the term "fusion protein" refers to a recombinant protein comprising at least one polypeptide chain obtained or obtainable by genetic fusion, e.g., by the genetic fusion of at least two gene fragments encoding separate functional domains of distinct proteins.

[0031] The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.

[0032] In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (k). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain contains two domains, the variable domain (VL) and the constant domain (CL). The heavy chain contains four domains, the variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The light chain (CL) and heavy chain (CH) constant region domains confer important biological properties such as antibody chain association, secretion, placental transport, complement fixation, and binding to Fc receptors (FcR).

[0033] Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portion of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is composed mainly of residues from the hypervariable or complementarity determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or affect the overall homogeneity and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequence intervening between the CDRs. Thus, the variable regions of the light and heavy chains typically comprise four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0034] Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter "Kabat et al."). The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in a sequence according to the SEQ ID NO:. The actual linear amino acid sequence may contain fewer or additional amino acids compared to the strict Kabat numbering, which corresponds to a shortening or insertion of framework or complementarity determining regions (CDRs), which are structural components of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. For the agonist antibodies described below, the CDRs were determined using a CDR discovery algorithm from www.bioinf.org.uk - see the section entitled "How to identify CDRs by looking at a sequence" in the antibody page. The predicted CDRs of some agonist antibodies, such as 11B6, 12E2, 12B4, CP (CP-870,893 from Pfizer) or 24A3 are described in the examples below.

[0035] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to a full-length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the ectodomain of CD40). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab fragments, V L , V H , C L and a monovalent fragment consisting of the CH1 domain; F(ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; V H and an Fd fragment consisting of the CH1 domain; L and V H Fv fragment consisting of domains; V H Examples of such fragments include dAb fragments consisting of the antigen-binding domains (Ward et al., 1989 Nature 341:544-546), or any fusion protein containing such an antigen-binding fragment.

[0036] In addition, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but V L and V H The domains can be joined using recombinant methods by synthetic linkers that allow them to be made into a single-chain protein that pairs to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0037] As used herein, the term "IgG Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. A human IgG heavy chain Fc region is generally defined as comprising the amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The C-terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody. Thus, a composition of antibodies of the invention may include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue.

[0038] The term “K assoc " or "K a " as used herein is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K dis " or "K d " as used herein is intended to refer to the off-rate of a particular antibody-antigen interaction.

[0039] The term “K D " as used herein means K d K a (i.e., K d / K a ) and expressed as a molar concentration (M). D Values ​​can be determined using methods well established in the art. D A method for determining is by using surface plasmon resonance, for example by using a biosensor system such as a Biacore® system.

[0040] As used herein, the term "binding specificity" refers to the K DIt refers to the ability of an antibody to detectably bind to an antigenic recombinant polypeptide, such as a recombinant CD40 polypeptide, with an affinity of 100 nM or less, 10 nM or less, 5 nM or less.

[0041] Antibodies that do not cross-react with a specific antigen are D is 100 nM or more, or K D is 1mM or more, or K D "Affinity" is intended to refer to an antibody that binds to its antigen with an affinity of 10 mM or greater, as measured, for example, using similar surface plasmon resonance (SPR) measurements as disclosed in the Examples. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding to these proteins in standard binding assays.

[0042] An isolated CD40 activating protein according to the present disclosure is a protein that has binding specificity for CD40 and activating or agonistic properties for the CD40 receptor. The CD40 activating protein may have cross-reactivity to other antigens, such as related CD40 molecules from other species. Furthermore, in certain embodiments, the isolated CD40 activating protein may be substantially free of other cellular material and / or chemicals.

[0043] The phrases "an antibody that recognizes an antigen" and "an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0044] Specificity may further be indicated by the affinity / avidity ratio for binding to a specific antigen (wherein the specific antigen is a CD40 polypeptide) versus non-specific binding to other unrelated molecules, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater. The term "affinity" as used herein refers to the strength of binding of an antibody to an epitope.

[0045] The present disclosure relates to the unexpected discovery that fusion proteins of CD40L and certain CD40 agonistic antibodies (e.g., derived from agonistic mAbs 11B6 or 12B4, as described in WO 2010 / 104748) exhibit superior CD40 activation properties compared to the administration of the corresponding agonistic antibodies alone or in combination with such agonistic antibodies and soluble CD40L (sCD40L).

[0046] "Humanized antibodies," as used herein, broadly include antibodies made by non-human cells having variable and constant regions that have been modified to closely resemble antibodies made by human cells, for example by modifying the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. Antibodies as used herein may contain amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). In particular, the term "humanized antibodies" includes antibodies that contain silent variants of the Fc IgG region.

[0047] In certain embodiments, the term "humanized antibody" includes antibodies having the six CDRs of a murine antibody, but with humanized framework and constant regions.

[0048] More specifically, the term "humanized antibody", as used herein, may include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0049] As used herein, a "CD40 agonist" antibody is intended to refer to an antibody that increases CD40-mediated signaling activity in the absence of CD40L in a cell-based assay, such as a B cell proliferation assay. Such assays are described in more detail in the Examples below.

[0050] As used herein, the term "silent" antibody refers to an antibody that does not show or has low ADCC activity. As used herein, the term "ADCC" or "antibody-dependent cellular cytotoxicity" activity refers to cell depletion activity. ADCC activity can be measured by ADCC assays described in the literature.

[0051] Silenced effector functions can be obtained by mutations in the Fc region of the antibody and are described in Art: Strohl 2009 (LALA & N297A); Baudino 2008, D265A (Baudino et al., J. Immunol. 181 (2008): 6664-69; Strohl, CO Biotechnology 20 (2009): 685-91). An example of a silent Fc IgG1 antibody contains the L234A and L235A mutations in the IgG1 Fc amino acid sequence.

[0052] As used herein, a protein or antibody having "CD40 activating" properties refers to a protein or antibody that can increase CD40-mediated signaling activity. In particular, as used herein, a protein having CD40 activating properties has at least one or more of the following properties: (i) induces proliferation of B cells, as measured in vitro by flow cytometric analysis, e.g., as measured by a B cell proliferation assay described in the Examples below, e.g., by analysis of replicative dilution of CFSE-labeled cells; (ii) induces secretion of cytokines such as IL-6, IL-12, or IL-15, as measured in vitro using the dendritic cell activation assay described in the Examples below.

[0053] In certain embodiments, the CD40-activating proteins of the present disclosure have at least the same activation properties as a soluble version of the CD40 receptor's natural ligand, CD40L.

[0054] In a particular embodiment, the CD40-activating protein comprises the CD40-binding domain of CD40L that is not in a trimeric form.

[0055] In certain embodiments, the CD40 activating proteins of the present disclosure are tetravalent with respect to CD40 binding.

[0056] In a specific embodiment, the CD40 activating protein comprises a bivalent CD40 agonist antibody having one monomeric CD40 binding domain of CD40L covalently or non-covalently bound to each arm of the bivalent antibody, preferably via the C-terminal portion of each arm of the bivalent antibody of either the light or heavy chain of each arm.

[0057] In other specific embodiments, the CD40 activating proteins of the present disclosure have at least similar activation properties as a reference CD40 agonist antibody, typically selected from among the following CD40 agonist (including partial agonist) antibodies: 11B6, 12B4, CP-870, 893 or 24A3, typically the 11B6 antibody or a humanized version.

[0058] In other specific embodiments, the CD40 activating proteins of the present disclosure have at least similar activation properties and may be at least 10-fold, at least 50-fold, or at least 100-fold more active than Mega sCD40L, a known trimeric version of soluble CD40L, to induce cytokine secretion as measured in a dendritic cell activation assay, as described in the Examples below.

[0059] As used herein, the term "Mega sCD40L" refers to a trimeric CD40 ligand molecule linked via the collagen domains of adiponectin / ACRP30 / AdipoQ, sold as MEGA CD40L® and also described in Kornbluth et al., 2012.

[0060] In other specific embodiments, the CD40 activating proteins of the present disclosure have at least the same activation properties as a combined composition of soluble CD40L with the same CD40 agonist antibody (or antigen-binding fragment thereof) present in the CD40 activating protein.

[0061] As used herein, the term "optimized" means that a nucleotide sequence is modified to use codons that are preferred in a production cell or organism, typically a eukaryotic cell, such as a Chinese Hamster Ovary cell (CHO) or a human cell, to encode an amino acid sequence. An optimized nucleotide sequence is engineered to retain, completely or as far as possible, the amino acid sequence originally encoded by the starting nucleotide sequence. An amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0062] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

[0063] The Needleman and Wunsch algorithm (NEEDLEMAN and Wunsch) can be used to determine the percent identity between two amino acid sequences.

[0064] Percent identity between two nucleotide or amino acid sequences can also be determined using algorithms such as, for example, EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extension penalty" of 0.5, a false "end gap penalty" of 0.5, an "end gap open penalty" of 10, and an "end gap extension penalty" of 0.5. In general, "percent identity" is a function of the number of matching positions divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 sequence positions are identical between the two sequences compared after alignment, the identity is 60%. Percent identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules that have the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications.

[0065] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0066] As used herein, "dendritic cell" (DC) refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their characteristic morphology and high levels of surface MHC-class II expression. These cells can be isolated from a number of tissue sources, conveniently from peripheral blood, as described in the Examples below.

[0067] CD40 Activation Proteins of the Disclosure The present disclosure provides at least the following protein domains: (i) a CD40 agonist antibody (αCD40) or an antigen-binding fragment thereof; and (ii) the CD40-binding domain of CD40L (CD40L), typically of SEQ ID NO: 14, or a functional fragment thereof having at least 90%, 95% or 100% identity to SEQ ID NO: 14. The present invention relates to a CD40 activating protein comprising the

[0068] In certain embodiments, the CD40 binding domain of CD40L (preferably in monomeric form) is covalently or non-covalently linked to the C-terminus of the light or heavy chain of said CD40 agonist antibody or antigen-binding fragment thereof, optionally via a linker, such as a peptide or chemical linker. In one embodiment, the CD40 binding domain of CD40L is non-covalently linked to the C-terminus of the light chain of said CD40 agonist antibody or antigen-binding fragment thereof.

[0069] In certain embodiments, the CD40 binding domain of CD40L is fused, optionally via a linker, e.g., a peptide linker, to the C-terminus of the light or heavy chain of said CD40 agonist antibody or antigen-binding fragment thereof. Typically, the CD40 binding domain of CD40L is fused, optionally via a linker, e.g., a peptide linker, to the C-terminus of the light chain of said CD40 agonist antibody or antigen-binding fragment thereof.

[0070] In other specific embodiments, the CD40 binding domain of CD40L is conjugated to a CD40 agonist antibody or antigen-binding fragment thereof using chemical coupling. Several methods are known in the art for binding or conjugating an antibody to its conjugate moiety. Examples of linker types used to attach a moiety to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers, such as valine-citrulline linkers. The linker can be selected to be susceptible to cleavage by, for example, low pH in the lysosomal compartment, or by proteases, such as proteases preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, D). For further discussion of linker types and methods for conjugating therapeutic agents to antibodies, see also Panowski et al., 2013, for a review on antibody drug conjugates.

[0071] In certain embodiments, the CD40 activating protein disclosed herein is an antibody-like protein comprising a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; CD40L is, for example, the CD40 binding domain of CD40 ligand comprising SEQ ID NO:14, or a functional fragment thereof having at least 90%, 95% or 100% identity to SEQ ID NO:14.

[0072] In a more particular embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy-PL-BP1, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; · BP1 is the first member of the binding pair BP1 / BP2 and allows non-covalent coupling with the second member of said binding pair BP2, BP1 / BP2.

[0073] In another particular embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a heavy chain of the formula αCD40Heavy-PL-CD40L and a light chain of the formula αCD40Light-PL-BP1, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; · BP1 is the first member of the binding pair BP1 / BP2 and allows non-covalent coupling with the second member of said binding pair BP2, BP1 / BP2.

[0074] Any binding pair known in the art, typically a binding pair of polypeptide domains, can be used for the above embodiments of DC40 activating proteins. Examples of such binding pairs BP1 / BP2 include, but are not limited to, dockering domain / cohesin domain, or mSA2 / biotin, as described below.

[0075] In a more particular embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy-PL-Doc, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; Doc is a dockerin domain or domains that allow non-covalent coupling to a cohesin fusion protein, or any functional variant thereof, as described, for example, in U.S. Patent Application Publication No. 20160031988A1 and U.S. Patent Application Publication No. 20120039916A1, including SEQ ID NO:111.

[0076] In another particular embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy-PL-mSA2, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; mSA2 is a monomeric streptavidin 2 domain to allow non-covalent coupling with a biotin label or fusion protein comprising, for example, SEQ ID NO: 168, or any functional variant thereof, as described in Lim et al. (Biotechnology Bioeng 2013, 110, 57-67).

[0077] In another specific embodiment, the CD40 activating protein disclosed herein is an antibody-like protein comprising a heavy chain of the formula αCD40Heavy-PL-CD40L and a light chain of the formula αCD40Light, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; CD40L is, for example, the CD40 binding domain of CD40 ligand comprising SEQ ID NO:14, or a functional fragment thereof having at least 90%, 95% or 100% identity to SEQ ID NO:14.

[0078] In another more specific embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a heavy chain of the formula αCD40Heavy-PL-CD40L and a light chain of the formula αCD40Light-PL-Doc, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; Doc is a dockerin domain or domains that allow non-covalent coupling to a cohesin fusion protein, or any functional variant thereof, as described, for example, in U.S. Patent Application Publication No. 20160031988A1 and U.S. Patent Application Publication No. 20120039916A1, including SEQ ID NO:111.

[0079] In another particular embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a heavy chain of the formula αCD40Heavy-PL-CD40L and a light chain of the formula αCD40Light-PL-mSA2, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; mSA2 is a monomeric streptavidin 2 domain to allow non-covalent coupling with a biotin label or fusion protein comprising, for example, SEQ ID NO: 168, or any functional variant thereof, as described in Lim et al. (Biotechnology Bioeng 2013, 110, 57-67).

[0080] Preferred embodiments of αCD40Light, αCD40Heavy, and CD40L are further described in the next sections.

