Antibodies to CD40 and compositions thereof

Abstract

The present disclosure provides antibodies that bind to CD40, and methods of using them to modulate immunity in a patient in need thereof and to treat autoimmune disorders or immunoinflammatory disorders.

Description

ANTIBODIES TO CD40 AND COMPOSITIONS THEREOFSEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 9, 2024, is named 027628_P1010_SL.xml and is 93,759 bytes in size.BACKGROUND OF THE INVENTION

[0002] CD40 is a 50 kDa type I transmembrane glycoprotein and a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is primarily expressed on a variety of immune cells, such as B cells, dendritic cells (DCs), monocytes and macrophages. The only known ligand of CD40 is CD40L (CD 154) is primarily found on activated CD4+T cells but also on B cells and platelets.

[0003] The CD40-CD40L interaction promotes clustering of CD40, which is required for strong induction of intracellular signaling and activation. Stimulation of the CD40 receptor induces proliferation and activation of B cells and CD40-CD40L interaction is shown to be critical for germinal center (GC) responses and isotype class switching.

[0004] While CD40 has a central role in generating effective immune responses, it also has a pathogenic role in several autoimmune diseases, including autoimmune thyroiditis, type 1 diabetes (T1D), inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).

[0005] In view of the role of CD40 in immune responses and their repercussions in autoimmune and immunoinflammatory disorders, there is a need for new and improved therapies that target CD40.SUMMARY OF THE INVENTION

[0006] The present disclosure provides anti-CD40 antibodies and antigen-binding portions thereof. In some embodiments, the anti-CD40 antibody binds specifically to amino acids 41-55 and / or amino acids 61-75 of SEQ ID NO: 97 (human CD40). In certain embodiments, the antibody further binds specifically to amino acids 73-76 of SEQ ID NO: 97. In some embodiments, the antibody binds specifically to one or more of amino acids 46, 66, 73, 74, 75 and 76 of SEQ ID NO: 97, e.g., to all of said amino acids. In certainembodiments, the antibody further binds specifically to amino acid 50, 78, or both.

[0007] The present disclosure also provides an isolated anti-CD40 antibody or an antigen-binding portion thereof, wherein the antibody binds to the same epitope as a reference antibody having a heavy chain variable domain (VH) and a light chain variable domain (VL) comprisingSEQ ID NOs: 1 and 2, respectively,SEQ ID NOs: 11 and 12, respectively,SEQ ID NOs: 21 and 22, respectively,SEQ ID NOs: 31 and 32, respectively,SEQ ID NOs: 41 and 42, respectively,SEQ ID NOs: 51 and 52, respectively,SEQ ID NOs: 61 and 62, respectively, orSEQ ID NOs: 71 and 72, respectively.

[0008] In some embodiments, the heavy chain of the anti-CD40 antibody comprises: i) heavy chain complementarity determining regions (H-CDR) 1-3 comprising SEQ ID NOs: 3-5, respectively; ii) a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1; iii) a VH comprising SEQ ID NO: 1; or iv) SEQ ID NO: 1 and any one of SEQ ID NOs: 81-83 (e g., SEQ ID NO: 83); and the light chain of the anti-CD40 antibody comprises: i) light chain complementarity determining regions (L-CDR) 1-3 comprising SEQ ID NOs: 6-8, respectively; ii) a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2; iii) a VL comprising SEQ ID NO: 2; or iv) SEQ ID NOs: 2 and 84.

[0009] The present disclosure also provides an anti-CD40 antibody or an antigen-binding portion thereof, wherein the antibody comprises H-CDR1-3 and L-CDR1-3 amino acid sequences of: a) SEQ ID NOs: 3-8, respectively; b) SEQ ID NOs: 13-18, respectively; c) SEQ ID NOs: 23-28, respectively; d) SEQ ID NOs: 33-38, respectively; e) SEQ ID NOs: 43-48, respectively;f) SEQ ID NOs: 53-58, respectively; g) SEQ ID NOs: 63-68, respectively; or h) SEQ ID NOs: 73-78, respectively.

[0010] In some embodiments, the anti-CD40 antibody comprises a VH amino acid sequence and a VL amino acid sequence that are at least 90% identical to the amino acid sequences of: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively; c) SEQ ID NOs: 21 and 22, respectively; d) SEQ ID NOs: 31 and 32, respectively; e) SEQ ID NOs: 41 and 42, respectively; f) SEQ ID NOs: 51 and 52, respectively; g) SEQ ID NOs: 61 and 62, respectively; or h) SEQ ID NOs: 71 and 72, respectively.

[0011] In some embodiments, the anti-CD40 antibody comprises a VH and a VL comprising: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively; c) SEQ ID NOs: 21 and 22, respectively; d) SEQ ID NOs: 31 and 32, respectively; e) SEQ ID NOs: 41 and 42, respectively; f) SEQ ID NOs: 51 and 52, respectively; g) SEQ ID NOs: 61 and 62, respectively; or h) SEQ ID NOs: 71 and 72, respectively.

[0012] In some embodiments, an anti-CD40 antibody herein is of isotype subtype IgGi. In certain embodiments, the antibody comprises a mutant Fc domain with reduced binding affinity for an Fc gamma receptor. The mutant Fc domain may have, for example, a) mutations L234A and L235A, or b) mutations L234A, L235E, and G237A, wherein the mutation positions are numbered according to Eu numbering.In certain embodiments, the antibody comprises a heavy chain constant region comprising SEQ ID NO: 82 or 83.

[0013] The present disclosure also provides an anti-CD40 antibody that comprises: a) a heavy chain (HC) comprising SEQ ID NOs: 1 and 83, and a light chain (LC)comprising SEQ ID NOs: 2 and 84; b) an HC comprising SEQ ID NOs: 11 and 83, and an LC comprising SEQ ID NOs: 12 and 84; c) an HC comprising SEQ ID NOs: 21 and 83, and an LC comprising SEQ ID NOs: 22 and 84; d) an HC comprising SEQ ID NOs: 31 and 83, and an LC comprising SEQ ID NOs: 32 and 84; e) an HC comprising SEQ ID NOs: 41 and 83, and an LC comprising SEQ ID NOs: 42 and 84; f) an HC comprising SEQ ID NOs: 51 and 83, and an LC comprising SEQ ID NOs: 52 and 84; f) an HC comprising SEQ ID NOs: 61 and 83, and an LC comprising SEQ ID NOs: 62 and 84; or f) an HC comprising SEQ ID NOs: 71 and 83, and an LC comprising SEQ ID NOs: 72 and 84.

[0014] The present disclosure also provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding portion herein and a pharmaceutically acceptable excipient.

[0015] The present disclosure also provides isolated nucleic acid molecule(s) comprising a nucleotide sequence that encodes the heavy chain sequence, and a nucleotide sequence that encodes the light chain sequence, of an anti-CD40 antibody or antigen-binding portion herein. In some embodiments, the nucleic acid molecule(s) comprise any one of SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, and 80.

[0016] The present disclosure also provides vector(s) comprising isolated nucleic acid molecule(s) herein. In some embodiments, the vector(s) further comprise expression control sequence(s).

[0017] The present disclosure also provides a host cell comprising a nucleotide sequence that encodes the heavy chain sequence, and a nucleotide sequence that encodes the light chain sequence, of an anti-CD40 antibody or antigen-binding portion herein.

[0018] The present disclosure also provides a method for producing an anti-CD40 antibody or an antigen-binding portion thereof, comprising- providing a host cell herein,- culturing said host cell under conditions suitable for expression of the antibody or antigenbinding portion, and- isolating the resulting antibody or antigen-binding portion.

[0019] The present disclosure also provides a method of treating an autoimmune or inflammatory condition in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-CD40 antibody or antigen-binding portion herein, or a pharmaceutical composition herein. Also provided is an anti-CD40 antibody or antigenbinding portion herein or pharmaceutical composition herein for use in treating an autoimmune or inflammatory condition in a patient in need thereof, and use of an anti-CD40 antibody or antigen-binding portion herein or pharmaceutical composition herein in the manufacture of a medicament for treating an autoimmune or inflammatory condition in a patient in need thereof. In certain embodiments, the patient is a human patient.

[0020] In some embodiments, the condition for treatment is selected from the group consisting of asthma, allergic asthma, chronic spontaneous urticaria (CSU), diabetes (e.g., type 1 diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), allergy, antibody -mediated rejection, organ graft rejection, graft-versus-host disease (GvHD), Addison’s disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, celiac disease, autoimmune alopecia, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpurajuvenile myositis, Kawasaki disease, inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, pemphigus vulgaris, thrombocytopenic purpura, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, vitiligo, and Vogt- Koyanagi-Harada Disease.

[0021] In certain embodiments, the condition is chronic spontaneous urticaria, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Sjogren’s syndrome, lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), Graves’ disease, inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, or vitiligo.

[0022] In particular embodiments, the disease is chronic spontaneous urticaria, atopic dermatitis, or pemphigus vulgaris.

[0023] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a set of line graphs showing the binding of anti-CD40 antibody 15906 and analogues of iscalimab and ravagalimab to human, marmoset, cynomolgus and mouse CD40 ECD receptor expressed on transiently transfected CHO-S cells. Mock transfected CHO-S cells were used as a negative control. Data are shown as means ± SD of triplicates.

[0025] FIG. 2 is a pair of line graphs showing the blocking by anti-CD40 antibody 15906 and analogues of iscalimab and ravagalimab of the binding of human CD40 ligand (CD40L) to human CD40 or marmoset CD40 expressed on CHO-S cells. Data are presented as duplicate means ± SEM. IgG1.3F, the isotype control, was used as a negative control.

[0026] FIG. 3 is a competition plot of epitope binning for anti-CD40 antibodies 15906 (“AID0001)”, 15837 (“31789-15837”), 16029 (“31809-16029”), and analogues of ravagalimab and iscalimab. The analyzed antibodies are grouped in three different bins.

[0027] FIG. 4 is a set of line graphs showing the activation of Fey receptor signaling upon treatment with antibody 15906 or analogues of iscalimab and KPL-404. Isotype control IgG1.3f was included as a negative control.

[0028] FIG. 5 is a set of line graphs showing neutrophil-induced ADCC of CD40- expressing Daudi cells upon treatment with antibody 15906 or an analogue of iscalimab. Isotype control IgG1.3f was included as a negative control. Error bars show SEM of duplicate measurements.

[0029] FIG. 6 is a line graph showing the percent specific lysis of CD40-expressing target B cells as measured by calcein release upon treatment with anti-CD40 antibodies or a negative control IgGl-LALA antibody in the presence of healthy donor peripheral blood mononuclear cells (PBMCs). Untreated lysis (with PBMCs present, no antibody) was deducted, and values normalized to maximal lysis. Spontaneous lysis (no PBMCs present) was deducted, and values normalized to maximal lysis. Lower horizontal dotted line represents untreated signal level (0) and upper dotted line represents spontaneous lysis. Error bars represent SEM of triplicate measurements.

[0030] FIG. 7 is a line graph showing the activation of a CD40 reporter cell line upon treatment with 15906 (a CD40 antagonist antibody) or 16040 (a CD40 agonist antibody). Isotype control IgG1.3F was included as a negative control.

[0031] FIG. 8 is a line graph showing the proliferation of primary B cells upon treatment with anti-CD40 antibody 15906 or with analogues to iscalimab and KPL-404. Isotype control IgG1.3F was included as a negative control. Error bars represent mean ± SEM of measurements.

[0032] FIG. 9 is a line graph showing activation of dendritic cells (DCs) by anti-CD40 antibody 15906 and an analogue to selicrelumab, as determined by measuring IL-12p40 levels. Isotype control IgG1.3f was included as a negative control. Error bars represent mean ± SEM of measurements.

[0033] FIG. 10 is a line graph showing the antagonistic activity of antibody 15906 and analogues to iscalimab and KPL-404 on proliferation of CD40 signaling reporter bioluminescence activity (counts per second) from a reporter cell line upon treatment for four hours. Isotype control IgG1.3f was included as a negative control. Error bars represent mean ± SEM of measurements.

[0034] FIG. 11 is a line graph showing the antagonistic activity of various anti-CD40 antibodies on proliferation of primary B cells upon treatment for four days, as measured by signaling reporter bioluminescence activity (counts per second).

[0035] FIG. 12 is a pair of line graphs showing the dose-dependent inhibition of IgG- secreting (Panel A) and IgE-secreting (Panel B) plasma cells by antibody 15906.

[0036] FIG. 13 is a set of line graphs showing the dose dependent blocking of T celldependent B cell activation upon treatment with antibody 15906. Isotype control IgG1.3f was included as a negative control.

