Antagonistic CD40 antibodies

Abstract

The disclosure features antagonistic CD40 polypeptides, such as antibodies and antigen-binding fragments thereof, and the use of these polypeptides to inhibit the downstream signaling of CD40. The antibodies and antigen-binding fragments thereof can be used to treat a wide variety of cancers, autoimmune disorders, neurological disorders, infectious diseases, and inflammatory diseases.

Description

[0001] ANTAGONISTIC CD40 ANTIBODIES

[0002] Sequence Listing

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 4, 2024, is named “00786-589WO2_Sequence_Listing_12_4_24” and is 68,486 bytes in size.

[0004] Background

[0005] Maintaining control of the cell-mediated and humoral immune responses is an important facet of healthy immune system activity. The aberrant regulation of lymphocyte driven immune responses (e.g., T cell and B cell driven immune reactions) has been associated with a wide array of human diseases, as the inappropriate mounting of an immune response against various self and foreign antigens plays a causal role in such pathologies as autoimmune disorders, asthma, allergic reactions, graft-versus-host disease, transplantation graft rejection, and a variety of other immunological disorders. These diseases are mediated by, e.g., T and B lymphocytes that exhibit reactivity against “self” antigens and those derived from non-threatening sources, such as allergens or transplantation allografts.

[0006] An important regulator of cell-mediated and humoral immune responses is CD40. CD40 is a 48 kDa type I integral membrane glycoprotein and a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF). CD40 belongs to the family of TRAF-binding receptors, which are TNFRSF members that contain motifs with four to six amino acids called TRAF-interacting motifs (TIMs) which recruit TRAF proteins. As mediators of CD40 signaling, TRAF proteins are adaptor molecules that activate multiple downstream signaling pathways such as, e.g., NFKB, Janus kinase (JAK), ERK, p38MAPK, and PI3K that help in cell survival, proliferation, and cytokine production (see Sonar et al. Front. Immunol. 6:364, 2015; which is incorporated herein by reference in its entirety). CD40 is expressed on a variety of cell types, including normal and neoplastic immune cells and tumor cells.

[0007] The ligand for CD40 is CD40L (also referred to as CD154, gp39, and TRAP) and is a member of the TNF superfamily. CD40L is a transmembrane protein that is predominantly expressed on activated CD4 + T cells and expressed on a small subset of CD8 + T cells (see, e.g., Van Kooten and Banchereau, J. Leukoc. Biol. 67: 2-17, 2000). Structural studies of unliganded CD40 have revealed CD40 exhibits an anti-parallel dimer conformation, which changes into a trimeric conformation upon binding with CD40L (Smulski et al. J. Biol. Chem. 288:10914, 2013, the disclosure of which is incorporated herein by reference in its entirety).

[0008] The interaction of CD40 and CD40L induces both humoral and cellular immune responses. For example, CD40 regulates this ligand-receptor pair and activates other antigen- presenting cells (APC), including B cells and dendritic cells (DC) (Toubi and Shoenfeld, Autoimmunity 37: 457-464, 2004). Activation of CD40 is one of the critical signals that allows full maturation of DCs into efficient APCs that drive T cell activation. Studies in mouse models have shown that CD40 signaling on dendritic cells also plays an important role in the generation of Th17 cells, underlying diseases such as arthritis and multiple sclerosis (lezzi et al. Proc. Natl. Acad. Sci. USA 106: 876-881 , 2009). Administration of an antagonistic anti-CD40L antibody in mice has been shown to be beneficial in several models of autoimmune diseases (Toubi and Shoenfeld, Autoimmunity 37: 457-464, 2004). Furthermore, antagonistic antibodies directed against CD40 have shown beneficial effects in inflammatory disease models (Laman et al. Eur. J. Immunol. 32: 2218-2228, 2002).

[0009] Thus, there are sufficient preclinical studies to provide evidence for the critical role of the CD40-CD40L interaction in driving an efficient T cell-dependent immune response. Blocking CD40 signaling is therefore recognized as a promising therapeutic strategy for suppressing pathogenic autoimmune responses in diseases such as rheumatoid arthritis, multiple sclerosis, or psoriasis. There remains a significant need for therapeutic agents that can intervene in the interaction of CD40-CD40L and be used to block CD40 signaling. There also exists a need for therapies leveraging such therapeutic agents that can diminish harmful immune responses while augmenting protective immune responses for use in treatments targeting such diseases as autoimmune disorders, infectious diseases, inflammatory diseases, neurological diseases, and cancer, among others.