[0081] In certain embodiments, the CD40 activating protein further comprises one or more antigens "Ag" fused or conjugated or coupled by non-covalent coupling to either the corresponding heavy or light chain of the CD40 agonist antibody or antigen-binding fragment thereof. The antigens may be directly conjugated to the polypeptide chain of the CD40 activating protein, e.g., to the C-terminus of the polypeptide chain of the CD40 activating protein, and optionally via a peptide linker such as FlexV1, f1, f2, f3, or f4, described below. They may also be coupled by non-covalent coupling, included in a cohesin fusion protein for coupling to a dockerin domain, and / or a biotin fusion protein for coupling to a monomeric streptavidin 2 domain.

[0082] As used herein, the term "antigen" or "Ag" refers to any antigen and various types (e.g., peptides, proteins, glycoproteins, polysaccharides, glycolipids, lipopeptides, etc.) that may be used in a vaccine, whether or not comprising the whole microorganism or a portion thereof. Thus, the term "antigen" refers to a molecule capable of initiating a humoral and / or cellular immune response in a recipient of the antigen. Antigens are usually key molecules encoded by pathogens that cause a disease for which vaccination is an advantageous treatment.

[0083] In certain embodiments, the Ag is a peptide concatemer. The Ag may also comprise a polynucleotide, the sequence of which is selected to encode an antigen whose expression is desired by the individual to whom the polynucleotide is administered, in the case of an immunization technique called DNA immunization.

[0084] Typically, as used herein, an Ag is selected from a viral or other infectious disease antigen, or a cancer antigen.

[0085] In certain embodiments, the Ag is selected from infectious disease antigens selected from bacterial, viral, parasitic, and fungal antigens.Typically, the Ag is at least one viral antigen.For example, the at least one viral antigen comprises a peptide from adenovirus, retrovirus, picornavirus, herpesvirus, rotavirus, hantavirus, coronavirus, togavirus, flavinvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus, papillomavirus, parvovirus, poxvirus, hepadnavirus, rotovirus, or sponge virus.In another aspect, the at least one viral antigen comprises a peptide from at least one of HIV, CMV, hepatitis A, B, and C, influenza; measles, polio, smallpox, rubella, respiratory syncytial virus, herpes simplex, varicella zoster, Epstein-Barr, Japanese encephalitis, rabies, influenza, or cold virus.

[0086] In certain embodiments, the viral antigens are selected from one or more of the following antigenic domains: HIV-1 Gag p24 (SEQ ID NO:45), Nef (SEQ ID NO:46), and Gag p17 (SEQ ID NO:47) (comprising a combination of three antigens called GNG, see detailed amino acid sequence below) or a combination of HPV16 E6 and HPV16 E7 antigens (HPV16 E6 / E7) (also called HPV, see also detailed amino acid sequence below).

[0087] The GNG sequence is represented by the following formula (from C-terminus to N-terminus): FlexV1-LE-gag17-VDf3-VD-nef-EF-f4-QF p24-6xHis] (flexible linker sequence) During the ceremony, FlexV1 is a peptide linker of SEQ ID NO: 15; LE is the dipeptide Leu-Glu; Gag17 has the following amino acid sequence: MGARASILSGGELDRWEKIRLRPGGNKQYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKS (SEQ ID NO: 47) is an HIV-1 viral antigen; VD is a dipeptide linker of valine and aspartic acid; f3 has the following amino acid sequence: TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO:53) is a peptide linker of the formula: nef has the following amino acid sequence: MGGKWSKRSVVGWPTVRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKVEEANEGENNSLLHPMSLHGMDDPEREVLVWKFDSRLAFHHMARELHPEYYKDC (SEQ ID NO: 46) is an HIV-1 viral antigen; EF is a dipeptide linker of glutamic acid and phenylalanine; f4 is a flexible linker TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO:54); QF is a dipetidine linker of glutamine and phenylalanine; P24 has the following amino acid sequence: AQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVG (SEQ ID NO: 45) is an HIV-1 viral antigen; 6-His is a C-terminal hexahistidine tag of HHHHHH.

[0088] The complete amino acid sequence of GNG consists of SEQ ID NO:48.

[0089] Alternative GNG sequences can be used, for example, using GNG sequences with the same HIV-1 peptide sequence but with other peptide linkers.

[0090] The HPVs were of the following formula: Flex-v1-HPV16E6-HPV16E7-f1 (wherein FlexV1 is as defined above, HPV16E6-HVP16E7 has the following amino acid sequence: MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVGDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEASMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCK (SEQ ID NO:55) having; f1 has the following amino acid sequence: ASSSVSPTTSVHPTPTSVPPTPTKSSPAS (SEQ ID NO:56) (wherein m is a flexible linker).

[0091] The complete amino acid sequence of HPV consists of SEQ ID NO:57.

[0092] In other specific embodiments, the viral antigen is selected from one or more of the following HIV antigenic domains: Gag p17(17-35) of SEQ ID NO: 16, Gag p17-p24(253-284) of SEQ ID NO: 17, and Nef(116-145) of SEQ ID NO: 18, Pol 325-344(RT158-188) of SEQ ID NO: 19, and Nef(66-97) of SEQ ID NO: 20.

[0093] In a preferred embodiment, said viral antigen is selected from the combination of the five HIV antigenic domains: Gag p17(17-35) of SEQ ID NO: 16, Gag p17-p24(253-284) of SEQ ID NO: 17, and Nef(116-145) of SEQ ID NO: 18, Pol 325-344(RT158-88) of SEQ ID NO: 19, and Nef(66-97) of SEQ ID NO: 20, and is represented by the following formula: FlexV1-gag p17(17-35)-f1-gag p17-p24(253-284)-f2-nef(116-145)-f3-nef(66-97)-f4-Pol 325-344(RT158-188) (wherein FlexV1, f1, f2, f3, and f4 are as described above). This is contained in the HIV5pep sequence.

[0094] A particular embodiment of the HIV5pep amino acid sequence is set forth in SEQ ID NO:112.

[0095] In other specific embodiments, the Ag is CEA, prostate specific antigen (PSA), HER-2 / neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC related proteins (mucins) (MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), MARCO-MART, cyclin B1, cyclin D1, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), prostate Ca The tumor associated antigen is selected from psm, prostate serum antigen (PSA), PRAME (melanoma antigen), β-catenin, MUM-IB (melanoma ubiquitous mutated gene product), GAGE ​​(melanoma antigen) 1, BAGE (melanoma antigen) 2-10, C-ERB2 (Her2 / neu), EBNA (Epstein-Barr virus nuclear antigen) 1-6, gp75, human papillomavirus (HPV) E6 and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In another embodiment, the Ag is selected from tumor-associated antigens, including antigens from leukemias and lymphomas, neural tumors such as astrocytoma or glioblastoma, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors, stomach cancer, colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such as cervical, uterine, ovarian, vaginal, testicular, prostate or penile cancer, bone tumors, vascular tumors, or cancers of the lips, nasopharynx, pharynx and oral cavity, esophagus, rectum, gallbladder, biliary tree, larynx, lungs and bronchi, bladder, kidney, brain and other parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia. Such tumor-associated antigens include patient-specific tumor-mutated proteins containing known or potential T-cell epitopes.

[0096] In a particular embodiment, the CD40 activating protein of the present disclosure refers to a composite protein comprising two heterodimers, each heterodimer consisting of one heavy and one light chain of amino acids stably associated together, for example, via one or more disulfide bonds. Typically, the heavy chain comprises at least the VH region, preferably at least the CH1-VH region of a CD40 agonist antibody, and the light chain comprises at least the VL region, preferably at least the CL-VL region of said CD40 agonist antibody. At least said heavy or light chain is fused or conjugated, optionally via a linker, for example a peptide linker, to at least the CD40 binding domain of CD40L.

[0097] In certain embodiments, the CD40 activating protein of the present disclosure comprises the heavy and light chains of a CD40 agonist IgG antibody comprising an isotype constant region or an IgG Fc region, preferably an IgG4 or a mutated silent IgG Fc.

[0098] In a more particular embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy-(PL-Ag)x. ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; ·Ag is a viral or cancer antigen, identical or different; x is an integer from 1 to 20, for example 3, 4, or 5; CD40L is the binding domain of the ligand of CD40 comprising SEQ ID NO: 14, or a functional fragment thereof having at least 90%, 95% or 100% identity to SEQ ID NO: 14; ·- is a covalent bond.

[0099] In another more specific embodiment, said CD40 activating protein disclosed herein is an antibody-like protein comprising a heavy chain of the formula αCD40Heavy-PL-CD40L and a light chain of the formula αCD40Light-(PL-Ag)x, ·αCD40Light is the light chain of a CD40 agonist antibody; ·αCD40Heavy is the heavy chain of the CD40 agonist antibody; PL is a bond or a peptide linker, identical or different, preferably FlexV1 of SEQ ID NO: 15; ·Ag is a viral or cancer antigen, identical or different; x is an integer from 1 to 20, for example 3, 4, or 5; CD40L is the binding domain of the ligand of CD40 comprising SEQ ID NO: 14, or a functional fragment thereof having at least 90%, 95% or 100% identity to SEQ ID NO: 14; ·- is a covalent bond.

[0100] In certain embodiments, the PL is preferably a peptide linker to ensure optimal activation properties and yields in cell production.

[0101] In a particular embodiment, -(PL-Ag)x is located at the carboxy terminus of the heavy chain of said CD40 activating antibody-like protein.

[0102] Typically, a schematic diagram of the above CD40 activating protein embodiments is shown in Figure 1. Preferred embodiments of αCD40Light, αCD40Heavy, and CD40L are further described in the following sections.

[0103] In certain embodiments, the peptide linker may incorporate a glycosylation site or introduce a secondary structure. Furthermore, these linkers may increase the efficiency of expression or stability of the fusion protein, and therefore the efficiency of antigen presentation to dendritic cells. Such linkers may include flexV1, f1, f2, f3 and / or f4 linkers. Examples of these and others are described in WO 2010 / 104747, the contents of which are incorporated herein by reference. In particular, flexV1 is the polypeptide of SEQ ID NO: 15.

[0104] In a more specific embodiment, said CD40 activating protein comprises the PAB3405 CD40 agonist antibody consisting of SEQ ID NO:1 and SEQ ID NO:2.

[0105] In another specific embodiment, said CD40 activating protein comprises the PAB3408 CD40 agonist antibody consisting of SEQ ID NO:3 and SEQ ID NO:4.

[0106] In another specific embodiment, said CD40 activating protein consists of a heavy chain polypeptide comprising SEQ ID NO:5 and a light chain comprising SEQ ID NO:6.

[0107] In another specific embodiment, said CD40 activating protein consists of a heavy chain polypeptide comprising SEQ ID NO:5 and a light chain comprising SEQ ID NO:8.

[0108] In another specific embodiment, said CD40 activating protein consists of a heavy chain polypeptide comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:10.

[0109] In another specific embodiment, said CD40 activating protein consists of a heavy chain polypeptide comprising SEQ ID NO:11 and a light chain comprising SEQ ID NO:12.

[0110] In another specific embodiment, said CD40 activating protein consists of a light chain polypeptide of SEQ ID NO:109 and a heavy chain polypeptide of SEQ ID NO:110.

[0111] CD40 activating proteins having an amino acid sequence that has at least 90%, such as at least 95%, 96%, 97%, 98%, or 99% identity to any one of the above defined amino acid sequences are also part of the disclosure.

[0112] CD40 agonist antibodies for use in preparing fusion proteins of the present disclosure One skilled in the art can use CD40 agonist antibodies already known in the art or can use antibody screening techniques to generate de novo novel CD40 activating antibodies.

[0113] More specifically, the CD40 agonist antibodies (or antigen-binding fragments thereof) for use in the CD40 activating proteins of the present disclosure have one or more of the following advantageous properties: (i) a K of 500 nM or less, e.g., between 50 and 500 nM, as measured by an SPR binding assay, e.g., as described in the Examples below. D binds to the CD40 ectodomain having (ii) induces proliferation of B cells, e.g., as measured in the B cell proliferation assay described in the Examples below, as measured in vitro by flow cytometric analysis; and / or (iii) induces secretion of cytokines such as IL-6, IL-12, or IL-15, as measured in vitro using the dendritic cell activation assay described in the Examples below.

[0114] In certain embodiments, the CD40 agonist antibody has one or more of the following properties: (i) a K of 500 nM or less, e.g., between 50 and 500 nM, as measured by SPR, e.g., as described in the Examples below. D binds to the CD40 ectodomain having (ii) B cell proliferation, as measured in a B cell proliferation assay using the agonistic antibody described above, can be further increased, typically by at least 10-fold, or at least 100-fold, in the presence of a suboptimal dose of soluble CD40L, e.g., using the assay described in the Examples below; and / or (iii) secretion of cytokines such as IL-6, IL-12, or IL-15 can be enhanced, typically by at least 10-fold, or at least 100-fold, or at least 1000-fold, in the presence of suboptimal doses of soluble CD40L, as measured in vitro in a dendritic cell activation assay, e.g., using the assay described in the Examples below.

[0115] In certain embodiments, a CD40 agonist antibody is an antibody that has CD40-mediated signaling activity in the absence of CD40L in a cell-based assay that is at least similar to the CD40-mediated signaling activity of a reference CD40 agonist antibody, when measured in the same cell-based assay, said reference CD40 agonist antibody typically being selected from among the CD40 agonist antibodies: mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6, as described below.

[0116] In certain embodiments, the CD40 agonist antibody does not compete with sCD40L for binding to CD40.

[0117] In certain embodiments, in the presence of a certain suboptimal amount (6 nM) of soluble human CD40L, the CD40 agonist antibody has an EC50 (as measured in the B cell proliferation assay described in the Examples) that is between 1 and 200 times, preferably between 1 and 150 times, or between 1 and 100 times, the EC50 measured for the CD40 agonist antibody mAb5 (CP-870,893).

[0118] In other specific embodiments, in the presence of a certain suboptimal amount (6 nM) of soluble human CD40L, the CD40 agonist antibody has an EC50 (as measured in the B cell proliferation assay described in the Examples) that is equal to or less than the EC50 of the mAb1 (11B6) antibody.

[0119] The relative EC50 values ​​as measured in the B cell proliferation assay are further described in the Examples (see tables (Tables 4 and 5) for Figures 2 and 3).