[0037] FIG. 14 is a line graph showing plasma concentration versus time for the in vivo single dose PK profiles of antibody 15906 administered at 0.1, 1, or 10 mg / kg to hFcRn mice.

[0038] FIG. 15 is a schematic showing the design of the experiment in Example 18. Keyhole limpet hemocyanin (KLH) is administered at the timepoints shown, with subcutaneous injection of 15906 antibody at the timepoints shown.

[0039] FIG. 16 is a scatter dot plot showing quantification of anti-KLH IgM by ELISA in mouse plasma from the KLH model. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares,3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0040] FIG. 17 is a scatter dot plot showing quantification of anti-KLH IgG by ELISA in mouse plasma from the KLH model. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0041] FIG. 18 is a scatter dot plot showing the percentage of B cell expressing CD40 and CD86 in splenocytes harvest at day 14 as shown in FIG. 15. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0042] FIG.19 is a scatter dot plot showing the concentration of sCD40 in plasma at day 14 as shown in FIG. 15. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0043] FIG. 20 is a schematic showing the design of the experiment in Example 19. Keyhole limpet hemocyanin (KLH) is administered at the timepoints shown, with subcutaneous injection of 15906 antibody, KPL-404 analogue or BMS986325 analogue at the timepoints shown.

[0044] FIG. 21 is a scatter dot plot showing quantification of anti-KLH IgM by ELISA in mouse plasma from the KLH model. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0045] FIG. 22 is a scatter dot plot showing quantification of anti-KLH IgG by ELISA in mouse plasma from the KLH model. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares,3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0046] FIG. 23 is a scatter dot plot showing the concentration of sCD40 in plasma at day 7, day 14 and day 21 according to the design as shown in FIG. 20. The group receiving treatment at a dose 0.3 mg / kg is represented by circles, 1 mg / kg is represented by squares and 3 mg / kg is represented by triangles. The group receiving antibody 15906 is represented at 0.3mg / kg by black circle, at Img / kg by black square and at 3mg / kg by black triangle; KPL- 404 analogue is represented at 0.3mg / kg by grey circle, at Img / kg by grey square and at 3mg / kg by grey triangle; BMS986325 analogue is represented at 0.3mg / kg by outline grey circle, at Img / kg by outline grey square and at 3mg / kg by outline grey triangle.

[0047] FIG. 24 is a scatter dot plot showing the concentration of sCD40 in plasma at day 0, day 7, day 14 and day 20 according to the design as shown in FIG. 20 in a group receiving antibody 15906. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.3 mg / kg is represented by black circles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles. Data are expressed as mean + / - SD.

[0048] FIG. 25 is a scatter dot plot showing the concentration of sCD40 in plasma at day 0, day 7, day 14 and day 20 according to the design as shown in FIG. 20 in a group receiving KPL-404. The group receiving vehicle is represented by white circles, the group receiving KPL-404 at dose 0.3 mg / kg is represented by black circles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles. Data are expressed as mean + / - SD.

[0049] FIG. 26 is a scatter dot plot showing the concentration of sCD40 in plasma at day 0, day 7, day 14 and day 20 according to the design as shown in FIG. 20 in a group receiving BMS986325. The group receiving vehicle is represented by white circles, the group receiving BMS986325 at dose 0.3 mg / kg is represented by black circles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles. Data are expressed as mean + / - SD.

[0050] FIG.27 is a schematic showing the design of the experiment in Example 20. Keyhole limpet hemocyanin (KLH) is administered at the timepoints shown, with subcutaneous injection of 15906 antibody at the timepoints shown.

[0051] FIG. 28 is a scatter dot plot showing quantification of anti-KLH IgG by ELISA in mouse plasma from the KLH model. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares,3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0052] FIG. 29 is a scatter dot plot showing quantification of anti-KLH IgM by ELISA in mouse plasma from the KLH model. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0053] FIG. 30 is a scatter dot plot showing the concentration of sCD40 in plasma at day 20 as shown in FIG. 27. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.

[0054] FIG. 31 is a scatter dot plot showing the percentage of B cell expressing CD40 in splenocytes harvest at day 20 as shown in FIG. 27. The group receiving vehicle is represented by white circles, the group receiving antibody 15906 at dose 0.1 mg / kg is represented by white squares, 0.3 mg / kg is represented by white triangles, 1 mg / kg is represented by black squares, 3 mg / kg is represented by black triangles, and 10 mg / kg is represented by black circles. Data are expressed as mean + / - SD.DETAILED DESCRIPTION OF THE INVENTION

[0055] The present disclosure provides new antibodies that specifically bind to CD40 (“anti-CD40 antibodies”), or antigen-binding portions thereof, that can be used to block the interaction of CD40 with its ligand CD40L. The present antibodies can modulate the immune system in a patient and may be used for treatment of autoimmune disorders or inflammatory disorders. The antibodies antagonize and do not agonize CD40 activity. The antibodies inhibit B cell proliferation and block the upregulation of CD25, CD69 and CD86.

[0056] Unless otherwise stated, “CD40” refers to human CD40. A human CD40 polypeptide sequence is available under UniProt Accession No. P25942, as shown below:MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETEC LPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACES CVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPI IFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEI NFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ ( SEQ ID NO : 97 )A cynomolgus monkey CD40 polypeptide sequence is available under UniProt Accession No. G7PG38, as shown below:MVRLPLQCVLWGCLLTAVYPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETEC LPCSESEFLDTWNRETRCHQHKYCDPNLGLQVQQKGTSETDTICTCEEGLHCTSESCES CVPHRSCLPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCRPWTSCETKDLVVQQA GTNKTDVVCGPQDRQRALVVIPICLGILFVILLLVLVFIKKVAKKPNDKVPHPKQEPQE INFPDDLPGSNPAAPVQETLHGCQPVTQEDGKESRISVQERQ ( SEQ ID NO : 98 )A marmoset CD40 polypeptide sequence is available under UniProt Accession No. Q3LRP1, as shown below:MFRLPLQCVLWGCLLSSVHPEPPTACREKQYLINSQCCSLCQPGWKLVNDCTEVTETEC LPCGKGEFLDTWNRETHCHQHKYCDPNLGLRVQQEGTSVTDNICVCKEGRHCTSKACES CVLYHSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCRPWTRCETKGLAEQQA GTDKTDAVCGPQNRLRLLVVIPIMLGILFAILLVLVFIKKVDRKPQDKAPCTKQIPQEI DDLPGPNPTPPVQETLHGCQPVAQEDGKESRISVQERQ ( SEQ ID NO : 99 )

[0057] The term “antibody” (Ab) or “immunoglobulin” (Ig), as used herein, refers to a tetramer comprising two heavy chains (HCs) (about 50-70 kDa) and two light chains (LCs) (about 25 kDa) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be subdivided further into regions of hypervariability, termed “complementarity determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FRs). Each VH and VL is composed of three CDRs (H-CDR herein designates a CDR from the heavy chain; and L-CDR herein designates a CDR from the light chain) and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers, and of FR and CDR regions, in the heavy or light chain may be in accordance with IMGT® definitions (Lefranc et al., Dev Comp Immunol. (2003) 27(1):55- 77); or the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J Mol Biol. (1987) 196:901-17; Chothia et al., Nature (1989) 342:878-83; MacCallum et al., J Mol Biol. (1996) 262:732-45; or Honegger and Pliickthun, J Mol Biol. (2001) 309(3):657-70.

[0058] The term “recombinant antibody” refers to a non-naturally occurring antibody that is expressed from a cell or cell line comprising the nucleotide sequence(s) that encode the antibody, wherein said nucleotide sequence(s) are not naturally associated with the cell.

[0059] The term “isolated protein,” “isolated polypeptide” or “isolated antibody” refers to a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, and / or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. Such naturally associated components may include, e.g., nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, cellular debris, growth medium, etc.

[0060] The term “affinity” refers to a measure of the attraction between an antigen and an antibody. The intrinsic attractiveness of the antibody for the antigen is typically expressed as the binding affinity equilibrium constant (KD) of a particular antibody-antigen interaction. An antibody is said to specifically bind to an antigen when the KD for the binding is < 1 pM, e.g., < 100 nM or < 10 nM. A KD binding affinity constant can be measured, e.g., by surface plasmon resonance (Biacore™) using the Biacore™ T200 system, the IBIS MX96 SPR system from IBIS Technologies, or the Carterra LSA SPR platform, or by bio-layer interferometry, for example using the Octet™ system from ForteBio.

[0061] The term “epitope” as used herein refers to a portion (determinant) of an antigen that is bound by an antigen-binding protein (e.g., an antibody or an antigen-binding portion thereof). For example, an epitope may refer to the portion of an antigen that specifically binds to an antigen-binding site of an antigen-binding protein (e.g., an antibody or antigenbinding portion thereof), wherein the antigen-binding site is known as a paratope. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics. An antigen may have more than one epitope.

[0062] An epitope may be “linear” or “conformational.” In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. A “functional” epitope refers to residues that directly contribute to the affinity of theantigen / antigen-binding protein interaction. Epitopes may be determined using methods such as, e.g., alanine scanning mutational analysis, peptide blot analysis, peptide cleavage analysis, crystallographic studies, NMR analysis, and hydrogen / deuterium exchange detected by mass spectrometry.

[0063] Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope using techniques well known in the art. For example, an antibody to a linear epitope may be generated, e.g., by immunizing an animal with a peptide having the amino acid residues of the linear epitope. An antibody to a conformational epitope may be generated, e.g., by immunizing an animal with a mini-domain containing the relevant amino acid residues of the conformational epitope. An antibody to a particular epitope can also be generated, e.g., by immunizing an animal with the target molecule of interest (e.g., CD40) or a relevant portion thereof, then screening for binding to the epitope. An antibody to a particular epitope also may be generated using phage display methods.

[0064] One can determine whether an antibody binds to the same epitope as or competes for binding to CD40 with an anti-CD40 antibody of the present disclosure by using methods known in the art, including, without limitation, competition assays, epitope binning, and alanine scanning. In some embodiments, one allows the antibody of the present disclosure to bind to the antigen under saturating conditions, and then measures the ability of the test antibody to bind to that antigen. If the test antibody is able to bind to the antigen at the same time as the reference antibody, then the test antibody binds to a different epitope than the reference antibody. However, if the test antibody is not able to bind to the antigen at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the antibody of the present disclosure. To test whether an antibody cross-competes with another antibody, one may use the competition method described above in two directions, i.e., determining if the known antibody blocks the test antibody and vice-versa. Competition or cross-competition experiments can be performed using, e.g., ELISA, RIA, Biacore™, SPR, Bio-Layer Interferometry or flow cytometry. For example, the experiments may be performed, e.g., using a Biacore™ T200, IBIS MX96, or Carterra LSA SPR instrument or the Octet™ system.

[0065] The term “human antibody” refers to an antibody in which the variable domains and constant region sequences are derived from human sequences. The term encompasses antibodies with sequences that are derived from human genes but have been modified, e.g., to decrease immunogenicity, increase affinity, and / or increase stability. Further, the term encompasses antibodies produced recombinantly in nonhuman cells, which may impartglycosylation not typical of human cells. The term also encompasses antibodies produced in transgenic nonhuman organisms with human antibody genes (e.g., OmniRat® rats).

[0066] The term “antigen-binding portion” or “antigen-binding fragment” of an antibody, or simply “antibody portion,” as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD40, or portion(s) thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” include (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen (e.g., a CDR3 peptide or a FR3-CDR3- FR4 peptide). Further, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as single chain Fv (scFv)). Also within the present disclosure are antigen-binding molecules comprising a VH and / or a VL. In the case of a VH, the molecule may also comprise one or more of a CHI, hinge, CH2, or CH3 region. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. Also contemplated are tribodies, tetrabodies, nanobodies (e.g., monovalent or bivalent nanobodies), minibodies, domain-specific antibodies, single domain antibodies, and domain- deleted antibodies.

[0067] Antigen-binding portions, such as Fab and F(ab’)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antigen-binding portions and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, e.g., as described herein.

[0068] The class (isotype) and subclass of anti-CD40 antibodies described herein may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA or Western blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant regions of the heavy and / or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

[0069] Unless otherwise indicated, the numbering of all antibody amino acid residues in this disclosure is according to the IMGT® numbering scheme.I. Anti-CD40 Antibodies and Binding Proteins

[0070] The present disclosure provides anti-CD40 antibodies and antigen-binding portions thereof, as well as binding proteins comprising said antibodies or antigen-binding portions. In a certain aspect, the antibodies disclosed herein are human antibodies generated from transgenic animals (e.g., rats) that are able to produce antibodies encoded by rearranged human antibody genes. In particular embodiments, the human antibodies may contain certain mutations, e.g., to change primer-derived mutations back to the germline sequence.