[0010] Summary of the Disclosure

[0011] CD40 antagonistic polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) inhibit the activation of human CD40 by binding (e.g., on the exterior surface of a cancer cell that expresses CD40, a myeloid-derived suppressor cell (MDSC), a T cell (e.g., a T-reg cell), a B cell, a monocyte, a neutrophil, a platelet, a granulocyte, a bone marrow derived lymphoid cell, or a parenchymal cell), thereby blocking CD40 from recruiting its cognate or natural ligand (e.g., CD40 ligand (CD40L)). The cognate or natural ligands of CD40 potentiate CD40 signaling by nucleating a trimer of CD40. This trimerization event brings individual CD40 proteins into proximity and initiates signaling. CD40 antagonist polypeptides (e.g., single-chain polypeptides, antibodies, and antibody fragments thereof) can antagonize this interaction by binding to CD40 and promoting the formation of an anti-parallel dimer conformation of CD40. The anti-parallel dimer conformation of CD40, which is stabilized by the binding of a CD40 antagonist polypeptide of the disclosure, reduces or inhibits binding of CD40 to the cognate or natural CD40 ligand and the formation of the active trimer conformation. Additionally, the disclosure features pharmaceutical compositions of the antagonistic polypeptides, as well as methods of treatment for a subject diagnosed with cancer, autoimmune disease, infectious disease, or inflammatory disease.

[0012] In a first aspect, the disclosure features an antibody or antigen-binding fragment thereof capable of specifically binding human CD40 and comprising a heavy chain complementarity determining region (CDR) 1 (CDR-H1 ), CDR-H2, and CDR-H3, and a light chain CDR 1 (CDR-L1 ), CDR-L2, and CDR-L3, in which the CDR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 1 -5 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 6-9 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 10-13 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L1 comprises the amino acid sequence of any one of SEQ ID NOs: 14-18 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L2 comprises the amino acid sequence of any one of SEQ ID NOs: 19-22 or a variant thereof with up to two conservative amino acid substitutions, and the CDR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 23-26 or a variant thereof with up to two conservative amino acid substitutions.

[0013] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof with up to two conservative amino acid substitutions, the CDR- L2 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof with up to two conservative amino acid substitutions, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof with up to two conservative amino acid substitutions.

[0014] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1 , the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 14, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 19, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 23.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 27, and a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 28.

[0016] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 27, and the light chain comprises the amino acid sequence of SEQ ID NO: 28.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof does not compete with antibody ABBV-323 for binding to CD40.

[0018] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof with up to two conservative amino acid substitutions, the CDR- L2 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof with up to two conservative amino acid substitutions, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof with up to two conservative amino acid substitutions.

[0019] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 2, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 7, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 11 , the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 15, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 20, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 24.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 29, and a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 30.

[0021] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the light chain comprises the amino acid sequence of SEQ ID NO: 30.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof does not compete with antibody ABBV-323 for binding to CD40.

[0023] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof with up to two conservative amino acid substitutions, the CDR- L2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof with up to two conservative amino acid substitutions, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof with up to two conservative amino acid substitutions.

[0024] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 3, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 8, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 12, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 16), the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 21 , and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 25.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 31 , and a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 32.

[0026] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 31 , and the light chain comprises the amino acid sequence of SEQ ID NO: 32.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof competes with antibody ABBV-323 for binding to CD40. In some embodiments, the antibody or antigen-binding fragment thereof competes with antibody ABBV-323 for binding to CD40 at an epitope within the N- terminal peptide of CD40.

[0028] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof with up to two conservative amino acid substitutions, the CDR- L2 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof with up to two conservative amino acid substitutions, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof with up to two conservative amino acid substitutions.

[0029] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13, the CDR-L1 comprises the amino acid sequence of SEQ ID NO:

[0030] 17, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 33, and a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 34.

[0032] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34.

[0033] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof with up to two conservative amino acid substitutions, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof with up to two conservative amino acid substitutions, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof with up to two conservative amino acid substitutions, the CDR- L2 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof with up to two conservative amino acid substitutions, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof with up to two conservative amino acid substitutions.