[0120] Various methods of screening antibodies to select novel CD40 agonist antibodies have been described in the art. These methods can be divided into in vivo systems, such as transgenic mice that can produce fully human antibodies upon antigen immunization, and in vitro systems that consist of generating antibody DNA coding libraries, expressing the DNA libraries in a suitable system for antibody production, selecting clones that express antibody candidates that bind to the target by affinity selection criteria, and recovering the corresponding coding sequences of the selected clones.

[0121] These in vitro techniques are known as display technologies and include, but are not limited to, phage display, RNA or DNA display, ribosome display, yeast or mammalian cell display. They are well described in the art (for reviews, see, e.g., Nelson et al., 2010 Nature Reviews Drug discovery, "Development trends for human monoclonal antibody therapeutics" (Advance Online Publication) and Hoogenboom et al., Methods in Molecular Biology 178:1-37, O'Brien et al., eds., Human Press, Totowa, NJ, 2001). In one particular embodiment, human recombinant CD40 agonistic antibodies are isolated using phage display methods for screening of human recombinant antibody libraries with CD40 binding and agonistic properties.

[0122] V H and V L A repertoire of genes or relevant CDR regions can be cloned separately by polymerase chain reaction (PCR) or synthesized by a DNA synthesizer and randomly recombined in a phage library, which can then be screened for antigen-binding clones. Such phage display methods for isolating human antibodies are established in the art or are described in the Examples below. See, for example, U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081 to Griffiths et al.

[0123] In certain embodiments, human antibodies against CD40 can be identified using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[0124] The HuMAb Mouse® (Medarex, Inc.) contains a human immunoglobulin gene minilocus encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al., 1994 Nature 368(6474):856-859).

[0125] In another embodiment, human CD40 agonist antibodies can be generated using mice carrying human immunoglobulin sequences on a transgene and transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome, such mice are referred to herein as "KM mice" and are described in detail in PCT Publication WO 02 / 43478 by Ishida et al.

[0126] Monoclonal antibodies (mAbs) can also be produced by conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization techniques of Kohler and Milstein, 1975 Nature 256: 495. Many techniques for producing monoclonal antibodies can be used, e.g., viral or oncogenic transformation of B lymphocytes.

[0127] The animal system for preparing hybridomas is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0128] Chimeric or humanized antibodies can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and can be engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the mouse variable region can be linked to a human constant region using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the mouse CDR region can be inserted into a human framework using methods known in the art. See, for example, U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. Please refer to the number.

[0129] It is further contemplated that the monoclonal antibodies may be further screened or optimized for their CD40 agonist properties as defined above. In particular, it is contemplated that the monoclonal antibodies may have one, two, three, four, five, six or more modifications in the amino acid sequence of one, two, three, four, five or six CDRs of the monoclonal or humanized antibodies provided herein. It is contemplated that the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 of the VJ or VDJ region of the light or heavy chain variable region of the antibody may have insertions, deletions or substitutions with conserved or non-conserved amino acids. Such amino acids that may be substituted or may constitute a substitution are disclosed above.

[0130] CD40 agonist antibodies known in the art for use in preparing the CD40 activating proteins of the present disclosure include recombinant CD40 agonist antibodies mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6, which are structurally characterized by their variable heavy and light chain amino acid and nucleotide sequences as set forth in Tables 1 and 2 below.

[0131] [Table 1]

[0132] [Table 2]

[0133] Other CD40 agonist antibodies that may be used include any chimeric or humanized antibody comprising the six CDRs of mAb1, mAb2, mAb3, mAb4, mAb5 or mAb6 defined above.

[0134] Exemplary amino acid sequences of VH CDR1 (also referred to as HCDR1), VH CDR2 (also referred to as HCDR2), VH CDR3 (also referred to as HCDR1), VL CDR1 (also referred to as LCDR1), VL CDR2 (also referred to as LCDR2), and VL CDR3 (also referred to as HCDR3) of several CD40 agonist antibodies according to the present disclosure are shown in Table 3.

[0135] In Table 3, the CDR regions of the antibodies of the disclosure are depicted using Kabat numbering (Kabat et al., 1992, hereafter "Kabat et al.").

[0136] [Table 3]

[0137] In certain embodiments, the CD40 agonist antibody is selected from the following antibodies: (i) a humanized antibody comprising the six CDRs of mAb1, mAb2, mAb3, mAb4, mAb5, or mAb6; (ii) a humanized antibody comprising the VH and VL domains of SEQ ID NO: 21 and SEQ ID NO: 22, respectively; (iii) an antibody that competes with at least one of the antibodies identified in (i) or (ii) for binding to CD40-expressing cells; (iv) an antibody that binds to the same epitope as one of the antibodies identified in (i) or (ii).

[0138] In particular, in certain embodiments, the CD40 agonist antibody binds to an epitopic region comprising or consisting of amino acid residues 36-59 of SEQ ID NO: 13 (CDR1 region of CD40). More specifically, it binds to an epitopic region comprising or consisting of amino acid residues 50-58 of CD40. In certain embodiments, the CD40 agonist antibody directly contacts at least the following amino acid residues: E56 and E58. In other embodiments, the CD40 agonist antibody directly contacts at least the following amino acid residues: D50 and E58.

[0139] Other examples of CD40 agonist antibodies are described in WO 2010 / 009346, WO 2010 / 104747 and WO 2010 / 104749. Other anti-CD40 agonist antibodies in development include: CP-870,893 - a fully human IgG2 CD40 agonist antibody developed by Pfizer. It has a cell count of 3.48 × 10 -10 K of M D It binds to CD40 but does not block the binding of CD40L (see, e.g., U.S. Pat. No. 7,338,660).

[0140] SGN-40 is a humanized IgG1 antibody developed by Seattle Genetics from the mouse antibody clone S2C6, which was generated using a human bladder carcinoma cell line as an immunogen. It has a 1.0 × 10 -9 It binds to CD40 at the KD of M and acts by enhancing the interaction between CD40 and CD40L, thus exhibiting a partial agonist effect (Francisco JA et al., Cancer Res, 60: 3225-31, 2000).

[0141] Also, US Patent Application Publication No. 20120301488A1 by APEXIGEN describes another anti-CD40 agonistic mAb. US Patent Application Publication No. 20170342159 by AbbVie Biotherapeutics Inc. describes another agonistic antibody. CDX-1140 is an agonistic CD40 antibody by Celldex, another agonistic antibody.

[0142] Any other known antibodies can potentially be combined with binding CD40L to increase their biological activity using the methods disclosed in this application.

[0143] Nucleic acid molecules encoding the CD40-activating proteins of the present disclosure Also disclosed herein are nucleic acid molecules that encode the CD40-activating proteins of the present disclosure.

[0144] Exemplary nucleic acid molecules are those that encode the variable light and heavy chain amino acid sequences of the CD40 activating antibody-like proteins disclosed in the previous section, using the genetic code and, optionally, taking into account codon bias depending on the host cell type.

[0145] Typically, a nucleic acid molecule encoding a CD40 activating protein of the present disclosure comprises the coding sequence for the CD40 agonist antibody of SEQ ID NO:1 and SEQ ID NO:2, for example the nucleic acid of SEQ ID NO:33 and SEQ ID NO:34, respectively.

[0146] In a specific embodiment, the nucleic acid molecule encoding a CD40 activating protein comprises the coding sequence for the CD40 agonist antibody consisting of SEQ ID NO:3 and SEQ ID NO:4, eg, the nucleic acid of SEQ ID NO:35 and SEQ ID NO:36, respectively.

[0147] In another specific embodiment, the nucleic acid molecule encoding a CD40-activating protein comprises a sequence encoding a heavy chain polypeptide comprising SEQ ID NO:5, and a sequence encoding a light chain polypeptide comprising SEQ ID NO:6, e.g., the nucleic acids of SEQ ID NO:37 and SEQ ID NO:38, respectively.

[0148] In another specific embodiment, the nucleic acid molecule encoding a CD40-activating protein comprises a coding sequence for a heavy chain polypeptide comprising SEQ ID NO:5, and a coding sequence for a light chain comprising SEQ ID NO:8, e.g., the nucleic acids of SEQ ID NO:39 and SEQ ID NO:37, respectively.

[0149] In another specific embodiment, the nucleic acid molecule encoding a CD40-activating protein comprises a coding sequence for a heavy chain polypeptide comprising SEQ ID NO:9, and a coding sequence for a light chain comprising SEQ ID NO:10, e.g., the nucleic acids of SEQ ID NO:41 and SEQ ID NO:42, respectively.

[0150] In another specific embodiment, the nucleic acid molecule encoding a CD40-activating protein comprises a coding sequence for a heavy chain polypeptide comprising SEQ ID NO:11, and a coding sequence for a light chain comprising SEQ ID NO:12, e.g., the nucleic acids of SEQ ID NO:43 and SEQ ID NO:44, respectively.

[0151] In another specific embodiment, the nucleic acid molecule encoding a CD40-activating protein comprises a coding sequence for a heavy chain polypeptide comprising SEQ ID NO:110, and a coding sequence for a light chain comprising SEQ ID NO:109, e.g., the nucleic acids of SEQ ID NO:114 and SEQ ID NO:113, respectively.

[0152] Nucleic acids encoding the CD40-activating proteins of the present disclosure having a nucleotide sequence that has at least 90%, e.g., at least 95%, 96%, 97%, 98%, or 99% identity to any one of the above defined nucleotide sequences, are also part of the present disclosure.

[0153] The present disclosure also relates to nucleic acid molecules derived from the latter sequence that are optimized for protein expression in mammalian cells, such as CHO or HEK cell lines.

[0154] The nucleic acid may be present in whole cells, cell lysates, or may be in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (Ausubel et al., 1988). The nucleic acid of the present disclosure may be, for example, DNA or RNA, or may or may not contain intron sequences. In one embodiment, the nucleic acid may be present in a vector, such as a phage display vector, or in a recombinant plasmid vector.

[0155] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For example, once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by conventional recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, into Fab fragment genes, or into scFv genes.

[0156] Generation of transfectomas producing CD40-activating protein The CD40 activating proteins of the present disclosure can be produced in host cell transfectomas, for example, using a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, 1985).

[0157] For example, to express a CD40 activating protein, DNA encoding said CD40 activating protein can be obtained by standard molecular biology or biochemistry techniques (e.g., DNA chemical synthesis, PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest) and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the gene is linked to the vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the CD40 activating protein. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. If the CD40 activating protein comprises separate polypeptides, for example one sequence encoding the heavy chain of a CD40 activating antibody-like protein disclosed in the above section and another sequence encoding the light chain of said CD40 activating antibody-like protein, the genes encoding the heavy and light chains can be inserted into separate vectors, or more typically, both genes can be inserted into the same expression vector. The gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites on the protein gene and vector, or blunt end ligation if no restriction sites are present).

[0158] A signal peptide can also be used for secretion of the polypeptide from the expressing cell, such as an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

[0159] Furthermore, the recombinant expression vectors disclosed herein have regulatory sequences that control the expression of CD40 activating protein in host cells. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the respective genes. It is understood by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the desired expression level of the protein, and the like. Regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that induce high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or P-globin promoter, may be used. Still further, there are also regulatory elements that are composed of sequences from different sources, such as the SV40 early promoter and the SRa promoter system containing the long terminal repeat of human T-cell leukemia virus type 1.

[0160] Furthermore, the recombinant expression vector of the present disclosure can carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0161] For expression of CD40 activating proteins, expression vectors are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, etc. In theory, it is possible to express the binding proteins of the present disclosure in either prokaryotic or eukaryotic host cells. Expression of recombinant proteins in eukaryotic cells, such as mammalian host cells, yeast or filamentous fungi, is contemplated because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0162] In one particular embodiment, a cloning or expression vector according to the present disclosure comprises one or more nucleic acids operably linked to a suitable promoter sequence, as described in the previous section.

[0163] Mammalian host cells for expressing recombinant antibodies of the disclosure include dhfr-CHO cells (described in Urlaub and Chasin, 1980), used with DHFR selection marker, CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells and SP2 cells, e.g., GS CHO cell line, and Chinese Hamster Ovary (CHO cells), such as GS Xceed™ Gene Expression System (Lonza) or HEK cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient for expression of the antibody in the host cell, and optionally secreting the antibody into the culture medium in which the host cell is grown. The antibody can be recovered and purified, e.g., after secretion, from the culture medium using standard protein purification methods.

[0164] In one particular embodiment, the host cell of the present disclosure is a host cell transfected with an expression vector comprising a coding sequence for a CD40 activating protein, as disclosed in the previous section.

[0165] The latter host cells can then be further cultured under conditions suitable for the expression and production of said CD40 activating protein.

[0166] Pharmaceutical Compositions In another aspect, the disclosure provides a composition, eg, a pharmaceutical composition, containing a CD40-activating protein formulated together with a pharma- ceutically acceptable carrier.

[0167] The pharmaceutical compositions disclosed herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include a CD40 activating protein of the present disclosure in combination with at least one antiviral agent, anti-inflammatory agent, vaccine adjuvant, and / or another anti-proliferative agent.

[0168] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier must be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous route. Depending on the route of administration, the active compound, i.e., CD40 activating protein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0169] Sterile phosphate-buffered saline is one example of a pharma- ceutically acceptable carrier. Other suitable carriers are known to those skilled in the art (Remington and Gennaro, 1995). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like.

[0170] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc.

[0171] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, and the like.

[0172] Preferably, the pharmaceutical compositions contain pharma- ceutically acceptable vehicles for injectable preparations, which may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of such salts) or may be dried, in particular lyophilized, compositions that allow the constitution of an injectable solution, if added, by the addition of, optionally, sterile water or saline.

[0173] The dose used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, the pathology involved or as a function of the desired duration of treatment.

[0174] To prepare a pharmaceutical composition, an effective amount of CD40 activating protein may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0175] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders or lyophilisates for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0176] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0177] Pharmaceutically acceptable salts which can be used in the formulation include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, as well as organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0178] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0179] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent with various other ingredients as listed above, as necessary, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients required from above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredient from the previously sterile-filtered solution.

[0180] Where use of DMSO as a solvent would result in extremely rapid penetration and delivery of high concentrations of active agent to small tumor areas, preparation of more or more concentrated solutions for direct injection is also contemplated.