[0071] In some embodiments, the anti-CD40 antibody or antigen-binding portion competes or cross-competes for binding to human CD40 with, or binds to the same epitope of human CD40 as, an antibody comprising HC and LC sequences that comprise: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively; c) SEQ ID NOs: 21 and 22, respectively; d) SEQ ID NOs: 31 and 32, respectively; e) SEQ ID NOs: 41 and 42, respectively; f) SEQ ID NOs: 51 and 52, respectively; g) SEQ ID NOs: 61 and 62, respectively; or h) SEQ ID NOs: 71 and 72, respectively.

[0072] The assignment of CDR regions for the anti-CD40 antibodies herein may be in accordance with any method known in the art, such as IMGT® , Kabat, Chothia, Martin, Contact, or AHo definitions, or any combination of any of these definitions (Kabat plus Chothia, for example). Also contemplated are sets of CDRs wherein each of H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 may individually be specified according to any of the methods for defining CDRs herein (e.g., H-CDR1 specified by the Kabat definition, H-CDR2 specified by the Chothia definition, etc.)

[0073] In some embodiments, the anti-CD40 antibody or antigen-binding portion has H- CDR1-3 comprising SEQ ID NOs: 3-5, 13-15, 23-25, 33-35, 43-45, 53-55, 63-65, or 73-75, respectively.

[0074] In some embodiments, the anti-CD40 antibody or antigen-binding portion has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, or 71. In certain embodiments, any variations from the selected sequence are in the framework regions (FRs).

[0075] In some embodiments, the anti-CD40 antibody or antigen-binding portion has a VHcomprising SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, or 71.

[0076] In some embodiments, the anti-CD40 antibody has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, or 71 (optionally wherein any variations from the selected sequence are in the FRs); and a heavy chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 81-83.

[0077] In some embodiments, the anti-CD40 antibody comprises a VH amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11, and a heavy chain constant region amino acid sequence of any one of SEQ ID NOs: 81-83.

[0078] In some embodiments, the anti-CD40 antibody or antigen-binding portion has L- CDR1-3 comprising SEQ ID NOs: 6-8, 16-18, 26-28, 36-38, 46-48, 56-58, 66-68, or 76-78, respectively.

[0079] In some embodiments, the anti-CD40 antibody or antigen-binding portion has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, or 72. In certain embodiments, any variations from the selected sequence are in the FRs.

[0080] In some embodiments, the anti-CD40 antibody or antigen-binding portion has a VLcomprising SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, or 72.

[0081] In some embodiments, the anti-CD40 antibody has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, or 72 (optionally wherein any variations from the selected sequence are in the FRs); and a light chain constant region amino acid sequence thatis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84.

[0082] In some embodiments, the anti-CD40 antibody comprises a VL amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, or 72, and a light chain constant region amino acid sequence of SEQ ID NO: 84.

[0083] In certain embodiments, the anti-CD40 antibody or antigen-binding portion comprises any one of the above-described heavy chain sequences and any one of the abovedescribed light chain sequences.

[0084] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of: a) SEQ ID NOs: 3-8, respectively; b) SEQ ID NOs: 13-18, respectively; c) SEQ ID NOs: 23-28, respectively; d) SEQ ID NOs: 33-38, respectively; e) SEQ ID NOs: 43-48, respectively; f) SEQ ID NOs: 53-58, respectively; g) SEQ ID NOs: 63-68, respectively; or h) SEQ ID NOs: 73-78, respectively.

[0085] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively; c) SEQ ID NOs: 21 and 22, respectively; d) SEQ ID NOs: 31 and 32, respectively; e) SEQ ID NOs: 41 and 42, respectively; f) SEQ ID NOs: 51 and 52, respectively; g) SEQ ID NOs: 61 and 62, respectively; or h) SEQ ID NOs: 71 and 72, respectively.In certain embodiments, any variations from the selected sequences are in the FRs.

[0086] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that comprise: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively;c) SEQ ID NOs 21 and 22, respectively;SEQ ID NOs 31 and 32, respectively;SEQ ID NOs 41 and 42, respectively; f) SEQ ID NOs 51 and 52, respectively; g) SEQ ID NOs 61 and 62, respectively; or h) SEQ ID NOs 71 and 72, respectively.

[0087] In some embodiments, the anti-CD40 antibodies of the present disclosure have the“LALA” mutations (L234A / L235A) in the Fc region. These mutations hinder binding of the antibodies to human FcyR (Fc gamma receptors). Additionally or alternatively, the anti- CD40 antibodies may have a G237A mutation in the Fc region. “IgG1.3f’ refers to the presence of the L234A, L235E, and G237A mutations, which are known to reduce effector function of the Fc region of IgGl antibodies (WO 88 / 07089). All Fc region positions are defined according to Eu numbering unless otherwise stated.

[0088] In some embodiments, the anti-CD40 antibody of the present disclosure comprises: a) an HC comprising SEQ ID NOs: 1 and 81 and an LC comprising SEQ ID NOs: 2 and 84; b) an HC comprising SEQ ID NOs: 11 and 81 and an LC comprising SEQ ID NOs: 12 and 84; c) an HC comprising SEQ ID NOs: 21 and 81 and an LC comprising SEQ ID NOs: 22 and 84; d) an HC comprising SEQ ID NOs: 31 and 81 and an LC comprising SEQ ID NOs: 32 and 84; e) an HC comprising SEQ ID NOs: 41 and 81 and an LC comprising SEQ ID NOs: 42 and 84; f) an HC comprising SEQ ID NOs: 51 and 81 and an LC comprising SEQ ID NOs: 52 and 84; g) an HC comprising SEQ ID NOs: 61 and 81 and an LC comprising SEQ ID NOs: 62 and 84; or h) an HC comprising SEQ ID NOs: 71 and 81 and an LC comprising SEQ ID NOs: 72 and 84.

[0089] In some embodiments, the anti-CD40 antibody of the present disclosure comprises: a) an HC comprising SEQ ID NOs: 1 and 82 and an LC comprising SEQ ID NOs: 2and 84; b) an HC comprising SEQ ID NOs: 11 and 82 and an LC comprising SEQ ID NOs: 12 and 84; c) an HC comprising SEQ ID NOs: 21 and 82 and an LC comprising SEQ ID NOs:22 and 84; d) an HC comprising SEQ ID NOs: 31 and 82 and an LC comprising SEQ ID NOs: 32 and 84; e) an HC comprising SEQ ID NOs: 41 and 82 and an LC comprising SEQ ID NOs: 42 and 84; f) an HC comprising SEQ ID NOs: 51 and 82 and an LC comprising SEQ ID NOs: 52 and 84; g) an HC comprising SEQ ID NOs: 61 and 82 and an LC comprising SEQ ID NOs:62 and 84; or h) an HC comprising SEQ ID NOs: 71 and 82 and an LC comprising SEQ ID NOs: 72 and 84.

[0090] In some embodiments, the anti-CD40 antibody of the present disclosure comprises: a) an HC comprising SEQ ID NOs: 1 and 83 and an LC comprising SEQ ID NOs: 2 and 84; b) an HC comprising SEQ ID NOs: 11 and 83 and an LC comprising SEQ ID NOs:12 and 84; c) an HC comprising SEQ ID NOs: 21 and 83 and an LC comprising SEQ ID NOs:22 and 84; d) an HC comprising SEQ ID NOs: 31 and 83 and an LC comprising SEQ ID NOs:32 and 84; e) an HC comprising SEQ ID NOs: 41 and 83 and an LC comprising SEQ ID NOs: 42 and 84; f) an HC comprising SEQ ID NOs: 51 and 83 and an LC comprising SEQ ID NOs: 52 and 84; g) an HC comprising SEQ ID NOs: 61 and 83 and an LC comprising SEQ ID NOs:62 and 84; or h) an HC comprising SEQ ID NOs: 71 and 83 and an LC comprising SEQ ID NOs: 72 and 84.

[0091] The present disclosure also provides an anti-CD40 antibody or an antigen-bindingportion thereof that competes or cross-competes for binding to CD40 with, or binds to the same epitope of CD40 as, antibody 15906, 15837, 15825, 15834, 15949, 16071, 16344 or 16029.

[0092] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 15906, 15837, 15825, 15834, 15949, 16071, 16344 or 16029.

[0093] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the VH and VL, respectively, of antibody 15906, 15837, 15825, 15834, 15949, 16071, 16344 or 16029. In certain embodiments, any variations from the VH and VL sequences of the selected antibody are in the FRs.

[0094] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that are the VH and VL, respectively, of antibody 15906, 15837, 15825, 15834, 15949, 16071, 16344 or 16029.

[0095] In some embodiments, the anti-CD40 antibody of the present disclosure is antibody 15906, 15837, 15825, 15834, 15949, 16071, 16344 or 16029, or an antibody with the same amino acid sequences as said antibody.

[0096] Also contemplated by the present disclosure is an anti-CD40 antibody or an antigen-binding portion thereof, wherein said anti-CD40 antibody- competes or cross-competes for binding to human CD40 with, or binds to the same epitope of human CD40 as, an antibody with an HC that comprises SEQ ID NO: 1 and an LC that comprises SEQ ID NO: 2;- comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 3-8, respectively;- comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) in amino acid sequence to SEQ ID NOs: 1 and 2, respectively (optionally wherein any variations from SEQ ID NOs: 1 and 2 are in the FRs);- comprises a VH and a VL that comprise the amino acid sequences of SEQ ID NOs: 1 and 2, respectively;- comprises an HC comprising the amino acid sequences of SEQ ID NOs: 1 and 83 and an LC comprising the amino acid sequences of SEQ ID NOs: 2 and 84;- comprises an HC comprising the amino acid sequences of SEQ ID NOs: 1 and 82 and anLC comprising the amino acid sequences of SEQ ID NOs: 2 and 84; or- comprises an HC comprising the amino acid sequences of SEQ ID NOs: 1 and 81 and an LC comprising the amino acid sequences of SEQ ID NOs: 2 and 84.

[0097] In some embodiments, the anti-CD40 antibody- competes or cross-competes for binding to CD40 with, or binds to the same epitope of CD40 as, antibody 15906;- comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 15906;- comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the VH and VL, respectively, of antibody 15906 (optionally wherein any variations from the VH and VL of antibody 15906 are in the FRs);- comprises a VH and a VL that are the VH and VL, respectively, of antibody 15906; or- is antibody 15906, or an antibody with the same amino acid sequences as said antibody.

[0098] In some embodiments, a “variant” antibody or antigen-binding portion, having amino acid substitutions (which may be conservative or non-conservative) from an antibody or antigen-binding portion exemplified herein, does not have substantially altered biologic activity from the exemplified antibody or antigen-binding portion. For example, the variant antibody or antigen-binding portion may retain at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the binding affinity of the parent antibody or antigen-binding portion, or may exceed the binding affinity of the parent antibody or antigen-binding portion. In some embodiments, a variant antibody or an antigen-binding portion thereof may have mutations, e.g., that increase its half-life, alter its immunogenicity, provide a site for covalent or non- covalent binding to another molecule, etc. In certain embodiments, the variant antibody or antigen-binding portion thereof may have mutations in its FRs (e.g., in one, two, three, four, five, six, seven, or eight of its FRs). In certain embodiments, the variant antibody or antigenbinding portion thereof may have mutations in its CDRs (e.g., in one, two, three, four, five, or six of its CDRs). In certain embodiments, the variant antibody or antigen-binding potion thereof may have mutations in its constant regions.

[0099] The class of an anti-CD40 antibody described herein may be changed or switched with another class or subclass. In some embodiments of the present disclosure, a nucleic acid molecule encoding the VL or VH of the antibody is isolated using methods well known in the art such that it does not include nucleic acid sequences encoding CL or CH, respectively.The nucleic acid molecules encoding VL or VH then are operatively linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class or subclass ofimmunoglobulin molecule. This may be achieved using a vector or nucleic acid molecule that comprises a CL or CH sequence, as described above. For example, an anti-CD40 antibody that was originally IgM may be class switched to IgG. Further, the class switching may be used to convert one IgG subclass to another, e.g., from IgGi to IgG?. A K light chain constant region can be changed, e.g., to a light chain constant region, or vice-versa.

[0100] The anti-CD40 antibody of the present disclosure can be an IgG, an IgM, an IgE, an IgA, or an IgD molecule, but is typically of the IgG isotype, e.g., of IgG subclass IgGi, IgG?aor IgG?b, IgG? or IgG . In some embodiments, the antibody is of the isotype subclass IgGi.

[0101] In some embodiments, the anti-CD40 antibody may comprise at least one mutation in the Fc region. A number of different Fc mutations are known, where these mutations alter, e.g., the antibody’s effector functions or half-life. For example, in some embodiments, the anti-CD40 antibody comprises at least one mutation in the Fc region that reduces effector function.

[0102] In some embodiments, the anti-CD40 antibody or antigen-binding portion of the present disclosure is antagonistic.

[0103] In some embodiments, the anti-CD40 antibody or antigen-binding portion binds to human CD40 with a KDof 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, or 0.1 nM or less (e.g., 70 nM or less, 20 nM or less, or 2 nM or less) as measured by surface plasmon resonance (e.g., as described in Example 4).

[0104] In some embodiments, the anti-CD40 antibody or antigen-binding portion does not bind to cynomolgus CD40. In other embodiments, the anti-CD40 antibody or antigenbinding portion binds to cynomolgus CD40 with a KD of 30, 25, 20, 15, 10, 5, or 1 nM or less (e.g., 15 nM or less) as measured by surface plasmon resonance (e.g., as described in Example 4).

[0105] In some embodiments, the anti-CD40 antibody or antigen-binding portion binds to marmoset CD40.

[0106] In some embodiments, the anti-CD40 antibody or antigen-binding portion does not bind to mouse CD40.

[0107] In some embodiments, the anti-CD40 antibody or antigen-binding portion does not activate any of receptors Fcyla, Fcyllb, FcyIIa-H / -R, and FcyIIIa-V / -F overexpressed on cells incubated with CD40-expressing Daudi cells (e.g., as determined using assays such as those described in Example 7).

[0108] In some embodiments, the anti-CD40 antibody or antigen-binding portion (e.g., inIgG1.3f format) does not induce neutrophil-mediated killing of CD40-expressing Daudi cells (e.g., as determined using assays such as those described in Example 8).

[0109] In some embodiments, the anti-CD40 antibody or antigen-binding portion (e.g., in IgG1.3f format) does not induce PBMC-mediated killing of B cells (e.g., as determined using assays such as those described in Example 9).

[0110] In some embodiments, the anti-CD40 antibody or antigen-binding portion does not demonstrate agonistic activity. For instance, in certain embodiments, the antibody or antigen-binding portion does not activate a CD40 reporter cell line (e.g., as determined using assays such as those described in Example 10). In certain embodiments, the antibody or antigen-binding portion does not induce B cell proliferation (e.g., as determined using assays such as those described in Example 11). In certain embodiments, the antibody or antigenbinding portion does not activate dendritic cells (e.g., as determined using assays such as those described in Example 12).[OHl] In some embodiments, the anti-CD40 antibody or antigen-binding portion demonstrates antagonistic activity. For instance, in certain embodiments, the antibody or antigen-binding portion inhibits proliferation of a CD40 reporter cell line, for example, by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (e.g., as determined using assays such as those described in Example 13). In certain embodiments, the antibody or antigen-binding portion inhibits proliferation of B cells, for example, by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (e.g., as determined using assays such as those described in Example 14). In certain embodiments, the antibody or antigen-binding portion blocks plasma cell differentiation of B cells in PBMCs, for example, by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (e.g., as determined using assays such as those described in Example 15). In certain embodiments, the antibody or antigen-binding portion blocks upregulation of CD25, CD69, and / or CD86 (e.g., all three) on B cells, for example, by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (e.g., as determined using assays such as those described in Example 16).

[0112] In some embodiments, the anti-CD40 antibody or antigen-binding portion binds to an epitope in the cysteine-rich domains (CRDs) of human CD40. In certain embodiments, the epitope is located in CRD1 and CRD2. For example, the epitope may be in CRD1 and partially in CRD2. In some embodiments, the epitope comprises, is comprised by, or overlaps with the contact surface of the receptor-ligand interaction. In certain embodiments, the epitope comprises human CD40 residues C41-T55, P61-T75, or both. In certain embodiments, the epitope comprises 1, 2, 3, 4, 5, 6, 7, or all 8 of human CD40 residues K46, D50, E66, R73, E74, T75, H76, and H78 (in any combination). In certain embodiments, theepitope comprises 1, 2, 3, 4, 5, or all 6 of human CD40 residues K46, E66, R73, E74, T75, and H76 (in any combination). In particular embodiments, the epitope comprises human CD40 residues K46, E66, R73, E74, T75, and H76.

[0113] In certain embodiments, the epitope is determined using the methods described in Examples 5 and 6 below. In certain embodiments, the anti-CD40 antibody or antigenbinding portion binds to a different epitope of human CD40 than, and / or does not compete for binding to human CD40 with, ravagalimab and / or iscalimab (or analogues thereof).

[0114] In some embodiments, an anti-CD40 antibody or antigen-binding portion described herein has at least one (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20) of the following properties: a) binds to human CD40 with a KD of 70 nM or less as measured by surface plasmon resonance (SPR); b) binds to marmoset CD40; c) does not bind to cynomolgus and / or mouse CD40; d) blocks binding of human CD40L to human CD40 expressed on CHO-S cells; e) does not activate any of receptors Fcyla, Fcyllb, FcyIIa-H / -R, and FcyIIIa-V / -F overexpressed on cells incubated with CD40-expressing Daudi cells; f) does not induce neutrophil-mediated killing of CD40-expressing Daudi cells; g) does not induce PBMC-mediated killing of B cells; h) does not demonstrate agonistic activity; i) does not activate a CD40 reporter cell line; j) does not induce B cell proliferation; k) does not activate dendritic cells; l) demonstrates antagonistic activity; m) inhibits proliferation of a CD40 reporter cell line; n) inhibits proliferation of B cells; o) blocks plasma cell differentiation of B cells in PBMCs; p) blocks upregulation of CD25, CD69, and CD86 on B cells; q) recognizes an epitope of CD40 in cysteine-rich domain (CRD)l and partially in CRD2; r) binds to a human CD40 epitope comprising residues C41-T55 and / or P61-T75; s) binds to an epitope comprising human CD40 residues K46, E66, R73, E74, T75, and / or H76 (e.g., all six of said residues), optionally further comprising D50 and / or H78; andt) does not bind to the same epitope as ravagalimab and / or iscalimab.

[0115] In some embodiments, the anti-CD40 antibody or antigen-binding portion may comprise, for instance, all of properties a)-t) (e.g., antibody 15906), at least properties a), c), g), n), and t), (e.g., antibody 15837), at least properties a), g), n), and t) (e.g., antibody 16029), or at least property n) (e.g., antibodies 15825, 15834, 15949, 16071, and 16344).

[0116] In some embodiments, the present disclosure provides an anti-CD40 binding protein that comprises, or has the binding specificity of, the anti-CD40 antibody or antigenbinding portion described herein. In some embodiments, the binding protein is a multispecific (e.g., bispecific) binding molecule that has the binding specificity of an anti- CD40 antibody described herein (e.g., may comprise the six CDRs or the VH and VL of said anti-CD40 antibody). In some embodiments, the multispecific binding molecule additionally has the binding specificity of a) another, distinct anti-CD40 antibody, e.g., an antibody that targets different epitope(s) on the same protein(s), or b) another, distinct antibody that targets a different protein, such as another cell surface molecule whose activity mediates the immune response or a disease condition such as an inflammatory or autoimmune disease.Multispecific binding molecules are known in the art, and examples of different types of multispecific binding molecules (e.g., bispecific binding molecules) are given elsewhere herein.

[0117] In some embodiments, the anti-CD40 binding protein is a fusion antibody or an immunoadhesin.

[0118] In some embodiments, the anti-CD40 binding protein is an immunoconjugate, wherein the anti-CD40 antibody or antigen-binding portion thereof is conjugated to another molecule, e.g., a therapeutic moiety. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins.

[0119] In some embodiments, the anti-CD40 binding protein is a small modular immunopharmaceutical (SMIP).

[0120] In some embodiments, the anti-CD40 binding protein is a chimeric antigen receptor (CAR). Such a CAR may be used in CAR-T therapy, in which T cells are engineered to express the CAR targeting CD40.II. Nucleic Acid Molecules and Vectors

[0121] The present disclosure also provides nucleic acid molecules and sequences encoding anti-CD40 antibodies or antigen-binding portions thereof described herein, or binding proteins described herein. In some embodiments, different nucleic acid moleculesencode the heavy chain and light chain amino acid sequences of the anti-CD40 antibody or antigen-binding portion. In other embodiments, the same nucleic acid molecule encodes the heavy chain and light chain amino acid sequences of the anti-CD40 antibody or antigenbinding portion.

[0122] A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single- and double-stranded forms.

[0123] In some embodiments, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain sequence, or a nucleotide sequence that encodes the light chain sequence, or both, of an anti-CD40 antibody or antigenbinding portion thereof described herein. In some embodiments, the present disclosure provides a set (e.g., a pair) of nucleic acid molecules comprising a nucleotide sequence that encodes the heavy chain sequence, and a nucleotide sequence that encodes the light chain sequence, of an anti-CD40 antibody or antigen-binding portion thereof described herein.

[0124] The present disclosure also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more nucleotide sequences recited herein, e.g., to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, and 80. The term “percent sequence identity” in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48, or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (see, e.g., Pearson, Methods Enzymol. (1990) 183:63-98; Pearson, Methods Mol. Biol. (2000) 132: 185-219; Pearson, Methods Enzymol. (1996) 266:227-58 (1996); and Pearson, J. Mol.Biol. (1998) 276:71-84; incorporated herein by reference).

[0125] In some embodiments, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, and 80.

[0126] In some embodiments, the present disclosure provides a nucleic acid molecule or a set (e.g., a pair) of nucleic acid molecules comprising the nucleotide sequences of a) SEQ ID NOs: 9 and 10; b) SEQ ID NOs: 19 and 20; c) SEQ ID NOs: 29 and 30; d) SEQ ID NOs: 39 and 40; e) SEQ ID NOs: 49 and 50; f) SEQ ID NOs: 59 and 60; g) SEQ ID NOs: 69 and 70; or h) SEQ ID NOs: 79 and 80.

[0127] In any of the above embodiments, the nucleic acid molecules may be isolated. Nucleic acid molecules referred to herein as “isolated” or “purified” are nucleic acids which (1) have been separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin; and / or (2) do not occur in nature.

[0128] In a further aspect, the present disclosure provides a vector suitable for expressing one or both of the chains of an antibody or antigen-binding portion thereof as described herein. The term “vector”, as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

[0129] The present disclosure provides vectors comprising nucleic acid molecules that encode the heavy chain sequence, the light chain sequence, or both the heavy and light chain sequences, of an anti-CD40 antibody or antigen-binding portion thereof as described herein. In certain embodiments, a vector of the present disclosure comprises a nucleic acid molecule described herein, or a pair or set of nucleic acid molecules described herein. In certain embodiments, the present disclosure provides a set (e.g., a pair) of vectors comprising a set or pair of nucleic acid molecules described herein. The present disclosure further provides vectors comprising nucleic acid molecules encoding binding proteins, fusion proteins,modified antibodies, etc. as described herein. The vectors may further comprise an expression control sequence.

[0130] The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.III. Making of Antibodies and Binding Proteins

[0131] The anti-CD40 antibodies and antigen-binding portions thereof of the present disclosure, or related binding proteins as described herein, may be produced recombinantly using isolated nucleic acid molecules such as expression constructs. The encoding sequences for each polypeptide chain may be cloned into a single vector or cloned into separate vectors (e.g., a pair or set of vectors).

[0132] The antibodies or antigen-binding portions thereof or binding proteins may be produced in host cells, e.g., mammalian host cells, using appropriate expression constructs. Mammalian cell lines available as hosts for expression include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells, and yeast cell lines. In certain embodiments, the cell lines are not derived from a human embryo. Cell lines may be selected based on their expression levels. The antibodies, antigen-binding portions, or binding proteins may be isolated and purified from the host cell culture using well known methods, such as centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0133] In some embodiments, a host cell described herein comprises nucleic acid molecule(s) comprising a nucleotide sequence that encodes the heavy chain sequence, or a nucleotide sequence that encodes the light chain sequence, or both, of an anti-CD40 antibody or antigen-binding portion thereof described herein. In certain embodiments, the host cell comprises nucleic acid molecule(s) encoding both the heavy and light chain sequences. In certain embodiments, the host cell comprises nucleic acid molecule(s) encoding only the heavy or light chain sequence, such chains being useful, e.g., as intermediates for the expression of an antibody or antigen-binding fragment that includes said heavy or light chain.