[0181] If formulated, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like can also be used.

[0182] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for each subject.

[0183] The CD40 activating protein can be formulated in a therapeutic mixture to contain about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 milligrams, or 1.0 to about 10 milligrams per dose, and multiple doses can also be administered.

[0184] Vaccine Compositions The present disclosure also relates to a vaccine comprising a CD40 activating protein of the present disclosure and a pharma- ceutically acceptable vehicle.

[0185] As used herein, the term "vaccine" is intended to mean a composition that can be administered to a human or animal to induce an immune response; this immune response can result in the production of antibodies or simply the activation of certain cells, in particular antigen-presenting cells, T-lymphocytes and B-lymphocytes. In certain embodiments, the vaccine is capable of producing an immune response in a patient that leads to the production of neutralizing antibodies with respect to the antigens provided in the vaccine. The vaccine can be a composition for prophylactic or therapeutic purposes or both.

[0186] A vaccine may comprise an effective amount of the CD40 activating protein of the present disclosure dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium. Such compositions may also be referred to as inocula. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition. The vaccine compositions of the present disclosure may comprise classic pharmaceutical preparations. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0187] In addition, if desired, the vaccine may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, and / or adjuvants that enhance the effectiveness of the vaccine. Examples of adjuvants that may be effective include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE, and RIBI, which are three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene / Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants, and Kill A. Additionally, immunomodulators such as lymphokines (e.g., IFN-[gamma], IL-2 and IL-12) or synthetic IFN-[gamma] inducers such as poly I:C or poly ICLC (Hiltonol) can be used in combination with the adjuvants described herein.

[0188] In certain embodiments, the adjuvant may be selected from among cytokine adjuvants such as Poly ICLC, CpG, LPS, Immunoquid, PLA, GLA, or IFNα. In other embodiments, the adjuvant may be a Toll-like receptor agonist (TLR). Examples of TLR agonists that may be used include TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, or TLR9 agonists.

[0189] Vaccine preparations of CD40-activating protein as active immunogenic ingredient can be prepared as injections, either as liquid solutions or suspensions; solid forms suitable for solution in liquid or suspension before infection can also be prepared. Preparations can be emulsified and encapsulated in liposomes. The active immunogenic ingredient is often mixed with a carrier that is pharma- ceutically acceptable and compatible with the active ingredient.

[0190] Methods of using the CD40 activating proteins of the present disclosure By eliciting an immune response against antigens present on the CD40 activating proteins, the CD40 activating proteins of the present disclosure may be useful as drugs, particularly for treating or preventing cancer or infectious disorders.

[0191] In some embodiments, the CD40 activating proteins may be used in a method of treating or preventing a viral infection or a cancer disorder in a subject comprising administering to the subject a CD40 activating protein of the present disclosure.

[0192] Yet another aspect relates to a method for inducing and / or enhancing a B cell and / or T cell response against a viral or tumor associated antigen in a subject in need thereof, comprising administering to a subject in need thereof a CD40 activating protein or vaccine of the present disclosure.

[0193] A further aspect relates to a method of inducing an IgG-binding antibody response against an antigen in a subject in need thereof comprising administering a CD40-activating protein of the present disclosure or a vaccine composition of the disclosure.

[0194] In some embodiments, the method further comprises administration of an immunostimulatory agent, hi some embodiments, the immunostimulatory agent is administered sequentially or simultaneously with the vaccine or therapeutic composition.

[0195] In some embodiments, the immunostimulant is mixed with the vaccine composition extemporaneously prior to injection of the vaccine composition into a subject.

[0196] Additionally, the methods of the present disclosure may also include administration of one or more adjuvants. The adjuvant may be directly or indirectly bound or conjugated to one or more of the vaccine components, such as the antigen or CD40 activating protein. In other embodiments, the adjuvant may be provided or administered separately from the vaccine composition. In certain embodiments, the adjuvant is a cytokine adjuvant, such as poly-ICLC, CpG, LPS, Immunoquid, PLA, GLA, or IFNα. In other embodiments, the adjuvant may be a Toll-like receptor agonist (TLR). Examples of TLR agonists that may be used include TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, or TLR9 agonists.

[0197] In some embodiments, administration comprises intradermal, intramuscular, or subcutaneous administration.

[0198] In some embodiments, a viral vaccine, e.g., a CD40 activating protein comprising a viral antigen, is used in a method of enhancing an immune response to at least one viral epitope comprising administering to a patient a viral vaccine described herein.

[0199] In some embodiments, such viral vaccines are used to prevent healthy subjects from becoming infected with the virus (prophylactic treatment), including, for example, administering such a viral vaccine of the present disclosure to a healthy subject not infected with the virus. In other embodiments, the viral vaccines of the present disclosure are used in a method of treating a patient at an early stage of viral infection, including administering the viral vaccine to the patient.

[0200] It is contemplated that the at least one viral antigen elicits at least one of a humoral and / or a cellular immune response in a host, preferably a human patient or a primate.

[0201] Administration of the vaccine or pharmaceutical composition according to the present disclosure is via any common route, so long as the target tissue is available via that route, to maximize delivery of the antigen to the site for maximum (or in some cases minimal) immune response. Administration of the vaccine is generally via orthotopic, intradermal, mucosal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Other areas of delivery include oral, nasal, buccal, rectal, vaginal or topical. The vaccine of the present disclosure is preferably administered parenterally, by injection, for example, either subcutaneously or intramuscularly.

[0202] Vaccines or pharmaceutical compositions of the present disclosure can be administered in a manner compatible with the dosage formulation, and in an amount that is prophylactically and / or therapeutically effective. The amount administered will depend on the subject to be treated, including, for example, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection or treatment desired. Suitable dosage ranges are in the range of about 0.1 mg to 1000 mg, e.g., in the range of about 1 mg to 300 mg, or in the range of about 10 mg to 50 mg, of several hundred micrograms of active ingredient per vaccination.

[0203] Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be particular to each subject. It will be apparent to those skilled in the art that a therapeutically effective amount of a CD40 activating protein of the present disclosure will depend, among other things, on the administration schedule, the unit dose of antigen administered, whether the CD40 activating protein is administered in combination with other therapeutic agents, the immune status and health of the recipient, and the therapeutic activity of the particular CD40 activating protein.

[0204] Vaccines can typically be administered in a single dose schedule or a multiple dose schedule. A multiple dose schedule can include, for example, 1-10 separate doses as an initial course of vaccination, followed by subsequent time intervals required to maintain or enhance the immune response, for example, a second dose at 1-4 months, and a second dose several months later, if necessary. Periodic booster immunizations at intervals of 1-5 years are desirable to maintain a desired level of protective immunity, usually for 3 years. Immunizations can be followed over a course of immunization by in vitro proliferation assays of peripheral blood lymphocytes (PBLs) co-cultured with antigen, and by measuring the levels of IFN-[gamma] released from primed lymphocytes. Assays can be performed using conventional labels, such as radionucleotides, enzymes, fluorescent labels, etc. These techniques are known to those skilled in the art and can be found in U.S. Pat. Nos. 3,791,932, 4,174,384, and 3,949,064.

[0205] Vaccines may be provided in one or more "unit doses". A unit dose is defined as comprising a predetermined amount of vaccine calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The amount to be administered, and the particular route and formulation, are within the skill of those of ordinary skill in the clinical arts. The subject to be treated may also be evaluated, among other things, for the state of the subject's immune system and the protection desired. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. The amount of vaccine delivered may vary from about 0.001 to about 0.05 mg / kg body weight, e.g., 0.1 to 5 mg per subject.

[0206] Further aspects relate to kits comprising a CD40 activating protein of the present disclosure, a nucleic acid of the present disclosure, an expression vector of the present disclosure, or a host cell of the present disclosure; and, optionally, instructions for use of the kit. The kits can be used to perform the methods described herein. In some embodiments, the kits are for inducing a T cell response and / or a B cell response in a subject, and the kits comprise a CD40 activating protein of the present disclosure or a vaccine of the present disclosure.

[0207] The present disclosure is further illustrated by the following examples, which should not, however, be construed as limiting the scope of the invention. EXAMPLES

[0208] 1. Method MDDC Preparation Method - Protocol 1M human blood monocytes / mL were cultured in 6-well plates (2mL / well) in RPMI medium + 10% FBS + 10ng / mL human IL-4 + 100ng / mL human GM-CSF. Half of the medium was replaced on days 2 and 4 to maintain the same concentrations of IL-4 and GM-CSF. Cells were harvested on day 5 by gentle washing without scraping and seeded in 200μL at 100,000 cells per well in 96-well v-bottom plates. Typically, 1M DCs were derived from 2M monocytes. Different concentrations of anti-CD40 mAb or anti-CD40 IgG4 fusion protein and 10ng / mL IL-4 and 100ng / mL GM-CSF were added and after 24 or 48 hours, the supernatants were tested for secreted cytokines and the cells were stained for cell surface activation markers.

[0209] Surface Plasmon Resonance (SPR) Binding Assay - Protocol Surface plasmon resonance (SPR) assay binding measurements were performed on a SensiQ Pioneer instrument (SensiQ Technologies, Inc., Oklahoma City, OK, USA). Protein A or Protein G (100 μg / mL in 10 mM NaAc pH 4.5) was immobilized using amine coupling chemistry on a COOH2 or COOH5 sensor chip at 25° C. according to the manufacturer's recommended protocol. The running buffer was 10 mM HEPES, 3.4 mM EDTA, 0.005% Tween 20, 8.8 g / L NaCl, pH 7.5. Subsequently, anti-CD40 mAb was injected at a concentration of 125 nM using channel 1 (fast injection, 10 μL / min for 4 min); a dilution series of cohesin-human CD40 ectodomain protein (P3398) (25, 12.5, 6.25, 3.125, 1.6, 0.8 nM, 25 μL / min for 2 min) was injected using channels 1-2; finally, the surface was regenerated by injection of 20 mM NaOH for 1 min (25 μl). Binding data were analyzed with Qdat software (SensiQ Technologies, Inc.).

[0210] T Cell Expansion - Protocol After thawing and washing, 2M PBMCs were cultured in 1 mL cRPMI + 10% AB serum in 24-well flat-bottom plates at 37 °C for 1 h. 2 The cells were cultured at 37°C in the presence of aCD40 abs. Cells were treated with aCD40 abs or controls at different concentrations (1 nM, 0.1 nM and 0.01 nM). Conditions were performed in triplicate to obtain sufficient T cells at the end of the culture. On day 2, 1 mL of cRPMI + 10% AB serum and IL-2 at a final concentration of 100 U / mL was added to each well. Half of the medium was replaced with fresh IL-2 added on days 4 and 6 without doubling the concentration. Cells were rested until day 10, at which point they were harvested and washed twice in PBS with 2 mM EDTA. Cells were then resuspended in a volume of cRPMI + 10% AB serum that allowed equal distribution of cells between the required conditions, in a final volume of 200 μL per condition, counted and plated in O2 in 50 mL tubes. 2 The mixture was rested O / N at 37°C in the presence of

[0211] On day 11, cells were seeded into V-bottom 96-well plates and 2 Cells were restimulated with 2 μM peptide or control in the presence of 0.1 μM β-lactamase for 1 h at 37°C. After 1 h, 0.175 μL of GolgiStop and 0.45 μL of Brefeldin were added to each well in a volume of 50 μL of cRPMI + 10% AB serum, and cells were incubated for an additional 4 h. Cells were then spun down and stained with the following antibodies: αCD3 BV711, αCD4 Pe-Cy7, αCD8 Pacific Blue, αCD56 Pe-Cy5, αCD16 APC-H7, αCD45 Pacific Orange, αNKG2C Alexa Fluor 700, αNKG2D PECF594, αCD69 FITC, αTNF-α APC, αINF-γ PE. Aqua was used as a viability dye. After staining, cells were resuspended in BD fixative and analyzed on a BD LSR II flow cytometer.

[0212] B Cell Proliferation Assay - Protocol Human PBMCs were thawed with benzonide 1:10 in RPMI medium (1 μL in 5 mL), cells were washed 2× in PBS, resuspended in PBS to have Cf 10M / mL, then stained with CSFE Cf 1.25 μM (Ci=5 mM) for 7 min at RT in the dark. 10 mL of FBS was added, cells were left in the cold for 5 min, then washed 2× in PBS, resuspended in RPMI medium with 10% FBS, and 1M cells / mL were distributed per well. Human IL-4 (10 ng / mL) and human IL-21 (5 ng / mL) were added to the cells with various amounts of anti-CD40 mAb or anti-CD40 IgG4 fusion protein for 6 days. CD19 APC: 1 μl; CD27 APC-H7: 1 μl; CD38 PE-Cy7: 0.5 μl; Live / Dead Aqua: 1 μl.

[0213] Dendritic Cell Activation Assay - Protocol 1M human blood monocytes / mL were cultured in 6-well plates (2mL per well) in RPMI medium + 10% FBS + 10ng / mL human IL-4 + 100ng / mL human GM-CSF. Half of the medium was replaced on days 2 and 4 to maintain the same concentrations of IL-4 and GM-CSF. Cells were harvested on day 5 by gentle washing without scraping and seeded at 100,000 cells / well in 200μL in 96-well v-bottom plates. Typically, 1M DCs were derived from 2M monocytes. Different concentrations of anti-CD40 mAb or anti-CD40 IgG4 fusion protein and 10ng / mL IL-4 and 100ng / mL GM-CSF were added and after 24 or 48 hours, the supernatants were tested for secreted cytokines and the cells were stained for cell surface activation markers.

[0214] T cell proliferation assay using HIV5 long peptide: ICS-protocol All cultures are performed in 10% AB (unfiltered) in cRPMI: cRPMI Hepes (1M) [12.5ml per 500ml] NEAA (10X) [5ml per 500ml] 2ME (1000X) [450 μl per 500 ml; 50 uM final] Sodium pyruvate (10X) [5ml per 500ml] Pen Strep (10,000U / 10,000U) [5ml per 500ml] Adjust pH to 7.4 with NaOH

[0215] Thaw cells in 10% AB cRPMI (use 50 U benzonase in the first dilution of cells) Wash 2x with 1x PBS, 2mM EDTA. Resuspend cells in 10% AB cRPMI in a 50ml loose-capped tube at a concentration of 2x10e6 / ml and store cells at 37°C. 2 Leave at 5% overnight. Next day (Day 0): Re-count and adjust to 2x10e6 / ml (per well)* based on viability / density.