[0134] Host cells used to produce the antibodies, antigen-binding portions, or binding proteins are also termed “recombinant host cells.” A “recombinant host cell” (or simply “host cell”), as used herein, means a cell into which a recombinant expression construct has been introduced. By definition, a recombinant host cell does not occur in nature. A protein produced from a recombinant host cell is a recombinant protein.IV. Pharmaceutical Compositions and Uses

[0135] Another aspect of the present disclosure is a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) an anti-CD40 antibody or antigen-binding portion thereof or binding protein of the present disclosure. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient. A “pharmaceutically acceptable excipient” may include appropriate solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. Examples of pharmaceutically acceptable excipients are water and saline (e.g., phosphate-buffered saline).

[0136] The pharmaceutical compositions herein may be used to treat an inflammatory or autoimmune disease. In some embodiments, a method of treating a disease (such as an inflammatory or autoimmune disease) in a subject comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion disclosed herein. In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of asthma, allergic asthma, chronic spontaneous urticaria (CSU), diabetes (e.g., type 1 diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), allergy, antibody-mediated rejection, organ graft rejection, graft-versus-host disease (GvHD), Addison’s disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, celiac disease, autoimmune alopecia, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid,Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpurajuvenile myositis, Kawasaki disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, thrombocytopenic purpura, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, vitiligo, and Vogt- Koyanagi-Harada Disease.

[0137] In some embodiments, the antibodies or antigen-binding portions thereof, compositions, or binding proteins of the present disclosure may be used to treat CSU, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Sjogren’s syndrome, lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), Graves’ disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis or vitiligo.

[0138] In some embodiments, the antibodies or antigen-binding portions thereof, compositions, or binding proteins of the present disclosure may be used to treat CSU, atopic dermatitis or pemphigus vulgaris.

[0139] Treat,” “treating,” and “treatment” refer to a method of alleviating or abrogating a biological disorder and / or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition.

[0140] “Therapeutically effective amount” refers to the amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated.

[0141] The anti-CD40 antibodies or antigen-binding portions thereof, antibody compositions, or binding proteins of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with the anti-CD40 antibodies or antigen-binding portions thereof, antibody compositions, or binding proteins of the present disclosure may include at least one additional therapeutic treatment (combination therapy), e.g., another immunomodulatory agent.

[0142] The pharmaceutical compositions herein may be delivered to the patient through parenteral administration. In particular, parenteral administration is contemplated to include,but is not limited to, subcutaneous, intraperitoneal, intramuscular, or intravenous injection (e.g., intravenous infusion). Particular embodiments include the intravenous route (e.g., intravenous infusion) and the subcutaneous route (e.g., subcutaneous injection).V. Diagnostic Uses

[0143] The antibodies and antigen-binding portions or binding proteins of the present disclosure also are useful in diagnostic processes (e.g., in vitro or ex vivo). For example, the antibodies, antigen-binding portions, or binding proteins can be used to detect and / or measure the level of CD40 in a biological sample from a patient (e.g., a tissue sample, or a blood sample). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistochemistry. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the antibodies, antigen-binding portions, or binding proteins described herein.

[0144] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater thanor less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0145] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any compound disclosed herein can be used in any of the treatment method here, wherein the individual to be treated is as defined anywhere herein. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0146] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLESExample 1: Cloning of Anti-CD40 Antibodies from Rat B Cells

[0147] Antibodies against human CD40 were isolated from an antibody repertoire derived from OmniRat® rats (Osborn et al., J Immunol. (2013) 190(4): 1481-90), a transgenic rat strain from Ligand Pharmaceuticals Inc. that produces antibodies with fully human idiotypes. Cloning of rat-derived antibody genes from single-cell sorted antibody-secreting B cells (ASC) was performed by means of Symplex™ antibody discovery technology (Meijer et al., J Mol Biol. (2006) 358(3):764-72).

[0148] Antibody repertoire constructs encoding fully human immunoglobulins in IgGl ,3f or IgGl-LALA format (see below) were transfected into HEK293 cells. Cell culture supernatants were screened for binding to CD40 expressed on the surface of CHO cells using flow cytometry in a high-throughput format. CD40 reactive clones were analyzed by DNA sequencing and antibody-encoding DNA sequences were extracted. Selected antibody clones were expressed and tested functionally as described below.

[0149] Missense mutations in the amino termini of heavy and light chains that were introduced by the use of degenerate primers in the Symplex™ cloning of the antibodyencoding cDNA fragments were corrected back to germline sequence (“germlined”). Table 1 shows the heavy and light chain variable domain nucleotide sequences of the germlined antibodies designated 15906, 15837, 15825, 15834, 15949, 16071, 16344, and 16029. The correction process involved amino terminal sequence correction to germline as well as codon usage optimization. The targets for matching to human germline sequences were identified by BLAST® homology searches for the heavy chain and light chain variable regions.Table 1: Anti-CD40 Antibody Variable Domain Nucleotide Sequences

[0150] Amino acid sequences of the variable domains of antibodies 15906, 15837, 15825, 15834, 15949, 16071, 16344, and 16029 are shown in Table 2, where the CDRs annotated according to the IMGT® system are underlined.Table 2: Anti-CD40 Antibody Variable Domain Amino Acid Sequences

[0151] Amino acid sequences of the complementarity-determining regions (CDRs) of antibodies 15906, 15837, 15825, 15834, 15949, 16071, 16344, and 16029 as defined by the IMGT® system are shown in Table 3, with SEQ ID NOs under each sequence in parentheses.Table 3: Anti-CD40 Antibody CDR Amino Acid Sequences

[0152] Amino acid sequences of the constant regions (CH and CL, respectively) of antibodies 15906, 15837, 15825, 15834, 15949, 16071, 16344, and 16029 are shown inTable 4. “IgG1.3f’ refers to the presence of L234A, L235E, and G237A mutations in the heavy chain (numbered according to the Eu numbering scheme), which are known to reduce effector function of the Fc region of IgGl antibodies (WO 88 / 07089). “IgGl LALA” refers to the presence of “LALA” mutations in the heavy chain (L234A / L235A, numbered according to the IMGT® numbering scheme) that are known to reduce effector function of the Fc region of IgGl antibodies (Hezareh et al., J Virol. 75(24): 12161-68 (2001); Hessell et al., Nature 449(7158): 101-04 (2007)).Table 4: Constant Region Amino Acid Sequences

[0153] Table 5 shows SEQ ID NO information for antibodies 15906, 15837, 15825, 15834, 15949, 16071, 16344, and 16029, including the SEQ ID NOs of the CDRs according to the IMGT® system (nt: nucleotide; aa: amino acid).Table 5: Anti-CD40 Antibody SEQ ID NOs

[0154] Table 6 shows full-length heavy and light chain amino acid sequences with their corresponding SEQ ID NOs for analogues of select anti-CD40 antibodies.Table 6: Heavy and Light Chain Amino Acid Sequences of Anti-CD40 Antibody AnaloguesHC: heavy chain LC: light chainExample 2: Direct Binding of Anti-CD40 Antibodies to CHO-S Cells Transiently Transfected with Human, Marmoset, Cynomolgus, or Mouse CD40

[0155] This example describes in vitro binding of anti-CD40 antibody 15906 to CHO-S cells transiently expressing human, marmoset, cynomolgus, or mouse (UniProt Accession No. P27512) CD40 receptor. The isotype control, IgG1.3F, was included as a negative control. Iscalimab and ravagalimab analogues were included as comparators.

[0156] CHO-S cells were transiently transfected with a human CD40 extracellular domain (ECD), marmoset CD40 ECD or cynomolgus CD40 ECD construct. Mock transfected CHO-S cells were included in the assay as negative control. Antibodies were tested for binding to the transfected cells in a 12-point titration curve for each antibody ranging from 10 pg / mL to 0.2 ng / mL, and every concentration was assayed in triplicate.

[0157] In a separate experiment, CHO-S cells were transiently transfected with a mouse CD40 ECD construct and mock transfected cells used as a negative control. Antibodies were tested for binding to the transfected cells in a 12-point titration curve for each antibody ranging from 30 pg / mL to 0.6 ng / mL, and every concentration was assayed in triplicate.

[0158] The anti-CD40 antibodies were incubated for 30 minutes at 4°C with hamster cell line CHO-S transiently expressing CD40 receptors. The cells were washed twice and subsequently incubated for an additional 20 minutes with AF647-conjugated secondary antihuman IgG (H+L) antibody. After washing, antibody binding to the cells was detected using a high-throughput flow cytometer iQue® Screener PLUS (Sartorius) measuring the GeoMean of AF647 signal in each well.

[0159] Anti-CD40 antibody 15906 bound to human and marmoset CD40 ECD protein, while it had low or no binding to cynomolgus or mouse CD40 ECD (FIG. 1). Notably, antibody 15906 bound to human CD40 receptor with the highest potency (ECso in nM) and efficacy of the antibodies tested. The IgGl control showed no binding to CHO-S cells expressing human, marmoset, mouse or cynomolgus CD-40.

[0160] Efficacy and ECso values for binding of anti-CD40 antibodies to CHO-S cells transfected with human, marmoset, mouse or cynomolgus CD-40 were calculated using GraphPad Prism 10 software.Example 3: In vitro Blocking of Binding Between Human CD40 Ligand (CD40L) and Human or Marmoset CD40 Receptor Expressed on CHO-S Cells by Anti-CD40 Antibodies

[0161] This example describes the ability of anti-CD40 antibodies (particularly 15906) to block the binding of human CD40 ligand (CD40L) to human or marmoset CD40 receptor extracellular domain (ECD) transiently expressed on CHO-S cells. The isotype control, IgG1.3f, was included as a negative control.

[0162] Hamster CHO-S cells transiently transfected with human or marmoset CD40 receptor ECD were encoded with different intensities of violet encoder dye (Sartorius) to allow detection of each population seeded in the same well. After seeding the cells, the anti- CD40 antibodies were serially diluted in 3-fold dilutions from 30 mg / mL to 0.5 ng / mL and subsequently were added to the cells in each well and incubated for 30 minutes at 4°C. After washing, 500 ng / mL haemagglutinin (HA)-tagged human CD40L was added to the wells and incubated for 30 minutes at 4°C. After washing, FITC-conjugated anti-HA antibody (diluted 1 :200) was added to the wells and incubated for a further 20 minutes in the dark at 4°C. After a final washing step, binding of CD40L to CD40 receptor expressed on CHO-S cells was detected using a high-throughput flow cytometer on iQue® Screener PLUS (Sartorius) measuring FITC Geomean in each well. A 12-point curve was generated for each antibody and every concentration was analyzed in triplicate. Data were transferred to Excel and binding curves were generated using GraphPad Prism® software.

[0163] The anti-CD40 antibody 15906 fully blocked the binding of human CD40L to human CD40 receptor expressed on CHO-S (FIG. 2). Notably, the blocking capacity of 15906 was superior or equivalent to the reference antibody analogues tested (ravagalimab and iscalimab).

[0164] As expected, the IgG1.3f control antibody did not block binding of CD40L to human CD40 receptor in CHO-S cells, while in human CD40-transfected cells a partial blocking effect was observed. Despite this mild effect, it is possible to discriminate between blocking and non-blocking antibodies for the human CD40 receptor.Example 4: Measurement of Antibody Binding Kinetics Towards Human and Cynomolgus CD40 Extracellular Domain

[0165] This example demonstrates the binding of anti-CD40 antibodies to recombinant human and cynomolgus CD40 extracellular domains (ECDs) as measured by Surface Plasmon Resonance (SPR).

[0166] The anti-CD40 antibodies were tested for binding to recombinant human and cynomolgus CD40 (ACRO Biosystems) by Surface Plasmon Resonance (SPR) using Carterra® LSA™. An HC30M (Carterra®) chip was functionalized by goat anti -human Ig Fc (Southern Biotech) using amine-coupling. The chip was activated by freshly prepared 0.4 M EDC, 0.1 M sulfo-NHS, and 0.1 M MES, pH 5.5 (1 : 1 :1 v / v / v) for 5 minutes, coupled with 75 pg / mL anti-human Ig Fc in 10 mM sodium acetate, pH 4.5, for 10 minutes, and excess reactive esters were quenched for 3 minutes by injection of 1 M ethanolamine, pH 8.5. The instrument was primed in running buffer (PBS pH 7.4, 0.01% Tween-20, 0.5 mg / ml BSA). After priming and washing, antibodies were captured onto individual spots of the chip for 12 minutes as replicates (n=8). Kinetic analysis was performed by applying kinetic titration series of each antigen as increasing concentrations. Antigen association was performed for 10 minutes, and antigen dissociation was recorded for 15 minutes. After each cycle of antigen injections, the surface was regenerated by 0.45% H3PO4 for 2x20 s and washed for 5 minutes in running buffer. Binding responses were processed and analyzed using Carterra’s KIT software tool. Processed data were fitted to a simple Langmuir 1 : 1 binding model for calculation of the on-rate (konor ka), off-rate (koir or kd) and affinity (KD) constants.