[0216] Day 0: Seed cells in 24-well plates: Approximately 6 wells of targets per test molecule condition (so there are enough cells at the end of T cell expansion. A "cells only" set without test molecule is also needed as a control for restimulation) (Typically, if testing 4 different test molecules and a negative cell-only control, this requires 6x10e7 donor PBMCs) Seed cells into 24-well plates at 2 x 10e6 / well (1 ml at set-up)* in 10% AB cRPMI Add test molecule at 1 nM "final"** This can be done in a 50μl volume and added to 1ml of cells. · Or adjust cell volume for "equal" addition of cells and test molecule (500μl + 500μl)*. · **A range of test molecules was tested between 30nM and 0.1nM; for our comparative vaccine evaluation, 1nM was ultimately used. · Make fresh from concentrated stocks; don't store low-concentration proteins.

[0217] Day 2: After culture setup and protein stimulation, add 1 ml of 10% AB cRPMI containing IL2 (to give a final IL-2 concentration in the well of 100 U / ml).

[0218] Day 4: Remove 1 ml and add 1 ml of 100 U / ml IL2 in 10% AB cRPMI.

[0219] Day 6: Remove 1 ml and add 1 ml of 100 U / ml IL2 in 10% AB cRPMI.

[0220] Day 8: Harvest all pools for each test molecule condition (varies by test molecule, cell number, and type / morphology). Wash off IL2 (wash 2x with 1x PBS, 2mM EDTA). Resuspend cells in 10% AB cRPMI in a 50ml loose-cap tube at a concentration of 2x10e6 / ml and incubate cells at 37°C with CO. 2 Leave at 5% overnight.

[0221] Day 9: Filter (cells aggregated with CD40L constructs) and count and dispense equal amounts of cells (per total cells accumulated per donor) to allow restimulation with peptide. (Cell numbers / viability vary per PBMC donor; peptide conditions remain the same.) Typically, cells are around 1-5 million per testing time point, depending on the donor. Cells are seeded in 100 μl volume. Set up peptide stimulation in a 96-well V-bottom plate: 100 μl cells (approximately 1-5 x 10e6 per 100 μl) + 100 μl peptide (or solvent / control SEB) in a V-bottom 96-well plate: (split into 7 conditions between each donor) Solvent (highest volume for highest amount of peptide used), 10 μM peptide (2 μM–10 μM can be used), 2 μg / ml SEB. 1 h, 37 °C, CO 2 5%. 37°C, CO 2 After 1 hour stimulation at 5%, 50 μl of 10% AB cRPMI media containing 0.175 μl GolgiStop / 0.45 μl Brefeldin A: (BD GolgiStop, Cat 51-2092KZ; Brefeldin A, Cat 420601) is added. 37°C, CO 2 After 4 h at 5%, proceed to intracellular staining.

[0222] Intracellular staining protocol: 96-well V-bottom plates followed by peptide restimulation and Golgi / BFA block: Wash cells (1x w / 200μl 1x PBS): (Cfg 1600 RPM for 10 minutes; move plate to remove wash solution) Resuspend cells in 50μL Aqua* (1μl Aqua / 50μl 1X×PBS per sample required) for 20 minutes at 4°C. Wash cells (1x in 200μl with FACS buffer): (Cfg 1600 RPM for 10 minutes; move plate to remove wash solution) Stain cells in a cocktail of cell surface markers (αCD3 Per-CP 3 μL, αCD4 PE-Cy7 0.5 μL and αCD8 Pacific Blue 1 μL) in a total volume of 50 μL / sample of FACS buffer for 30 min on ice. Wash cells (2x with FACS buffer): (Cfg 1600 RPM for 10 min; move plate to remove wash solution) · Cells are suspended in 200 μL of Cytofix / Cytoperm** solution for 20 min at 4°C. · The cells are then spun and then washed 2x in 1x filtered (0.45μm) Perm / Wash** solution. · Stain cells in a cocktail of anti-cytokines (1 μL αTNFα APC and 2 μL αINFγ PE) in a total volume of 50 μL Perm / Wash** / Sample. Incubate at RT for 30 min Wash cells (2x with Perm / Wash* buffer): (Cfg 1600 RPM 10 minutes, move plate to remove wash solution) Resuspend in BD fixative (approximately 200 μl per sample). **BD Fixation / Transmission Kit Catalog #554714 FACS buffer: PBS + 2% FCS or BSA + 2mM EDTA BD Fixative Stabilizing Fixative 3x Concentrate:1:3 in Water Catalog #338036 *Aqua Live / Dead Invitrogen L34966 (reconstitute 50 μl DMSO per tube, use 1 μl / sample)

[0223] CD40 Receptor Clustering Assay - Protocol ExpiCHO-S cells (Thermo Fisher) stably expressing human CD40-eGFP or human CD40-mCherrry fusion proteins were used as a model to study CD40 cluster formation. Cells were incubated at a concentration of 1E6 cells / mL in CD CHO / M5 medium (Gibco) in 6-well plates with 25 mm diameter round cover slides (Electron Microscopy Science) at 37°C in the presence of 10 nM anti-CD40 antibody. After 1 h, the cover slides were gently washed twice with PBS and then resuspended in 1% PFA (Thermo Fisher) for 10 min at room temperature. After two further washes in PBS, the cover slides were finally mounted on ultra-frozen microscope slides (Fisherbrand) using ProLong Gold antifade reagent with DAPI (Invitrogen). Slides were left overnight at room temperature in the dark. The next day, slides were imaged with a Leica TCS SP5 confocal microscope and subsequently analyzed with ImageJ software.

[0224] Anti-CD40 mAb Internalization Assay Protocol CHO cells stably transfected with the CET 1019 HS-puro-SceI vector (Millipore Sigma) carrying the human CD40 cDNA insert (NM_001250.6 residues 31-864, C928) were grown in CD CHO / M5 medium (Gibco) with puromycin selection to obtain stably transfected bulk. Cells were incubated in 1% BSA (250K / 50 μl) in V-bottom 96-well plates with 100 nm of each test mAb (C3808, residues 1044–1213 of WP_065674352.1 fused to the flexV1 Doc Var1 module (Flamar et al., 2012) non-covalently associated at its H-chain C-terminus with a cohesin-mCherry fusion protein, fused to the cohesin domain from cellulosomal scaffolding protein A [Hungateiclostridium thermocellum] with a f1 flexible linker, AVY25163.1 residues 580–608 to mCherry, after the codon encoding ML, and the encoded secreted protein was visualized using the C-tag affinity matrix CaptureSelect™ (Thermo The antibodies were dispersed in cell culture medium containing the ANF29837.1 residues 330-562 (preceding the KEPEA sequence used for purification, Fisher Scientific, 191307005). The antibodies tested saturate CD40 binding sites on these cells at 100 nM (data not shown). At 30 min intervals, the labeled antibody complexes were added to the cells kept at 37°C in a cell culture incubator, and at the last (zero) time point, an equal volume of ice-cold PBS was added to all time points by centrifugation at 1600 rpm for 6 min with removal of liquid by shaking. Then, 110 μl of cold PBS was added to one time course train (for total binding analysis) and 100 μl of ice-cold 0.1 M glycine, 0.1 M NaCl pH 2.5 was added to a parallel time course train (i.e., acid stripping treatment to selectively remove cell surface-bound mAb). After 1 min, 10 μl of 1M Tris HCl pH 9 was added to the acid-treated rows to neutralize the acid, and a further 100 μl of cold PBS was added to the entire column, followed by centrifugation at 1600 rpm for 6 min with removal of liquid by shaking.Note that this period of acid treatment does not impair mCherry fluorescence (data not shown). After a final wash in PBS, cells were resuspended in 100 μl PBS and 75 μl was dispensed into a Black Fluor Micro2 plate (Thermo Fisher) for fluorescence reading at Ex 570_Em 625 nM in a SpectraMax Paradigm instrument (Molecular Devices).

[0225] Example sequence naming conventions: 11B6-5 PAB3405 ·rAB-pIRES2[manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-] ·rAB-IRES2-CI2[manti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C] C3677 rAB-pIRES2[manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C] SEQ ID NO:1 EVQLVQSGAEVKKPGASVKISKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVT VSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS- + C3862 rAB-IRES2-CI2 C3682 rAB-IRES2-CI2[m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C] Sequence number 2 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS- PAB3408 11B6-8 · rAB-pIRES2[m anti-CD40-11B6.1C3-VH-v3-LV-hIgG4H-C] · rAB-IRES2-CI2[m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C] C3678 rAB-pIRES2[m anti-CD40-11B6.1C3-VH-v3-LV-hIgG4H-C] Sequence number 3 EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATSYNQNFKDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTV TVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS C3682 rAB-IRES2-CI2[manti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C] SEQ ID NO:4 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS

[0226] From these two, two variants were derived that were tagged with CD40L: PAB3470 ·rAB-cetHS-puro[manti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40 ligand] ·rAB-cetHS-puro[manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C] C3724rAB-cetHS-puro [m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40 ligand] SEQ ID NO: 5 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECASQTPTNTISVTPTNNSTPTNNSNPKPNPASMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL +C3725 rAB-cetHS-puro [m anti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C] SEQ ID NO: 6 EVQLVQSGAEVKKPGASVKISCKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS-

[0227] PAB3471 · rAB-cetHS-puro [m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40 ligand] · rAB-cetHS-puro [m anti-CD40-11B6.1C3-VH-v3-LV-hIgG4H-C] C3724 rAB-cetHS-puro [m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40 ligand] SEQ ID NO: 5 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECASQTPTNTISVTPTNNSTPTNNSNPKPNPASMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL +C3726 rAB-cetHS-puro[m anti-CD40-11B6.1C3-VH-v3-LV-hIgG4H-C] Sequence number 8 EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATSYNQNFKDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS

[0228] We selected PAB3470 to attach HIV-5pep. PAB3499 (no CD40L) ·rAB-cetHS-puro[manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158] ·rAB-cetHS-puro[manti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C] C3735 rAB-cetHS-puro[manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158] SEQ ID NO:9 EVQLVQSGAEVKKPGASVKISCKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASEKIRLRPGGKKKYKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTITPTATTKPASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSITVAATAPTVTPTVNATPSAAASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS +C3739 rAB-cetHS-puro[m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C] SEQ ID NO: 10 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS

[0229] PAB3498 · rAB-cetHS-puro[m anti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158] · rAB-cetHS-puro[m antibody-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40 ligand] C3735 rAB-cetHS-puro[m anti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158] Sequence number 11 EVQLVQSGAEVKKPGASVKISCKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASEKIRLRPGGKKKYKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTITPTATTKPASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSITVAATAPTVTPTVNATPSAAASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS +C3524 rAB-cetHS-puro[m anti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40 ligand] SEQ ID NO: 12 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHV PWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGECASQTPTNTISVTPTNNSTPTNNSNPKPNPASMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL SEQ ID NO: 13: CD40L MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ leader or signal sequence 1-20; ectodomain residues 21–193; Transmembrane sequence 194–215; Cytoplasmic sequence 216-277.

[0230] This is the isoform selected as the "canonical" sequence, as reported by UniProtKB-P25942 (TNR5_HUMAN), which also describes sequence variants.

[0231] Variable domain sequences of other CD40 antibodies: HC and KC The amino acid at the end of the H chain V region is usually a lysine, but more typically is replaced with a serine (as in the CP sequence, this does not affect activity). 12B4 HC [manti-CD40_12B4.2C10_H-LV-hIgG4H-C]

[0232] [ka]

[0233] 12B4 KC [manti-CD40_12B4.2C10_K-LV-hIgGK-C]

[0234] [ka]

[0235] 12E12 HC [manti-CD40_12E12.3F3_H-V-hIgG4H-C]

[0236] [ka]

[0237] 12E12 KC [manti-CD40_12E12.3F3_K-V-hIgGK-C]

[0238] [ka]

[0239] 12E12 H2 humanized HC [h anti-CD40VH2-LV-hIgG4H-C]

[0240] [ka]

[0241] 12E12 H3 HIT HC [h anti-CD40 VH3-LV-hIgG4H-C]

[0242]

change

[0243] 12E12 K2ヒトKC [h anti-CD40VK2-LV-hIgGK-C]

[0244]

change

[0245] Pfizer HC[m anti-hCD40_CP870893H-LV-hIgG4H-C]

[0246]

change

[0247] Pfizer KC [m anti-hCD40_CP870893K-LV-hIgGK-C]

[0248]

change

[0249] 24A3 HC [manti-hCD40_24A3.3F1_H-LV-hIgG4H-C]

[0250]

change

[0251] 24A3 KC [manti-hCD40_24A3.3F1_K-LV-hIgGK-C]

[0252]

change

[0253] 11B6 hCD406 humanized HC fused to a dockerin domain - which when paired with 11B6 hCD40L humanized KC [mAnti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C-Flex-v1-hCD40Ligand] creates humanized 11B6-CD40L-dockerin for non-covalent coupling to any cohesin-antigen fusion. Complete sequence (C3724) FlexV1 (underlined) hCD40L (marker)

[0254] [ka]

[0255] 11B6 hCD40L humanized HC [manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-ChermoDockerin] (C3737) (ChermoDockerin) (marker)

[0256] [ka]

[0257] Flexible linker amino acid sequence: QTPTNTISVTPTNNSTPTNNSNPKPNP (flexV1, SEQ ID NO: 15) ASSSVSPTTSVHPTPTSVPPTPTKSSPAS (f1, SEQ ID NO:56) PTSTPADSSTITPTATPTATPTIKG (f2, SEQ ID NO: 130) TVTPTATATPSAIVTTITPTATTKP (f3, SEQ ID NO:53) TNGSITVAATAPTVTPTVNATPSAA (f4, SEQ ID NO: 54)

[0258] 2. Results Some agonists, including partial agonist anti-CD40 antibodies, can synergize with soluble CD40L to activate CD40. Antigen activated CD4 + When CD40L expressed on helper T cells engages with CD40L secreting the cytokines interleukin-4 and interleukin-21, it drives B cell proliferation, an event that is typically restricted to the germinal centers of lymphoid organs. We tested a matched panel of anti-human CD40 antibodies formatted as human IgG4 and human kappa light chains for their effect on driving human peripheral B cell proliferation in the presence of IL-4 and IL-21 (Figure 2). These antibodies covered a >100-fold range of agonistic effects in the order CP>12E12>12B4>11B6>24A3 (CP is CP-870,893, an antibody from Pfizer Inc. that has been tested in various clinical trials (Vonderheide et al., 2013)).

[0259] Repeating this assay in the presence of a fixed suboptimal concentration of soluble CD40L (sCD40L) did not affect the dose response of the 12B4 and 12E12 antibodies, slightly increased the potency of the CP antibodies, but greatly increased their potency through synergy (>100-fold) with the 11B6 and 24A3 antibodies (Figure 2). The synergistic cooperation of these two weak CD40 agonist signals suggests that the interaction of these specific mAbs with CD40 enhances the productive interaction of CD40L or vice versa. Synergy between substances activating different receptors on a single cell is a well-known phenomenon, for example, cooperation can be observed between agonist anti-CD40 mAbs and FcRII (Dahan et al., 2016), but in this case our two activators are acting on the same receptor type.

[0260] [Table 4]

[0261] Sentinel dendritic cells (DCs) process and present antigenic peptides in major histocompatibility molecules (MHC) to cognate antigen-specific T cells when exposed to foreign antigens and pathogen-derived danger signals (Hivroz et al., 2012). CD40 expressed on dendritic cells interacts with CD40L expressed on neighboring antigen-activated T cells, an event that is critical for initiating immunity, in part, through increasing the expression of cell surface DC activation molecules (e.g., CD68 and HLA) and inducing secretion of inflammatory cytokines by DCs (Ma and Clark, 2009). Therefore, we assayed a panel of anti-CD40 human IgG4 mAbs for their ability to initiate cytokine secretion on mature human monocyte-derived DCs (MDDCs). Similar to the B cell proliferation assay, these antibodies induced cytokine production over a similar >1000-fold efficacy range, with a similar ranking CP>12B4≧12E12>11B6>24A3 (Figure 3). The dose response of the 12B4 and 12E12 antibodies was not affected when assayed at suboptimal doses of sCD40L. However, low dose sCD40L increased the potency of the CP antibodies by approximately 100-fold, while strong synergy (>100-fold) on DC activation was again observed between sCD40L and the 11B6 and 24A3 antibodies (Figure 3). The difference in efficacy between the antibodies alone and those with sCD40L was larger than observed in the B cell proliferation assay, indicating a greater potential for cooperation between these two agonist types on MDDC.

[0262] [Table 5]

[0263] All of the panel of anti-human CD40 mAbs we studied bound with relatively high affinity to CD40 as measured by surface plasmon resonance (SPR), with the order of on-rates being 11B6>12B4>12E12>24A3>CP and the order of off-rates being 12B4>11B6>24A3>12E12>CP (Table 1). Previous studies with agonistic anti-CD40 antibodies have not shown a clear correlation between these kinetic parameters and activation potency (Hagar et al., 2003), and the SPR data for the mAbs in this study are consistent with this conclusion.

[0264] [Table 6]

[0265] One possible mechanism of synergy between sCD40L and anti-CD40mAb activation of CD40 could be through their access to separate sites on the CD40 ectodomain: the agonistic anti-CD40L mAb CDX-1140 is known to interact with CD40 at a site distinct from CD40L, and similar synergy is observed between sCD40L and this mAb (He et al., 2016).

[0266] Thus, we tested our panel of anti-human CD40 IgG4 antibodies for their ability to inhibit binding of CD40 to CD40L expressed on the surface of L cells. Stoichiometric amounts of 12B4 and 12E12 antibodies prevented binding of CD40 to cell surface CD40L, whereas >20-fold higher levels of CP antibodies were required to even partially block binding to CD40L. On the other hand, 11B6 and 24A3 antibodies had minimal effect on binding to CD40L, even at the highest mAb doses (Figure 4).

[0267] These data were consistent for these mAbs either as the original murine antibodies or reformatted as hIgG4. Based on their affinity constants (Table 4), these antibodies all fully occupied the CD40 binding site at the critical discriminatory concentration of 1 μg / ml, where 12E12 and 12B4 mAbs completely blocked CD40L binding. These data indicate that 12B4 and 12E12 mAbs bind to a site on CD40 that is absolutely required for CD40-CD40L interaction, whereas CP, 11B6 and 24A3 mAbs bind to a CD40 site with minimal interference with CD40-CD40L interaction.

[0268] Thus, the synergy between sCD40L and anti-CD40 mAbs on B cell and DC activation is associated with the simultaneous access of both these agonists to different portions of CD40L.

[0269] Antigen fusion to agonistic anti-CD40 mAbs can blunt the activating effect of CD40, but some anti-CD40 mAb-antigen fusions act synergistically with sCD40L to restore the activating capacity of CD40. Fusion of antigens to the C-terminus of chimeric or humanized agonistic antibodies can blunt or eliminate the agonistic properties of the parent antibody [Flamar et al., 2013]. A panel of anti-CD40 antibodies matched to the human IgG4 isotype, with or without concatenated strings of HIV-1 long T-cell epitope-rich peptides derived from the Gag, Nef, and Pol gene regions grafted onto the H and / or L chain C-terminus [Flamaer et al., 2013], was tested for their relative efficacy in inducing human B cell proliferation and human dendritic cell activation.

[0270] The agonists 11B6, 12B4, 12E12, and CP IgG4 mAbs, when grafted with five linked HIV-1 long peptide domains interspersed with glycosylated flexible linkers at their H chain C-termini, became very weak agonists for inducing B cell proliferation (Fig. 5). Addition of suboptimal levels of sCD40L restored the B cell proliferation capacity of 11B6-HIV5pep and CP-515pep to levels characteristic of similar "naked" anti-CD40 11B6 and CP mAbs incubated with sCD40L, but had no significant effect on 12B4-HIV5pep or 12E12-HIV5pep mAbs (Fig. 5).

[0271] These same four anti-CD40-HIV5pep fusion proteins also had very low potency for upregulating activation markers on MDDCs, and suboptimal levels of sCD40L enhanced the activity of 11B6-HIV5pep and CP-HIV5pep but had no effect on 12B4-HIV5pep or 12E12-HIV5pep fusion proteins (Figure 6). However, the degree of sCD40L enhancement was approximately 5- to 10-fold lower than that observed with the 11B6 and CP "naked" mAbs (Figure 6).

[0272] sCD40L cooperates with anti-CD40.HIV5pep to inhibit antigen-specific memory CD8 in PBMCs from HIV-1-infected individuals. + Expanding T cells The PBMC and DC-T cell co-culture system is useful for validating DC-targeted prototype vaccine constructs, in particular for selecting the best receptor for targeting cellular T cell responses (Yin et al., 2016), as well as for determining the CD4 receptor of selected fusion antigens. + and CD8 +These assays are useful in in vitro assays to confirm efficacy for eliciting broad T cell peptide specificity for both IL-1 and IL-2 T cell responses (Flamar et al., 2013). Based on such studies, CD40 targeting is particularly attractive, however, the potential contribution of CD40 activation with characteristic antigen internalization into early endosomal DC compartments has not been addressed (Chattergee et al., 2012; Yin et al., 2016).

[0273] We demonstrate that HIV-1 + The efficacy of anti-CD40 mAb fused to HIV5pep on HIV-1 antigen-specific T cell expansion was tested in donor PBMC cultures. A significant increase in antigen-specific CTL expansion was observed by co-administration of suboptimal levels of sCD40L with anti-CD40 11B6 and CP HIV5pep vaccines, but not with anti-CD40 12E12 and 12B4 HIV5pep vaccines (Figure 7).

[0274] CD40L fused to an agonistic anti-CD40 antibody can maximize CD40 activation The synergistic cooperation of sCD40L and agonistic anti-CD40 mAb may be a valuable property in vivo, for example, through allowing CD40L on activated T cells to access CD40 on DCs already occupied by the mAb. Alternatively, agonistic anti-CD40 mAb and sCD40L may be delivered simultaneously in vivo to provide potential therapeutic benefit through the enhanced CD40 activation observed in vitro.

[0275] Trimeric sCD40L has shown efficacy in preclinical studies (Stone et al., 2009) and may be available in the future for real-world clinical validation of the combination of sCD40L with mAbs that have synergistic effects with agonistic mAbs.

[0276] Here, we explored the novel concept of physically linking sCD40L to agonistic mAbs by direct fusion with the obvious potential advantage of establishing single-agent highly active agonists. To this end, the entire ectodomain of human CD40L was fused to the C-terminus of the L chain of an anti-CD40 mAb via a glycosylation-rich flexible linker sequence (termed flex V1, or ASQTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO: 15); Flamar et al., 2013). These "bivalent" anti-CD40-CD40L mAbs were efficiently expressed in 293 and CHO cells as homogeneous secreted products (data not shown).

[0277] The anti-CD40 IgG4 mAbs 11B6 and 12B4 fused to CD40L were tested for their efficacy in inducing B cell proliferation compared to non-fused mAbs. Both of these CD40L fusion mAbs were highly potent in this assay, consistent with the efficacy of the highly potent CPmAbs co-administered with suboptimal sCD40L (see FIG. 2). In both cases, CD40L adducts greatly increased the potency of the parent mAbs (>1,000-fold for 11B6 and >100-fold for 12B4), with the increase for 11B6 mAb being more than 10-fold more potent than when co-administered with sCD40L (FIG. 8). CD40L fused to a control IgG4 mAb was also active in inducing B cell proliferation, but approximately 10-fold less than the 11B6 and 12B4-CD40L fusion proteins (FIG. 8), highlighting the advantage of combining CD40L with anti-CD40 antibody binding via direct conjugation.

[0278] The entire panel of anti-CD40 IgG4 mAbs fused to CD40L was tested for their efficacy in inducing DC activation compared to unfused mAbs. Notably, direct linkage of CD40L to all mAbs except 12E12 dramatically increased their efficacy (i.e., maximal response) compared to the synergy observed by adding unlinked sCD40L to 11B6, CP, and 24A3 mAbs (Figure 9). Furthermore, consistent with the B cell proliferation assay mAb 12B4, which directly competes with CD40L for CD40 occupancy, also benefited greatly from CD40L fusion (Figure 9). Note that hCD40L directly fused to control (non-DC binding) IgG4 had minimal activity against MDDCs, suggesting that the CD40L-anti-CD40 mAb fusion approach is preferentially effective for DC activation.

[0279] Direct ligation of CD40L to the agonistic anti-CD40 mAb 11B6 increases affinity and activation efficacy We used SPR analysis to confirm the effect of CD40 fused to the L chain C-terminus on the CD40 binding kinetics of anti-CD40 11B6 and anti-CD40 12E12 mAbs by immobilizing them on a protein A / G surface and flowing soluble human CD40 ectodomain over them in liquid phase. The CD40L adduct on the anti-CD40 12E12 mAb did not significantly change the on or off rates of the antibody compared to the parental anti-CD40 12E12 mAb (data not shown). This was expected since the anti-CD40 12E12 mAb competed for the CD40L binding site on CD40 and a human IgG4 control mAb with CD40L fused to the L chain as well showed no detectable binding to CD40 in this format (data not shown). In contrast, CD40L adduct on anti-CD40 11B6 mAb significantly altered the antibody off-rate compared to the parental anti-CD40 11b6 mAb (data not shown). Specifically, the on-rate was only slightly affected, whereas the off-rate was reduced by approximately 15-fold, indicating cooperativity between anti-CD40 mAb and CD40L in binding to CD40.

[0280] The agonistic properties of anti-CD40 mAbs blunted by antigen fusion can be restored by fusing CD40L directly to the C-terminus of their light chains The agonistic properties of anti-CD40 mAbs can be reduced or eliminated through fusion to some antigens. For example, HIV5pep antigen fused to the H chain C-terminus greatly reduces agonistic properties when carried by 11B6, 12B4, or 12E12 mAb vehicles, whereas co-administered sCD40L enhances the activity of anti-CD40 11B6 HIV5pep fusion protein (Figures 5 and 6). Similarly, fusion of CD40L to the L chain of anti-CD40 11B6-HIV5pep protein also enhances the activity of B cell proliferation and MDDC cytokine production (Figure 10A). Figure 10B shows that anti-CD40 11B6 carrying HIV5pep antigen at the H chain C-terminus (11B6-HIV5pep in Figure L2) has minimal efficacy on CD40 activation as determined by B cell proliferation assay. However, addition of low doses of sCD40L (11B6-HIV5pep+sCD40L) or fusion of human CD40L to the C-terminus of the L chain (11B6-CD40L-HIV5pep) resulted in highly potent CD40 activation, to a greater extent than could be achieved by the CP-970,893 IgG4 antibody.

[0281] [Table 7]

[0282] Anti-CD40 11B6 mAb fused directly to CD40L is a superior agonist compared to the highly active dimer-trimer form of soluble CD40L MEGACD40L® (Mega sCD40L) is a widely used (Kornbluth et al., 2012) highly active protein in which two trimeric CD40 ligand molecules are artificially linked via the collagen domains of adiponectin / ACRP30 / AdipoQ (see Miconnet and Pantaleo, Vaccine 2008). Figure 10C shows the response of human MDDCs to a dose range of Mega sCD40L and anti-CD40 11B6-CD40L mAb with or without the HIV5pep antigen fused to the H chain C-terminus. Anti-CD40 11B6-CD40L was approximately 100-fold more active than Mega sCD40L and Mega sCD40L administered with 10 nM anti-CD40 11B6 to induce cytokine secretion in this assay, indicating that physical linkage of anti-CD40 11B40 to CD40L is essential for this very high activity. Importantly, anti-CD40 11B6-CD40L linked via the H chain C-terminus to the HIV5 pep antigen was also approximately 10-fold more active than Mega sCD40L.