[0167] The binding kinetics of anti-CD40 antibodies are presented in Table 7. Antibodies 15906, 16029, and 15837 demonstrate high affinities, ranging from single-digit to lower double-digit nanomolar levels, for binding to the extracellular domain of recombinant human CD40. Antibodies 15906 and 15837 show weak binding to recombinant cynomolgus CD40 ECD, preventing accurate data fitting under the tested conditions. By contrast, antibody 16029 shows good affinity for cynomolgus CD40 ECD. The affinities of the ravagalimab, iscalimab, KLP-404, and BMS986325 analogues fall within the sub-nanomolarto single-digit nanomolar range, displaying good cross-reactivity with cynomolgus CD40 ECD.Table 7: Binding Kinetics( kon, kOff, and KD) of Binding of Anti-CD40 Antibodies to Human and Cynomolgus CD40 ECD as Measured by SPRSD: standard deviation w.b. for weak bindingExample 5: Epitope Binning of Anti-CD40 Antibodies

[0168] This example measures paired competition of anti-CD40 monoclonal antibodies using surface plasmon resonance (SPR). Antibodies belonging to different epitope groups or bins recognize different epitope binding regions on the CD40 extracellular domain ECD.

[0169] Epitope binning was performed by SPR (Carterra LSA). An array was generated by amine-coupling of antibodies onto an HC200M chip at a dilution of 5 mg / mL in 10 mM acetate buffer at pH 4.0 for 12 minutes, followed by deactivation with 1 M ethanolamine at pH 8.5 for 3 minutes.

[0170] For the epitope binning experiments, antibodies immobilized on the chip were subjected to a 5 -minute exposure to 50 nM of recombinant human CD40-His(AcroBiosy stems), followed by injections of antibodies at a concentration of 15 pg / mL, both diluted in running buffer (PBS pH 7,2, 0,05% Tween 20). To maintain the chip surface integrity, regeneration was accomplished by 2x30-second injections of 10 mM glycine at pH2.0 after each antibody sample injection. For each 6 cycles, an injection sequence involving the antigen followed by a buffer-only injection was performed for normalization.

[0171] Analysis of the generated data was performed utilizing the Carterra Epitope analysis software. The data were normalized to the binding signal of the antigen-only injection, rendering the average signal as zero response units. Antibody responses were categorized employing a threshold window into three categories: “blockers” (binding signal falling below the lower threshold), or “sandwiching” (binding signal above the higher threshold) and subjected to an automated clustering process, generating a heatmap and competition plot.

[0172] Epitope binning was performed using a classical sandwich assay setup and measured by SPR. Anti-CD40 antibodies 15906, 15837, 16029, along with the ravagalimab and iscalimab analogues, were tested pairwise to assess their competitive binding to recombinant human CD40. The antibodies were grouped into three distinct epitope bins, as shown in FIG. 3. The ravagalimab analogue, shown in the literature as binding to the CRD2 domain of CD40 (Argiriadi et al., BMC Mol Cell Biol. (2019) 20(l):29) was placed in the same epitope group as the iscalimab analogue (designated as bin 1), indicating that they likely recognize a similar epitope. Antibodies 15837 and 16029 were categorized into a separate epitope group (bin 2) and were found to compete with the ravagalimab and iscalimab analogues. Antibody 15906 was classified into a third group (bin 3) and competed exclusively with the antibodies in bin 2 (15837 and 16029 antibodies).

[0173] In summary, the analysis demonstrated that antibody 15906 in bin 3 does not compete with the ravagalimab and iscalimab analogues in bin 1, indicating that these antibodies target distinct epitopes. Conversely, antibodies 15837 and 16029 were shown to bind overlapping epitopes with both 15906 and the ravagalimab and iscalimab analogues.Example 6: Epitope Mapping of Anti-CD40 Antibodies by Mutagenesis and Surface Plasmon Resonance

[0174] Linear and conformational epitopes were characterized using a mutagenesis approach combined with surface plasmon resonance (SPR). This example demonstrates that monoclonal anti-CD40 antibodies 15906, iscalimab analogue, and ravagalimab analogue recognize epitopes distributed at cysteine-rich domains (CRD) CRD1, CRD2 and partially CRD3, of the extracellular domains (ECD) of CD40. The epitopes of the antagonistic antibodies exhibit varying degrees of overlap with the CD40L binding surface with the specific residues depending on the individual antibody.

[0175] The protein sequences of human and rat (Rattus norvegicus) CD40 were downloaded from UniProt (Accession Nos. P25942, Q4QQW2 respectively) and aligned. To map linear epitopes, human CD40 ECD Fc fusion proteins were generated having ten amino acids sequentially exchanged with corresponding rat CD40 sequence in segments overlapping by five amino acids. Conformational epitopes were characterized by alanine scanning mutagenesis of CD40 ECD.

[0176] The cDNA coding for human CD40 ECD was synthesized and cloned into a vector containing CMV promoter and human Ig Fc sequence (residues P101-K330), resulting in fusion of Ig Fc to the CD40 ECD C-terminus. Wild type (wt) and mutated human CD40 Fc fusion constructs were generated by standard gene synthesis techniques and proteins were expressed transiently in an ExpiCHO™ expression system. After harvesting, supernatants were tested for binding to anti-CD40 Fabs by surface plasmon resonance (SPR) using the Carterra® LSA™ Platform (Carterra, USA). An HC200M chip (Carterra, USA) was functionalized with goat anti -human Ig Fc (Southern Biotech) by amine-coupling. The chip was activated with freshly prepared 0.4 M EDC, 0.1 M sulfo-NHS, and 0.1 M MES, pH 5.5 (1 : 1 :1 v / v / v) for 5 minutes, coupled with 75 pg / mL anti-human Ig Fc in 10 mM sodium acetate, pH 4.5, for 10 minutes, and excess reactive esters were quenched for 3 minutes by injection of 1 M ethanolamine, pH 8.5. The instrument was primed in running buffer (PBS pH 7.4, 0.01% Tween 20, 0.5 mg / mL BSA).

[0177] After priming and washing, CD40 fusion proteins in culture supernatants were captured onto individual spots of the chip for 12 minutes as duplicates. Fab analytes were each prepared in running buffer. Kinetic analysis was performed by applying a kinetic titration series of monomeric Fabs as increasing concentrations from 0.8 nM and up to 300 nM. Fab association was performed for 15 minutes, and dissociation was recorded for 15 minutes. After each cycle of Fab injections, the surface was regenerated by 0.45% H3PO4 for 2x20 s and washed for 5 minutes in running buffer.

[0178] Binding responses were analyzed in Carterra’s KIT software tool by referencing to spots of wild-type rat CD40, buffer blanked and aligned to the y-axis. Processed data was fitted to a simple Langmuir 1 : 1 binding model for calculation of the on-rate (konor ka), off- rate (koff or ka) and affinity (KD) constants. Mutations generating inactive proteins common for all Fabs were deselected and a 5-fold affinity reduction compared to the KD of antibodies binding to wild type CD40 was used to define the epitopes.

[0179] The linear and conformational epitopes of anti-CD40 antibody 15906, along with iscalimab analogue and ravagalimab analogues, were mapped to identify key amino acids ofthe CD40 receptor critical for antibody binding (Table 8). Mutations that resulted in significant binding loss (a reduction in affinity by 5-fold or more) or led to unique nonbinding antigens were used to define the epitope of each antibody.Table 8: Summary of Epitopes of 15906 and Ravagalimab and Iscalimab Analogues

[0180] CD40 is a member of the tumor necrosis factor receptor superfamily having four cysteine-rich domains (CRD), each stabilized by 1-2 disulfide bridges. The activation of CD40 signaling occurs upon binding to its ligand, CD40L (CD 154), a soluble protein that exists as a stable trimer. This interaction leads to a signaling complex comprising three CD40 receptors interacting with the trimeric CD40L with an interaction surface distributed along the CRD1-3 of the receptor (An et al., J Biol Chem. (2011) 286(13): 11226-35).

[0181] The epitope recognized by 15906 is primarily located at CRD1 and partially in CRD2 of the CD40 receptor domain most distant from the cell surface. Among the eight conformational epitope residues identified for 15906, K46, E66, R73, E74, T75, and H76 (bolded in Table 8) are part of the interaction interface of the CD40:CD40L complex, explaining the ability of 15906 to block CD40L binding.

[0182] The epitopes of the iscalimab and ravagalimab analogues are both located at CRD2 of CD40. The conformational epitope residues identified in this region are also part of the interaction surface between the CD40 receptor and its ligand, contributing to the antagonistic activity of these analogues. These findings are consistent with epitope binning data, showing that 15906 does not compete with the iscalimab and ravagalimab analogues.

[0183] In summary, epitope mapping analysis shows that 15906 binds to CRD1 and partially to CRD2. Both epitopes overlap with the receptor’s interaction interface with the CD40 ligand, effectively blocking ligand binding. These epitopes are distinct from those recognized by the ravagalimab and iscalimab analogues.Example 7: Test of Activation of Fey Receptors by Anti-CD40 Antibody in FcyR Reporter Assay

[0184] This example describes an in vitro test of activation of Fey receptor signaling by anti-CD40 antibody 15906. Target cells are CD40-expressing Daudi cells. Isotype control IgG1.3f was included as a negative control, and the iscalimab analogue and KPL-404 analogue were also included for comparison.

[0185] NfkB-Luciferase-expressing FcyR reporter cells overexpressing Fcyla, Fcyllb, FcyIIa-H / -R, or FcyIIIa-V / -F were incubated with target cells (CD40-expressing Daudi cells) and anti-CD40 antibodies with a 4-fold titration of the indicated antibodies starting from 166.67 nM. After 16 hours of incubation, activation of Fey receptors was measured by activation of NfkB-Luciferase activity as measured by Bio-Gio™.

[0186] The activation of Fey receptor signaling in reporter cell lines after treatment with anti-CD40 antibody 15906 and target Daudi cells is shown in FIG. 4. Antibody 15906 did not activate any of Fcyla, Fcyllb, FcyIIa-H / -R, and FcyIIIa-V / -F receptors. By contrast, the iscalimab analogue and KPL-404 analogue activated the Fcyla receptor.Example 8: Test of Neutrophil-Induced Antibody-Dependent Cellular Cytotoxicity by Anti-CD40 Antibodies

[0187] This example describes an in vitro test of induction of antibody-dependent cellular cytotoxicity (ADCC) by anti-CD40 antibody 15906. The target cells used were CD40- expressing Daudi cells and the effector cells used were neutrophils isolated from whole blood donors. Isotype control IgG1.3f was included as a negative control. The iscalimab analogue was also included for comparison.

[0188] Neutrophils isolated from whole blood from healthy donors were incubated with target cells (CD40-expressing Daudi cells labeled with51Cr) in a 50: 1 ratio and anti-CD40 antibodies with a 4-fold titration of the indicated antibodies starting from 13.33 nM (donors 1-3) or 166.67 nM (donors 4-6). After 16 hours of incubation, the supernatant was removed and the amount of51Cr released into the medium was measured with scintillation fluid as a measure of cell killing.

[0189] The effects on neutrophil-induced ADCC of CD40-expressing Daudi cells is shown in FIG. 5. Antibody 15906 did not induce neutrophil-based killing of the Daudi cells.Example 9: Assessment of ADCC (PBMC Cell-Mediated Killing) Elicited by Anti-CD40 Antibodies Using CD40-Positive Isolated Primary B Cells as Targets

[0190] This example describes in vitro functional evaluation of several anti-CD40 antibodies (15906, 15387, 16029) in the Fc-effector function attenuated format IgG1.3f, with the purpose of examining antibody-dependent cellular cytotoxicity (ADCC).

[0191] The antibodies were first evaluated for their ability to elicit PBMC -mediated ADCC. For this assay, isolated, primary B cells from healthy human donors with endogenous expression of CD40 were used as target cells. Antibodies representing analogues of anti-CD40 clinical candidates were included for comparison (ravagalimab analogue in the IgGl-LALA-QL format (L234A, L235A, T250Q and M428L Fc attenuating mutations)) and as a positive control (mitazalimab analogue in the IgGl Fc wild type (IgGl-wt) format) to demonstrate an assay window. An IgGl-LALA (L234A, L235A Fc attenuating mutations) antibody targeting an irrelevant antigen was used as a negative control.