[0283] [Table 8]

[0284] To test whether CD40L fusions to the C-terminus of agonistic anti-CD40 mAbs L chains could increase the potency of those agonists fused to other antigens, we compared agonistic activity when fused at the C-terminus of the H chain to linked HIV-1 Gag p24 Nef Gag p17 (referred to as GNG) or HPV 16 E6 / E7 (referred to as HPV) antigens, with or without CD40L fused directly to the L chain. These two antigens did not significantly blunt the low potency of B cell CD40 activation of the parental 11B6 mAb, but CD40 L chain fusions enhanced activation to levels equivalent to co-administered sCD40 (Figure 11). 11B6 mAb fused to GNG at the C-terminus of the H chain and to CD40L at the C-terminus of the L chain were equivalent for MDDC activation compared to 11B6-CD40L mAb without fusion antigen, and both 11B6 and 11B8-GNG strongly synergized with sCD40L, while 11B6-CD40L-GNG was equivalent in potency to 11B6-GNG+sCD40 (Figure 11). These data were similar for 11B6 fused to HPV antigens in that CD40L fused to the L chain restored the full agonist activity seen with 11B6-HPV+sCD40 (Figure 11). These 11B6-CD40L antigen fusions were equivalent to the strong agonist activity observed with 12B4-CD40L mAb, as were CD mAb+sCD40L. Interestingly, while 12E12-HPV mAb blocked sCD40L action, the anti-CD40 12E12-CD40L-HPV mAb format restored strong agonist activity (FIG. 11).

[0285] The sufficiently "high" agonistic activity observed in B cell proliferation with anti-CD40 11B6-CD40L-GNG protein was reproduced by the high cytokine secretion response of MDDCs to 11B6-CD40L-GNG, in contrast to the very weak agonistic activity of 11B6-GNG (Figure 12). Note that the efficacy of the 11B6-CD40L molecule is significantly higher than the standard full agonistic anti-CD40 CP IgG4 antibody.

[0286] Anti-CD40.Gag-17-Nef-Gag p24 vaccine directly linked to CD40L specifically inhibited memory CD8 + Increases T cell responses To directly test whether the improved agonistic properties of the anti-CD40 11B6-GNG mAb linked to CD40L affect its potency in HIV-1-specific T cell proliferation, HIV-1-infected donor PBMCs were incubated with low doses of anti-CD40 11B6-GNG-CD40L and various control GNG fusion mAbs for 9 days with IL-2 feeding and subsequently stimulated with a pool of peptides derived from Gag p17, Gag p24 and Nef. In both donors, 11B6-GNG mAb with directly linked CD40L or co-administered sCD40L significantly out-regulated Nef-specific CD8 T cells. + induced a T cell response (Figure 13).

[0287] To directly test whether the improved agonistic properties of the anti-CD40 11B6-HIV5pep mAb with linked CD40L affect its potency in HIV-1 specific T cell proliferation, HIV-1 infected donor PBMCs were incubated with low doses of anti-CD40 11B6-GNG-CD40L and various control GNG fusion mAbs for 9 days with IL-2 feeding and subsequently stimulated with individual long peptides corresponding to the five Gag p17, Gag p24, Nef and Pol epitope components of HIV5pep. The general trend in eliciting HIV-1 peptide specific T cell responses was that the 11B6-HIV5pep mAb directly linked to CD40L induced superior HIV-1 specific CD8+ T cell responses and less HIV-1 specific CD4 + The primary objective of this study was to induce a T cell response (Figure 14).

[0288] An in vitro culture system combining anti-CD40 DC-targeting vaccines with sCD40L has potential value in increasing the yield of ex vivo expanded CTLs, for example, for cell therapy applications. However, as an in vivo vaccine strategy, it is limited by the need to simultaneously administer two different protein drugs, which is associated with complex administration / pharmacokinetic, GMP production, and licensing issues. A novel bivalent antibody format that directly couples CD40L to anti-CD40 DC-targeting mAb solves this problem.

[0289] Expansion of HIV-1-specific T cells by CD40-targeted GNG antigen in HIV-1-infected donor PBMC cultures HIV-1 + Donor PBMCs were cultured for 9 days with a range of doses of anti-CD40-GNG fusion protein (from left to right: 1, 0.1, 0.01 nM) with or without low doses of sCD40L (100 ng / ml; 6 nM) and IL-2, followed by stimulation with pooled HIV-1 Gag p17, Nef, and Gag p24 peptides for 6 h and then analyzed by ICS. Data show that antigen-specific (A) CD4+ cells producing IFNγ+TNFα in response to peptide stimulation. + and (B) CD8 + The percentage of T cells at the end of culture is shown (see Figure 26). Similar data were observed with PBMC from two other donors (Figure 27).

[0290] Anti-CD40-CD40L targeting HIV5pep antigen preferentially expands abundant HIV-1-specific CD8+ T cell responses in HIV-1-infected donor PBMC cultures HIV-1 +Donor PBMCs were cultured with IL-2 and anti-CD40 HIV5prep fusion proteins (1 nM; 2 nM for KIH proteins) for 9 days, followed by stimulation with long peptides specific for each of the five HIV-1 gag, nef, and pol regions for 6 hours and then analyzed by ICS. Figure 28 shows collated data from experiments with each indicated protein pair tested in four donors. Each point represents the IFNγ increase for each long peptide-specific response elicited in each donor, comparing anti-CD40 11B6-CD40L-HIV5pep KIH Gen 2 and anti-CD40 11B6-CD40L-HIV5pep Gen 1 (Y-axis) with anti-CD40 12E12-HIV5pep KIH Gen 2 and anti-CD40 E1212-HIV5pep Gen 2 (X-axis). + +TNFα+ HIV5pep-specific CD8 + (Panel A, black squares) or CD4 (Panel B, open grey circles) values ​​are shown. Panel A highlights responses that were below 20% of expansion cultures and excludes the strong patient 2 response to the nef66 peptide (38±7% elicited by anti-CD40-11B6-CD40L-HIV5pep vaccine vs. 48±10% elicited by anti-CD40-HIV5pep vaccine). Panel B shows the entire data set.

[0291] Fusion of CD40L to anti-CD40 11B6 enhances adjuvant-free antibody responses in an in vivo mouse model To test the possibility of combining anti-CD40L fusions with anti-CD40 11B6 antibodies to increase vaccine efficacy, human CD40 transgenic mice were vaccinated with anti-CD40 11B6 delivery vehicles coupled to HIV-1 Engv gp140 with or without CD40L light chain fusions. Vaccination with anti-CD40 11B6-CD40L fused directly to gp140 was compared to vaccination with cohesin-gp140 fusion protein, anti-CD40 11B6-CD40L non-covalently bound to cohesin-gp140 fusion protein, and anti-CD40 11B6-CD40L non-covalently coupled to non-CD40 targeted cohesin-gp140. Both the antibody CD40 11B6-CD40L non-covalently coupled to the cohesin-gp140 fusion protein and anti-CD40 11B6-CD40L directly fused to gp140 induced anti-gp140 IgG titers that were detected as early as 1 week after a single vaccination, and both vaccines increased the response to a similar extent after two subsequent vaccinations (Figure 16). Vaccination with anti-CD40 11B6 non-covalently linked to cohesin-gp140 induced serum anti-gp140 IgG titers that were detected only after the second vaccination, and the response further increased after the third vaccination, although titers were significantly reduced compared to the two anti-CD40 11B6-CD40L-based vaccines. Non-targeted cohesin-gp140 did not induce detectable anti-gp140 IgG responses, even after three vaccinations with a threefold molar excess of gp140 compared to CD40-targeted vaccines. These results should be considered in the context of results in a non-human primate model, where anti-CD40 E12-gp140 vaccination induced only modest anti-gp140 IgG responses compared to the same vaccine co-administered with the adjuvant PolyIC (Zurawski et al., 2016). The mouse data suggest that CD40L linked to the anti-CD40 11B6-gp140 construct may confer "adjuvant-like" properties to this protein vaccine.

[0292] Anti-CD40 11B6-CD40L induces potent activation of CD40 in an in vivo mouse model Infusion of CP-870,893 in cancer patients undergoing chemotherapy induces immune activation detected by an increase in plasma inflammatory cytokines (i.e., cytokine release syndrome), increased B cell expression of costimulatory molecules, and transient depletion of B cells (Beatty et al., 2013). In these patients, dose-limiting toxicity was determined to be 0.2 mg / kg, whereas 0.3 mg / kg was the limit determined in patients receiving this anti-CD40 agonist alone (Vonderheide et al., 2016). To evaluate the biological activity of anti-CD40 11B6-CD40L in vivo, we tested the short-term (24 h) effects of CP-870,893 hIgG4 and anti-CD40 11B6-CD40L at a dose of 10 μg (approximately 0.5 mg / Kg). Wild-type or human CD40 BAC transgenic mice on a wild-type (Taconic strain) or CD40 KO (ImmuRx strain) C57BL / 6 background were injected (intraperitoneally) with the molar equivalent of CP-870,893 hIgG4 or anti-CD40 11B6-CD40L and sacrificed 24 hours later. Blood was collected for assay of cytokines (serum by Luminex®) and cells from PBMC, skin-draining lymph nodes and spleen were analyzed by flow cytometry. B cells were characterized by analysis of activation markers CD69, MHC-II, OX40L and CD86. The results are shown in Figure 17 and show that CP-870,893 hIgG4 has minimal or no activity in these tests, whereas anti-CD40 11B6-CD40L induces a strong B cell depletion associated with activation, as well as cytokine secretion, as detected in the blood. These data predict that, for example, if used clinically as an adjuvant in cancer therapy, very low doses of anti-CD40 11B6-CD40L will be required compared to CP-870,893.

[0293] Fusion of CD40L to anti-CD40 11B6 increases both the efficacy and potency of cell killing directed by the CD40 ectodomain fused to the FAS transmembrane and intracellular domains. FAS (CD95) belongs to the tumor necrosis factor receptor (TNF-R) family, contains an intracellular "death domain", and can trigger apoptosis in response to its physiological ligand, FASL (Strasser et al., 2009). We constructed a fusion protein expressing human CD40 ectodomain residues 21-193 fused to human FAS residues 187-350 and established stably transfected Chinese hamster ovary (CHO) cells expressing the CD40 ectodomain linked to the FAS transmembrane and intracellular domains. CD40 agonists induce killing of these cells as determined by loss of mitochondrial reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mossman, 1983). In this assay, anti-CD40 11B6 IgG4 and anti-CD40 12E12 IgG4 show similar efficacy (as determined by the maximal reduction in MTT reduction) and similar potency (EC50 of approximately 2.5 nM and 1 nM, respectively), whereas anti-CD40 11B6-CD40L IgG4 shows increased potency (i.e., a larger maximal reduction in MTT reduction) and significantly increased efficacy (EC 50 is approximately 2.5 pM) (Figure 18). This data reaffirms the conclusions from examination of the superiority of anti-CD4011B6-CD40L potency seen in B cell and DC assays.

[0294] Anti-CD40 11B6-CD40L enhances CD40-mediated clustering and internalization We compared the rate and extent of CD40-mediated internalization of anti-CD40 11B6-CD40L versus anti-CD40 12E12. Although these two antibodies are well matched in their binding to CD40 based on SPR analysis (K of 12 and 28 nM, respectively), D, FIG. 24), and their potency of CD40 activation is dramatically different (FIG. 9, FIG. 18). Because ligand engagement leads to the formation of cross-linked CD40 lipid rafts (clusters) on the cell membrane, an event followed by CD40 internalization and downstream signaling (Wang et al., 2015), we compared the ability of anti-CD40 11B6-CD40L versus anti-CD40 12E12 to cluster and internalize cell surface CD40.

[0295] First, we compared the ability of these two anti-CD40 antibodies to induce the formation of cross-linked CD40 lipid rafts (clusters) on the cell membrane. We used CHO cells expressing human CD40-eGFP fusion protein or human CD40-mCherry fusion as a model to visualize cluster formation by confocal microscopy. Treatment of cells with 10 nM anti-CD40 11B6-CD40L for 1 h at 37° C. induced stronger CD40 cluster formation compared to the same treatment with anti-CD40 12E12 (FIG. 23). This data is consistent with the increased signaling capacity of anti-CD40 11B6-CD40L antibody, since CD40 clustering is likely the initial trigger of CD40 activation.

[0296] Next, we assayed anti-CD40-mediated binding and internalization of non-covalently attached mCherry modules using CHO cells expressing human CD40 as a model. Binding of both antibodies was rapid, reaching saturation within about 30 min at 37° C. and 0° C. (FIG. 24A and FIG. 24B). Treatment of cells for 1 min in cold isotonic acid buffer (pH 2.5) removed about 75% of cell-associated anti-CD40 12E12 label when conjugation was performed at 0° C. (FIG. 24A). There was a trend toward increased label retention at 37° C. when conjugation was performed at 37° C. (29±3% remaining label vs. 26±3%, ns), likely reflecting less internalization. In contrast, when anti-CD40 11B6-CD40L conjugation was performed at 0 °C, acid stripping removed only about 60% of the cell-bound label versus 26 ± 3% observed with anti-CD40 E12 mAb, and this difference between the two mAbs was significant (p < 0.0001, Figure 24 B), indicating that internalization of anti-CD40 11B6-CD40L was readily detectable even at 0 °C. It increased by 50-90% over the time course (Figure 24 B). Thus, anti-CD40 11B6-CD40L was internalized to a much greater extent than anti-CD40 12E12, and significant internalization was detected even at 0 °C. The binding and internalization properties of the anti-CD4011B6 mAb, not fused to CD40L, were very similar to those of the anti-CD40 12E12 mAb, and thus are solely a consequence of the CD40L adduct (FIG. 24C).

[0297] Anti-CD40 11B6 recognizes a distinct epitope on CD40 than anti-CD40 12E12 and anti-CD40 CP. Yu et al. (2018) summarized findings regarding the relationship between agonistic anti-CD40 mAbs and their interaction sites with CD40. For example, anti-CD40 CP binds within the CD40 CRD1 region (i.e., no binding if deleted) and also loses binding if residues 23-37 are deleted or the RE at residues 27-28 is replaced with AA. Also, Singh et al. (1998) reported that individual substitutions of negatively charged residues Glu74, Asp84, and Glu117 in CD40 disrupt CD40L binding. This mapping shows distinct sites of interaction in CD40 for CD40L versus CP antibodies. Using the method of Wan et al., 2012, residues R27 and E28 were individually replaced with A and the mutated CD40 ectodomain was tested for binding to anti-CD40 CP, anti-CD40 12E12, and anti-CD40 11B6 (±CD40L). As expected, the R27A and E28A mutations abolished or greatly reduced binding to anti-CD40 CP mAb (Figure 19). However, these mutations did not affect binding of anti-CD40 11B6 or 12E12 mAb. The data clearly distinguish the binding site on CD40 of anti-CD40 CP mAb from anti-CD40 11B6 or 12E12 mAb.