[0192] CD19+B cells to be used as target cells were isolated from healthy human donorPBMCs (peripheral blood mononuclear cells) and were labeled with calcein for 60 minutes. PBMCs (from the same donors) as effector cells, together with antibodies, were added to empty wells of a clear bottom 384 well plate. Calcein loaded cells were added to wells at a 1 :20 (target: effector) ratio and plates incubated for 90 minutes. Calcein levels in supernatants were measured in a fluorescence plate reader. Specific lysis was calculated by subtracting untreated lysis (calcein-loaded cells and PBMCs added, but no antibody) and normalizing to maximum lysis (Triton X-100 lysis of calcein-loaded B cells). Spontaneous lysis represents lysis of target cells without PBMCs or antibody added.

[0193] ADCC activity is exemplified in FIG. 6 using a representative PBMC donor sample from a cohort of six healthy donors. Substantial killing activity was observed with the mitazalimab analogue in the IgGl-wt format. A small increase of killing was seen with the ravagalimab analogue, suggesting some interaction with Fc-receptors eliciting effector cell activity in spite of the Fc-attenuated IgGl-LALA-QL format. None of anti-CD40 antibodies (15906, 15387, 16029) in the Fc-effector function attenuated format IgG1.3f induced additional killing above the background levels observed with a negative control IgGl-LALA antibody.Example 10: Functional Activity of Anti-CD40 Antibody in CD40 Reporter Cell Assay as Test of Agonistic Activity

[0194] This example describes an in vitro functional evaluation of anti-CD40 antibody 15906 with the purpose of demonstrating a lack of agonistic activity. The antibody was evaluated for its ability to activate a CD40 reporter cell line. A CD40 agonist antibody, 16040, was included as a positive control. Isotype control IgG1.3F was included as a negative control.

[0195] The CD40 reporter cell line NfKB-LUC2P-U2OS was incubated with anti-CD40 antibodies with a 4-fold titration of the indicated antibodies starting from 50 pg / mL. After 4 hours of incubation with IL-21 and CD40L, the proliferation of the reporter cell line was measured by use of Bio-Gio™. Proliferation was calculated by normalizing to untreated samples.

[0196] The activation of the CD40 reporter cell line after treatment with anti-CD40 antibody 15906, is shown in FIG. 7. 15906 antibody did not activate the CD40 reporter cell line and did not demonstrate agonistic activity.Example 11: Functional Activity of Anti-CD40 Antibodies in B Cell Proliferation Assay as Test of Agonistic Activity

[0197] This example describes in vitro functional evaluation of anti-CD40 antibody 15906 to demonstrate a lack of agonistic activity. The antibody was evaluated for its ability to activate primary B cells. Iscalimab and KPL-404 analogues were included for comparison. Isotype control IgG1.3f was included as a negative control.

[0198] Primary B cells were isolated from blood from healthy donors and incubated with anti-CD40 antibodies with a 6-fold titration of the indicated antibodies starting from 100 pg / mL. After 4 days of incubation with IL-21, the proliferation of B cells was measured using CellTiter-Glo®. Proliferation was calculated by normalizing to untreated controls.

[0199] The induction of B cell proliferation after treatment with anti-CD40 antibodies is shown in FIG. 8. Anti-CD40 antibody 15906 did not induce B cell proliferation and lacked agonistic activity.Example 12: Functional Activity of Anti-CD40 Antibodies in Dendritic Cell Activation Assay as Test of Agonistic Activity

[0200] This example describes in vitro functional evaluation of anti-CD40 antibody 15906 to demonstrate a lack of agonistic activity. The antibody was evaluated for its abilityto activate dendritic cells (DCs). Selicrelumab analogue was included as a positive control. Isotype control IgG1.3f was included as a negative control.

[0201] Dendritic cells (DCs) were differentiated from CD14+monocytes by 8 days of culture with 20 ng / mL GM-CSF and 20 ng / mL IL-4. The DCs were activated by stimulating CD40 with anti-CD40 antibodies. After 48 hours of culture, DC activation was determined by harvesting supernatants and measuring IL-12p40 levels with ELISA.

[0202] The activation of DCs by anti-CD40 antibody 15906, is shown in FIG. 9.Antibody 15906 did not activate DCs upon treatment, which demonstrates lack of agonistic activity.Example 13: Functional Activity of Anti-CD40 Antibodies in a CD40 Reporter CellAssay

[0203] This example describes in vitro functional evaluation of anti-CD40 antibody 15906 with the purpose of demonstrating dose-dependent antagonistic activity. The antibody was evaluated for its ability to block proliferation of a CD40 reporter cell line. Iscalimab and KPL-404 analogues were included for comparison. Isotype control IgG1.3f was included as a negative control.

[0204] The CD40 reporter cell line NfKB-LUC2P-U2OS was incubated with anti-CD40 antibodies with a 4-fold titration of the indicated antibodies starting from 50 pg / mL. After 4 hours of incubation with IL-21 and CD40L, the proliferation of the reporter cell line was measured by use of Bio-Gio™. Proliferation was calculated by normalizing to untreated controls.

[0205] The inhibition of proliferation of the CD40 reporter cell line after treatment with anti-CD40 antibodies is shown in FIG. 10. 15906 inhibited proliferation of the CD40 reporter cell line in a dose-dependent manner. 15906 showed superior activity compared to the KPL-404 analogue and equal activity as compared to the iscalimab analogue.Example 14: Functional Activity of Anti-CD40 Antibodies in B cell Proliferation Assay

[0206] This example describes in vitro functional evaluation of anti-CD40 antibodies (15906, 15837, 15825, 15834, 15949, 16071, 16344 and 16029) with the purpose of demonstrating dose-dependent antagonistic activity. The antibodies were evaluated for theirability to block proliferation of primary B cells from healthy donors. The ravagalimab analogue was included for comparison.

[0207] Primary B cells were isolated from blood from healthy donors and incubated with anti-CD40 antibodies with a 2-fold titration of the indicated antibodies starting from 50 pg / mL. After 4 days of incubation with IL-21 and CD40L, the proliferation of B cells was measured by use of CellTiter-Glo®. Proliferation was calculated by normalizing to untreated controls.

[0208] The inhibition of B cell proliferation after treatment with anti-CD40 antibodies and the ravagalimab analogue is shown in FIG. 11. All anti-CD40 antibodies inhibited the proliferation of B cells in a dose dependent manner upon four days of culture.Example 15: Functional Activity of Anti-CD40 Antibody in Plasma Cell Differentation Assay

[0209] This example describes in vitro functional evaluation of anti-CD40 antibody 15906 with the purpose of demonstrating dose-dependent antagonistic activity. The antibody was evaluated for its ability to block plasma cell differentiation of B cells in PBMCs from healthy donors. The isotype control, IgG1.3f was included as a negative control. Iscalimab analogue and BMS986325 analogue were included for comparison.

[0210] PBMCs were isolated from blood from healthy donors and incubated with CD40L and IL-21 and 25 pg / mL of antibody 15906. After 4 days of culture, the differentiation of plasma cells was measured by use of FluoroSpot. PVDF filter 96-well plates were coated with anti-IgG or IgA monoclonal antibodies (mAbs) and 5xl03cells were transferred to wells and incubated overnight. The next day, plates were developed by adding fluorophore- conjugated detection mAbs to generate fluorescent spots on the bottom of the plates (each representing an IgG or IgA secreting plasma cell). Each concentration was assayed in either duplicate or triplicate.

[0211] PBMCs were isolated from blood from healthy donors and incubated with CD40L, IL-21, IL-4 and a 5-fold titration of the indicated antibodies starting from 50 pg / mL. After 5 days of culture, the differentiation of plasma cells was measured by use of ELISPOT. PVDF filter 96-well plates were coated with anti-IgE mAbs and 5xl05cells were transferred to wells and incubated overnight. The next day, plates were developed by adding biotinylated anti-IgE mAbs and streptavidin-HRP to generate dark spots on the bottom of theplates (each representing an IgE secreting plasma cell). Each concentration was assayed in duplicate.

[0212] The blocking of plasma cell differentiation after treatment with anti-CD40 antibodies is shown in FIG. 12 wherein 15906 blocked differentiation of IgG-secreting plasma cells (Panel A) and IgE-secreting plasma cells (Panel B) in a dose-dependent manner.Example 16: Functional Activity of Anti-CD40 Antibody in T Cell-Dependent B Cell Activation Assay

[0213] This example describes in vitro functional evaluation of anti-CD40 antibody 15906 with the purpose of demonstrating dose-dependent antagonistic activity. The antibody was evaluated for its ability to block T cell-dependent B cell activation in PBMCs from healthy donors. Isotype control IgG1.3f was included as a negative control.

[0214] PBMCs were isolated from blood from healthy donors and incubated with antibody 15906 with a 4-fold titration of the indicated antibodies starting from 25 pg / mL. After 18 hours of incubation with IL-21, the activation of B cells was measured by use of flow cytometry. The presence of activation markers CD25, CD69, and CD86 on B cells was analyzed.

[0215] The blocking of T cell-dependent B cell activation after treatment with antibody 15906 is shown in FIG. 13. 15906 blocked upregulation of CD25, CD69 and CD86 in a dose-dependent manner. As expected, the isotype control showed no effect.Example 17: Single Dose PK of Anti-CD40 Antibody in hFcRn Mice

[0216] This example shows the single dose PK of antibody 15906 administered intravenously to hFcRn transgenic mice.

[0217] Female hFcRn transgenic mice (n=28, Biocytogen, China) received 0.1 mg / kg, 1 mg / kg or 10 mg / kg of antibody 15906 via intravenous (IV) or intraperitoneal (IP) routes. Plasma samples were collected at specific time points post-dose and analysed using an automated affinity flow-through assay on the GyroLab xP platform. Biotinylated capture reagent (human CD40) was bound to streptavidin-coated beads which were pre-packed in microstructure columns of specially designed compact discs. Plasma samples were automatically transferred to these microstructures and bound antibody 15906 was detected using AlexaFluor labeled antibody (goat anti-human IgG antibody). Standards, quality controls (QCs), and diluted samples were analyzed in a final concentration of 10% matrix.The bioanalytical method was qualified for its intended purpose. The lower limit of quantification (LLOQ) was 3.8 ng / mL in neat matrix.

[0218] The initial antibody / drug level was a bit lower than the expected level for all doses with acceptable elimination (T% at 243 hours). The elimination profiles were linear between the doses as shown in FIG. 14. A single animal of the 10 mg / kg IV group had continuously lower exposure than the other mice of the group. This is expected to be an administration error. No animals had a PK profile indicating ADA, and ADA was therefore not assessed.Example 18: Evaluation of Anti-KLH IgG and IgM Production in KLH Mouse Model with Antibody 15906

[0219] As shown in FIG. 15, human CD40 knock-in mice (Biocytogen) were subcutaneously injected at Day 0 and Day 7 with keyhole limpet haemocyanin (KLH) emulsified in TiterMax Gold Adjuvant. Initiated on day 0, the mice were treated three times weekly with a total of 6 treatments by intraperitoneal injection of vehicle buffer or different doses (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg) of antibody 15906. Blood was collected at day 7, day 10 and day 14 post KLH injection and processed into plasma. Anti- KLH IgG and IgM quantification were performed with the ELISA assay IgG (KLHG-1) and IgM (KLHM-1) of Life Diagnostics. Splenocytes were analyzed by FACS for presence of CD19+ B cells, and percentage of human CD40+ or CD86+ on CD19+ B cells. At day 14, soluble CD40 (sCD40) was quantified in mouse plasma with the use of the immunoassay kit R-PLEX Human CD40 / TNFRSF5 Assay.

[0220] Human CD40 KI mice responded to antibody 15906 in a dose-dependent manner. Antibody 15906 dosed with 1 mg / kg and 3 mg / kg controlled the anti -KLH IgM production as effectively as 10 mg / kg antibody 15906 as shown in FIG. 16. During the treatment period a pronounced and dose-dependent inhibition of anti-KLH IgG was observed for 1 mg / kg, 3 mg / kg and 10 mg / kg as shown in FIG. 17.

[0221] No inhibition of anti-KLH IgG and IgM production was observed with the lowest dose at 0.1 mg / kg and 0.3 mg / kg antibody 15906 compared to vehicle-treated control group.