[0298] Epitope mapping of anti-CD40 agonist antibodies 12E12 and 11B6 Two approaches were used to define CD40 residues that are important for interaction / binding with the two claimed antibodies (12E12 and 11B6) and to differentiate them from another agonist antibody, CP-870,893 (herein referred to as CP). The first approach was based on the PepScan (a Netherlands-based company) "Precision Epitope Mapping" platform. The second approach was built on the PepScan data by performing Ala-scanning mutagenesis of selected hydrophilic residues of the human CD40 ectodomain coupled to antibody binding analysis.

[0299] Using these approaches, we aimed to identify differences in the epitopes on CD40 specific for these three antibodies.

[0300] Six 12E12 binding peptide regions were identified from Pepscan analysis, and Ala mutagenesis covered the charged residues in all but one of these peptides (none of which had a highly charged residue).

[0301] [ka]

[0302] Nine 11B6-binding peptide regions were highlighted, and Ala mutagenesis covered the charged residues in eight of these nine peptides (excluding those that did not have any highly charged residues).

[0303] [ka]

[0304] The data obtained from the Ala mutagenesis study are summarized in Figure 25. Underlined are the four CDR structural homology regions of CD40. The remaining number of some mutations is indicated. Grey coordinates indicate the residues that were changed. P is Pfizer CP mAb; E is 12E12; B is 11B6; L is 11B6-CD40L; - has no effect on any of the mAbs tested; lowercase letters under the sequence indicate reduced binding; the letters CAPS mean no binding.

[0305] Mutagenesis resulted in the reliable identification of epitopes for CP, 11B6, and 12E12 binding, even though all three mAbs have distinct epitope components in the CDR1 region. The CP interaction was disrupted by changes in residues R27 and E28 (verification of published information); the 11B6 interaction involved interactions with residues K29, as well as E56 and E58; the combined 11B6-CD40L binding was specifically inhibited by the K46A change; the 12E12 interaction was dependent on D50 and E58. Thus, the data show some overlap in the epitopes of these three mAbs, but clear differences in key contact sites.

[0306] Other Examples of Fusion Proteins of the Disclosure The following alternative fusion proteins A1-A5 were prepared: Figure 22 shows a schematic diagram depicting the structure of each fusion protein, with the antigen either at the C-terminus of the light or heavy chain and / or the presence of a flexible linker.

[0307] Example A1: Anti-CD40 11B6 with CD40L at the C-terminus of the L chain PAB3588 C3334 (SEQ ID NO: 131) × C3792 (SEQ ID NO: 133) [manti-CD40_11B6.1C3_H-LV-hIgG4H-C-Nhe-Not][manti-CD40_11B6.1C3_Syn_K-LV-hIgGK-C-hCD40 ligand]

[0308] Example A2: Anti-CD40 humanized 11B6 with Flex-CD40L at the C-terminus of the H chain PAB3618 C3823 (SEQ ID NO: 135) × C3739 (SEQ ID NO: 137) [manti-CD40-11B6.1C3-VH-v2-LV-IgG4H-C-Flex-v1-hCD40 ligand][manti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C]

[0309] Example A3: Anti-CD40 humanized 11B6 with Flex-CD40L at the C-terminus of the L chain PAB3475 C3724 (SEQ ID NO: 139) × C3726 (SEQ ID NO: 141) [manti-CD40-11B6.1C3-Vκ-v2-LV-IgGK-C-Flex-v1-hCD40 ligand][manti-CD40-11B6.1C3-VH-v3-LV-hIgG4H-C]

[0310] Example A4: Anti-CD40 humanized 11B6 with CD40L at the C-terminus of the H chain PAB3615 C3821 (SEQ ID NO: 143) × C3739 (SEQ ID NO: 137) [manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C-hCD40 ligand][manti-CD40-11B6.1C3-Vκ-v2-LV-hIgGK-C]

[0311] Example A5: Anti-CD40 humanized 11B6 with Flex-CD40L at the C-terminus of the L chain PAB3470 C3724 (SEQ ID NO: 139) × C3725 (SEQ ID NO: 145) [manti-CD40-11B6.1C3-Vκ-v2-LV-IgGK-C-Flex-v1-hCD40 ligand][manti-CD40-11B6.1C3-VH-v2-LV-hIgG4H-C]

[0312] Superagonist properties are observed regardless of positioning of CD40L at either the C-terminus of the light chain or the C-terminus of the heavy chain, and with or without a flexible linker connecting sequence. Human MDDCs were cultured with a range of doses (shown from left to right in Figure 20: 10, 1, 0.1, 0.01 nM) of each anti-CD40 11B6-CD40L isoform, and the extent of cytokine secretion was determined after 24 hours. Data in Figure 20 represent the sum of the values ​​for each cytokine tested, normalized to the highest value within the experiment and averaged over repeated responses in two different donors. Error bars are standard error of the mean. Schematics shown below each titration series indicate the L or H chain CD40L fusion isoform, with F indicating the presence of the Flex V1 linker. The results show that there were no significant differences between the dose values ​​in this series.

[0313] The superagonistic activity of CD40L fused to anti-CD40 antibodies is independent of CD40L positioning, as defined by the efficacy and potency of cell killing induced by CD40 ectodomain fused to the FAS transmembrane and intracellular domains. FAS (CD95) belongs to the tumor necrosis factor receptor (TNF-R) family, contains an intracellular "death domain" and can trigger apoptosis in response to its physiological ligand, FASL (Strasser et al., 2009). We constructed a fusion protein expressing human CD40 ectodomain residues 21-193 fused to human FAS residues 187-350 and established stably transfected Chinese hamster ovary (CHO) cells expressing the CD40 ectodomain linked to the FAS transmembrane and intracellular domains. CD40 agonists induce killing of these cells as determined by loss of mitochondrial reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mossman, 1983). FAS and CD40 belong to the same TNF-R family and the mechanism of receptor activation (extracellular) is similar, but the intracellular signaling pathways, i.e., apoptosis and activation of selected cytokines and cell surface markers, are distinct. This fusion construct provides a convenient surrogate assay format for the analysis of CD40 activation based on transfected CHO cells. In this assay, anti-CD40 11B6 IgG4 and anti-CD40 12E12 IgG4 showed similar efficacy (as determined by maximal reduction in MTT reduction) and similar potency (EC 50 ≈2.5 nM and 1 nM), whereas anti-CD40 11B6-CD40L IgG4 shows increased potency (i.e., more maximal reduction in MTT reduction) and a significant increase in efficacy (EC50≈2.5 pM). 50is approximately 2.5 pM) (FIG. 21). Thus, CD40L fusion to the partial agonist anti-CD40 11B6 mAb can greatly increase potency and efficacy against three different CD40-bearing cell types.

[0314] [Table 9A]

[0315] [Table 9B]

[0316] [Table 9C]

[0317] [Table 9D]

[0318] [Table 9E]

[0319] [Table 9F]

[0320] [Table 9G]

[0321] [Table 9H]

[0322] [Table 10A]

[0323]

Table 10B

[0324]

Table 10C

[0325]

Table 10D

[0326]

Table 10E

[0327]

Table 10F

[0328]

Table 10G

[0329] [Table 10H]

[0330]

Table 10I

[0331]

Table 10J

[0332]

Table 10K

[0333]

Table 10L

[0334]

Table 10M

[0335]

Table 10N

[0336]

Table 10O

[0337]

Table 10P

[0338]

Table 10Q

[0339]

Table 10R

[0340]

Table 10S

[0341]

Table 10T

[0342]

Table 10U

[0343]

Table 10V

[0344]

Table 10W

[0345] [Table 10X]

[0346]

Table 10Y

[0347]

Table 10Z

[0348]

Table 10a

[0349]

Table 10b

[0350]

Table 10c

[0351]

Table 10d

[0352]

Table 10e

[0353]

Table 10f

[0354]

Table 10g

[0355]

Table 10h

[0356]

Table 10i

[0357]

Table 10j

[0358]

Table 10k

[0359]

Table 10l

[0360]

Table 10m

[0361]

Table 10n

[0362]

Table 10o

[0363]

Table 10p

[0364]

Table 10q

[0365]

Table 10r

[0366]

Table 10s

[0367] (References) TIFF0007640466000077.tif235169TIFF0007640466000078.tif237170TIFF0007640466 000079.tif226170TIFF0007640466000080.tif235170TIFF0007640466000081.tif40166

Claims

1. At least the following protein domains: (i) CD40 agonist antibody (αCD40); and (ii) the CD40-binding domain of CD40L 1. A CD40 activating protein comprising: By activating CD40, it induces the proliferation of B cells and / or cytokine secretion from dendritic cells, The CD40 agonist antibody is an antibody comprising: a. a humanized antibody comprising an HCDR1 of SEQ ID NO:27, an HCDR2 of SEQ ID NO:28, an HCDR3 of SEQ ID NO:29, an LCDR1 of SEQ ID NO:30, an LCDR2 of SEQ ID NO:31, and an LCDR3 of SEQ ID NO:32; b. A humanized antibody comprising an HCDR1 of SEQ ID NO: 74, an HCDR2 of SEQ ID NO: 75, an HCDR3 of SEQ ID NO: 76, an LCDR1 of SEQ ID NO: 77, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 79; c. A humanized antibody comprising an HCDR1 of SEQ ID NO: 80, an HCDR2 of SEQ ID NO: 81, an HCDR3 of SEQ ID NO: 82, an LCDR1 of SEQ ID NO: 83, an LCDR2 of SEQ ID NO: 84, and an LCDR3 of SEQ ID NO: 85; d. a humanized antibody comprising an HCDR1 of SEQ ID NO: 86, an HCDR2 of SEQ ID NO: 87, an HCDR3 of SEQ ID NO: 88, an LCDR1 of SEQ ID NO: 89, an LCDR2 of SEQ ID NO: 90, and an LCDR3 of SEQ ID NO: 91; e. A humanized antibody comprising an HCDR1 of SEQ ID NO: 92, an HCDR2 of SEQ ID NO: 93, an HCDR3 of SEQ ID NO: 94, an LCDR1 of SEQ ID NO: 95, an LCDR2 of SEQ ID NO: 96, and an LCDR3 of SEQ ID NO:

97. is selected from the CD40 binding domain of CD40L is a monomeric fragment of CD40L comprising SEQ ID NO: 14; A CD40 activating protein in which the CD40 binding domain of CD40L is covalently linked directly or via a linker to the C-terminus of the light or heavy chain of the CD40 agonist antibody.

2. The CD40 agonist antibody specifically binds to human CD40 and has the following characteristics: (i) induces proliferation of B cells as measured in vitro by flow cytometric analysis; or (ii) induce cytokine secretion as measured in vitro by a dendritic cell activation assay; The CD40 activating protein of claim 1, comprising at least one of the following:

3. The CD40 activating protein of claim 1 or 2, wherein the CD40 binding domain of CD40L is a fragment of CD40L consisting of SEQ ID NO:

14.

4. 4. The CD40 activating protein of claim 1, wherein the CD40 binding domain of CD40L is fused to the C-terminus of the light or heavy chain of the CD40 agonist antibody.

5. 5. A CD40 activating protein according to any one of claims 1 to 4, comprising the heavy and light chains of a CD40 agonist IgG antibody.

6. 6. The CD40 activating protein of any one of claims 1 to 5, comprising a peptide linker between CD40L and the light or heavy chain of the CD40 agonist antibody.

7. The CD40 activating protein of claim 6, wherein the peptide linker between CD40L and the light or heavy chain of the CD40 agonist antibody is the flexible linker FlexV1 of SEQ ID NO:

15.

8. The CD40 agonist antibody is an antibody comprising: a. a humanized antibody comprising the VH and VL domains of SEQ ID NO:21 and SEQ ID NO:22, respectively; b. A humanized antibody comprising the VH and VL domains of SEQ ID NO:23 and SEQ ID NO:22, respectively; c. A humanized antibody comprising the VH and VL domains of SEQ ID NO:58 and SEQ ID NO:59, respectively; d. A humanized antibody comprising the VH and VL domains of SEQ ID NO:60 and SEQ ID NO:61, respectively; e. A humanized antibody comprising the VH and VL domains of SEQ ID NO:62 and SEQ ID NO:63, respectively; f. A humanized antibody comprising the VH and VL domains of SEQ ID NO:64 and SEQ ID NO:65, respectively.

8. The CD40 activating protein of any one of claims 1 to 7, selected from:

9. 9. The CD40 activating protein of any one of claims 1 to 8, wherein one or more antigens are fused to the heavy or light chain of the CD40 agonist antibody.

10. 10. The CD40 activating protein of claim 9, wherein one or more infectious disease or cancer antigens are fused to a heavy or light chain of the CD40 agonist antibody.

11. comprising a light chain of the formula αCD40Light-PL-CD40L and a heavy chain of the formula αCD40Heavy-(PL-Ag)x, αCD40Light is the light chain of said CD40 agonist antibody; αCD40Heavy is the heavy chain of said CD40 agonist antibody; PL is a bond or a peptide linker, which may be the same or different; Ag is a viral antigen or a cancer antigen, identical or different; x is an integer between 1 and 20; CD40L is the binding domain of the ligand of CD40 comprising SEQ ID NO: 14; 11. The CD40 activating protein of any one of claims 1 to 10, wherein - is a bond.

12. The CD40 activating protein of claim 11, wherein the peptide linker is FlexV1 of SEQ ID NO:

15.

13. 13. The CD40 activating protein of claim 11 or 12, wherein the viral antigen is selected from an HIV peptide antigen, a papillomavirus antigen, and a coronavirus antigen.

14. 14. A pharmaceutical composition comprising a CD40 activating protein according to any one of claims 1 to 13, and one or more pharma- ceutically acceptable excipients.

15. a) for use as a vaccine, b) for use in enhancing a T cell specific response in a subject; or c) for use in inducing B cell proliferation and / or inducing cytokine proliferation of dendritic cells in a subject; 15. The pharmaceutical composition of claim 14.

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