[0222] As shown in FIG. 15, splenocyte harvest at termination day 14, antibody 15906 induced a significant decrease of CD40 and CD86 expression on B cells as shown in FIG. 18, as well as a dose dependent increase of sCD40 in plasma as shown in FIG. 19.Example 19: Evaluation of Antibody 15906 and Competitors in KLH Mouse Model

[0223] As shown in FIG. 20, human CD40 knock-in mice (Biocytogen) were subcutaneously injected at day 0 and day 7 with KLH emulsified in TiterMax Gold Adjuvant. Treatment with antibody 15906, KPL-404 analogue or BMS986325 analogue at different doses (0.3 mg / kg,l mg / kg, and 3 mg / kg) or vehicle buffer were initiated at day 0 and performed three times weekly for three weeks. Blood sampling was collected at day -7, day 7, day 14, and day 20 and processed into plasma. Anti -KLH IgG and IgM quantification were performed with the ELISA assay IgG (KLHG-1) and IgM (KLHM-1) of Life Diagnostics. sCD40 was quantified in mouse plasma with a non-competitive assay for antibody 15906 with the use of the immunoassay kit R-PLEX Human CD40 / TNFRSF5 Assay. Levels of treatment antibodies in plasma at day 7, day 14 and day 20 were quantified.

[0224] As shown in FIG. 21 and FIG. 22, antibody 15906, KPL-404 analogue and BMS986325 analogue have similar effect at 1 and 3 mg / kg to reduce anti-KLH IgM and IgG production compared to vehicle buffer. No effect was observed at 0.3 mg / kg for neither antibody 15906, KPL-404 analogue nor BMS986325 analogue.

[0225] Plasma levels of antibody 15906 were dose-dependent with a profile similar to KPL-404 analogue, while there seems to an indication of lower levels of BMS986325 analogue at 1 mg / kg and 3 mg / kg compared to antibody 15906 and KPL-404 analogue as shown in FIG. 23.

[0226] Antibody 15906, KPL-404 and BMS986325 displayed a dose-dependent increase of sCD40 levels in the blood, with antibody 15906 showing the highest levels of sCD40 as shown in FIG. 24, FIG. 25 and FIG. 26.Example 20: Evaluation of Antibody 15906 in KLH Model with Delayed Treatment Onset

[0227] As shown in FIG. 27, human CD40 knock-in mice (Biocytogen) were subcutaneously injected at day 0 and day 7 with KLH. Treatment with vehicle buffer or antibody 15906 using different doses (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg) were initiated on day 7 and performed three times weekly for three weeks. Blood sampling was performed at, day 6, day 14, day 21 and day 27 and process into plasma. Anti-KLH IgG and IgM quantification were performed with the ELISA assay IgG (KLHG-1) and IgM (KLHM-1) of Life Diagnostics. sCD40 was quantified in mouse plasma collected on day 27 with a non-competitive assay for antibody 15906 with the use of the immunoassay kit R- PLEX Human CD40 / TNFRSF5 Assay. At sacrifice spleen was collected and splenocytes analyzed percentage of human CD40+ on CD 19+ B cells.

[0228] Challenging antibody 15906 with a delayed treatment schedule compared to Examples 18 and 19, still revealed a strong effect of antibody 15906 to reduce anti-KLH IgG and IgM in plasma. While the lowest dose of antibody 15906 (0.1 mg / kg and 0.3 mg / kg) showed limited effect compared to vehicle treated mice, antibody 15906 at 1 mg / kg, 3 mg / kg and 10 mg / kg showed a dose dependent inhibition of anti KLH IgG and IgM production as shown in FIG. 28 and FIG. 29. In parallel, a dose dependent increase of sCD40 in plasma and decrease of CD40 expression on B cells from splenocyte was observed as shown in respectively FIG. 30 and FIG. 31.

Claims

What is claimed is:

1. An isolated anti-CD40 antibody or an antigen-binding portion thereof, wherein the antibody binds specifically to amino acids 41-55 and / or amino acids 61-75 of SEQ ID NO: 97.

2. The anti-CD40 antibody or antigen-binding portion of claim 1, wherein the antibody further binds specifically to amino acids 76, 78 or both of SEQ ID NO: 97.

3. An isolated anti-CD40 antibody or antigen-binding portion thereof, wherein the antibody binds specifically to one or more of amino acids 46, 66, 73, 74, 75 and 76 of SEQ ID NO: 97, optionally wherein said antibody binds specifically to all of said amino acids.

4. The anti-CD40 antibody or antigen-binding portion of claim 3, wherein the antibody further binds specifically to amino acid 50, 78, or both, of SEQ ID NO: 97.

5. An isolated anti-CD40 antibody or an antigen-binding portion thereof, wherein the antibody binds to the same epitope as a reference antibody having a heavy chain variable domain (VH) and a light chain variable domain (VL) comprisingSEQ ID NOs: 1 and 2, respectively,SEQ ID NOs: 11 and 12, respectively,SEQ ID NOs: 21 and 22, respectively,SEQ ID NOs: 31 and 32, respectively,SEQ ID NOs: 41 and 42, respectively,SEQ ID NOs: 51 and 52, respectively,SEQ ID NOs: 61 and 62, respectively, orSEQ ID NOs: 71 and 72, respectively.

6. The anti-CD40 antibody or antigen-binding portion of claim 5, wherein a) the heavy chain of the anti-CD40 antibody comprises: i) heavy chain complementarity determining regions (H-CDR) 1-3 comprising SEQ ID NOs: 3-5, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1;57iii) a VH comprising SEQ ID NO: 1; or iv) SEQ ID NO: 1 and any one of SEQ ID NOs: 81-83; and b) the light chain of the anti-CD40 antibody comprises: i) light chain complementarity determining regions (L-CDR) 1-3 comprising SEQ ID NOs: 6-8, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2; iii) a VL comprising SEQ ID NO: 2; or iv) SEQ ID NOs: 2 and 84.

7. An anti-CD40 antibody or an antigen-binding portion thereof, wherein the antibody comprises H-CDR1-3 and L-CDR1-3 amino acid sequences of: a) SEQ ID NOs: 3-8, respectively; b) SEQ ID NOs: 13-18, respectively; c) SEQ ID NOs: 23-28, respectively; d) SEQ ID NOs: 33-38, respectively; e) SEQ ID NOs: 43-48, respectively; f) SEQ ID NOs: 53-58, respectively; g) SEQ ID NOs: 63-68, respectively; or h) SEQ ID NOs: 73-78, respectively.

8. The anti-CD40 antibody or antigen-binding portion of claim 7, wherein the antibody comprises a heavy chain variable domain (VH) amino acid sequence and a light chain variable domain (VL) amino acid sequence that are at least 90% identical to the amino acid sequences of: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively; c) SEQ ID NOs: 21 and 22, respectively; d) SEQ ID NOs: 31 and 32, respectively; e) SEQ ID NOs: 41 and 42, respectively; f) SEQ ID NOs: 51 and 52, respectively; g) SEQ ID NOs: 61 and 62, respectively; or h) SEQ ID NOs: 71 and 72, respectively.

9. The anti-CD40 antibody or antigen-binding portion of claim 7, wherein the antibody comprises a VH and a VL comprising: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 11 and 12, respectively; c) SEQ ID NOs: 21 and 22, respectively; d) SEQ ID NOs: 31 and 32, respectively; e) SEQ ID NOs: 41 and 42, respectively; f) SEQ ID NOs: 51 and 52, respectively; g) SEQ ID NOs: 61 and 62, respectively; or h) SEQ ID NOs: 71 and 72, respectively.

10. The anti-CD40 antibody of any one of claims 1-9, wherein the antibody is of isotype subtype IgGi.

11. The anti-CD40 antibody of claim 10, wherein the antibody comprises a mutant Fc domain with reduced binding affinity for an Fc gamma receptor.

12. The anti-CD40 antibody of claim 11, wherein the mutant Fc domain has a) mutations L234A and L235A (Eu numbering), or b) mutations L234A, L235E, and G237A (Eu numbering).

13. The anti-CD40 antibody of any one of claims 1-9, wherein the antibody comprises a heavy chain constant region comprising SEQ ID NO: 82 or 83.

14. An anti-CD40 antibody that comprises: a) a heavy chain (HC) comprising SEQ ID NOs: 1 and 83, and a light chain (LC) comprising SEQ ID NOs: 2 and 84; b) an HC comprising SEQ ID NOs: 11 and 83, and an LC comprising SEQ ID NOs: 12 and 84; c) an HC comprising SEQ ID NOs: 21 and 83, and an LC comprising SEQ ID NOs: 22 and 84; d) an HC comprising SEQ ID NOs: 31 and 83, and an LC comprising SEQ ID NOs: 32 and 84; e) an HC comprising SEQ ID NOs: 41 and 83, and an LC comprising SEQ ID NOs: 42and 84; f) an HC comprising SEQ ID NOs: 51 and 83, and an LC comprising SEQ ID NOs: 52 and 84; g) an HC comprising SEQ ID NOs: 61 and 83, and an LC comprising SEQ ID NOs: 62 and 84; or h) an HC comprising SEQ ID NOs: 71 and 83, and an LC comprising SEQ ID NOs: 72 and 84.

15. A pharmaceutical composition comprising the anti-CD40 antibody or antigen-binding portion of any one of claims 1-14 and a pharmaceutically acceptable excipient.

16. Isolated nucleic acid molecule(s) comprising a nucleotide sequence that encodes the heavy chain sequence, and a nucleotide sequence that encodes the light chain sequence, of the anti-CD40 antibody or antigen-binding portion of any one of claims 1-14.

17. The isolated nucleic acid molecule(s) of claim 16, wherein the nucleic acid molecule(s) comprise any one of SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, and 80.

18. Vector(s) comprising the isolated nucleic acid molecule(s) of claim 16 or 17, wherein the vector(s) further comprise an expression control sequence.

19. A host cell comprising a nucleotide sequence that encodes the heavy chain sequence, and a nucleotide sequence that encodes the light chain sequence, of the anti-CD40 antibody or antigen-binding portion of any one of claims 1-14.

20. A method for producing an anti-CD40 antibody or an antigen-binding portion thereof, comprising providing a host cell of claim 19, culturing said host cell under conditions suitable for expression of the antibody or antigen-binding portion, and isolating the resulting antibody or antigen-binding portion.

21. A method of treating an autoimmune or inflammatory condition in a patient in needthereof, comprising administering to the patient a therapeutically effective amount of the anti- CD40 antibody or antigen-binding portion of any one of claims 1-14 or the pharmaceutical composition of claim 15.

22. The anti-CD40 antibody or antigen-binding portion of any one of claims 1-14, or the pharmaceutical composition of claim 15, for use in treating an autoimmune or inflammatory condition in a patient in need thereof.

23. Use of the anti-CD40 antibody or antigen-binding portion of any one of claims 1-14, or the pharmaceutical composition of claim 15, in the manufacture of a medicament for treating an autoimmune or inflammatory condition in a patient in need thereof.

24. The method of claim 21, the anti-CD40 antibody or antigen-binding portion or the pharmaceutical composition for use of claim 22, or the use of claim 23, wherein the condition is selected from the group consisting of asthma, allergic asthma, chronic spontaneous urticaria (CSU), diabetes (optionally type 1 diabetes or latent autoimmune diabetes), lupus (optionally systemic lupus erythematosus or lupus nephritis), arthritis (optionally rheumatoid arthritis), allergy, antibody-mediated rejection, organ graft rejection, graft-versus-host disease (GvHD), Addison’s disease, ankylosing spondylitis, anti -glomerular basement membrane disease, autoimmune hepatitis, celiac disease, autoimmune alopecia, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, juvenile myositis, Kawasaki disease, inflammatory bowel disease (optionally Crohn’s disease or ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, pemphigus vulgaris, thrombocytopenic purpura, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, vitiligo, and Vogt-Koyanagi-Harada Disease.

25. The method of claim 21, the anti-CD40 antibody or antigen-binding portion or the pharmaceutical composition for use of claim 22, or the use of claim 23, wherein the condition is chronic spontaneous urticaria, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Sjogren’s syndrome, lupus (optionally systemic lupus erythematosus or lupus nephritis), arthritis (optionally rheumatoid arthritis), Graves’ disease, inflammatory bowel disease(optionally Crohn’s disease or ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, or vitiligo.

26. The method of claim 21, the anti-CD40 antibody or antigen-binding portion or the pharmaceutical composition for use of claim 22, or the use of claim 23, wherein the condition is chronic spontaneous urticaria, atopic dermatitis, or pemphigus vulgaris.

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