[0034] In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13, the CDR-L1 comprises the amino acid sequence of SEQ ID NO:

[0035] 18, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 35, and a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 36.

[0037] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 35, and the light chain comprises the amino acid sequence of SEQ ID NO: 36.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds an epitope comprising five or more of amino acids 124-132 of SEQ ID NO: 37 and / or five or more of amino acids 143-152 of SEQ ID NO: 37, or an epitope with at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprising the amino acid sequence LLIY (SEQ ID NO: 38) bound to the N- terminus of the CDR-L2.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof inhibits signaling associated with CD40.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof binds CD40 with a Kd of between about 0 and about 10 nM or with a Kd of between about 0.001 pg / ml and about 50 pg / ml. In some embodiments, the antibody or antigen-binding fragment thereof binds CD40 with a Kd of between about 0 and about 1 nM.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof binds CD40 to form an antibody-antigen complex with a kOn of between about 104M-1s-1and about 106M-1s-1.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof binds CD40 to form an antibody-antigen complex, in which the complex dissociates with a kotf of no greater than about 10_3s-1.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a singlechain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem scFv (taFv).

[0045] In some embodiments, the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG isotype. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG 1 , lgG2, lgG3, or lgG4 isotype. In some embodiments, the antibody or antigen-binding fragment thereof is an lgG2 isotype.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region that lacks a cysteine residue at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region that lacks a cysteine residue at position 232 of the amino acid sequence of the lgG2 hinge region. In some embodiments, the antibody or antigenbinding fragment thereof comprises a human lgG2 hinge region that lacks a cysteine residue at position 233 of the amino acid sequence of the lgG2 hinge region.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having an amino acid other than cysteine at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having an amino acid other than cysteine at position 232 of the amino acid sequence of the lgG2 hinge region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having an amino acid other than cysteine at position 233 of the amino acid sequence of the lgG2 hinge region.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having a serine residue at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having a serine residue at position 232 of the amino acid sequence of the lgG2 hinge region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having a serine residue at position 233 of the amino acid sequence of the lgG2 hinge region.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region comprising an amino acid substitution or deletion at one or both of cysteine residues 232 and 233. In some embodiments, the lgG2 hinge region comprises an amino acid substitution at one or both of cysteine residues 232 and 233. In some embodiments, the lgG2 hinge region comprises an amino acid substitution at cysteine residue 232. In some embodiments, the lgG2 hinge region comprises an amino acid substitution at cysteine residue 233. In some embodiments, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the lgG2 hinge region comprises a C232S substitution. In some embodiments, the lgG2 hinge region comprises a C233S substitution.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises antigenbinding sites separated from one another by a distance of at least about 133 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of at least about 134 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of at least about 139 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of at least about 150 A.

[0051] In some embodiments, the antigen-binding sites are separated from one another by a distance of from about 133 A to about 150 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of from about 133 A to about 145 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of from about 133 A to about 139 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of from about 134 A to about 139 A.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof comprises a framework region from a human antibody or chimeric antibody.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof promotes the formation of and / or stabilizes an anti-parallel dimer conformation of CD40.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof destabilizes a trimeric conformation of CD40. In some embodiments, the antibody or antigen-binding fragment thereof reduces secretion of a soluble version of CD40, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the soluble version of CD40.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof inhibits expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11 , TRAF2, TRAF3, relB, and clAP2 / BIRC3, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the one or more genes.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof inhibits NFKB activation, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of NFKB activity, reduces or inhibits the proliferation of a population of T-reg cells, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the proliferation of the population of T-reg cells, induces the proliferation of a population of CD8+ effector T cells, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the proliferation of the population of CD8+ effector T cells, reduces or inhibits the proliferation of a population of cancer cells, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the proliferation of the population of cancer cells, inhibits CD40 signaling in proliferating cells, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of CD40 signaling in proliferating cells, does not inhibit CD40 signaling in resting cells, reduces or inhibits the proliferation of a population of myeloid-derived suppressor cells (MDSCs), e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the proliferation of the population of MDSCs, selectively reduces or inhibits the proliferation of a population of T-reg cells expressing CD25Hi, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the proliferation of the population of T-reg cells expressing CD25Hi, and / or reduces or inhibits the proliferation of a population of T-reg cells in the presence of a ligand for CD40, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the proliferation of the population of T-reg cells in the presence of the ligand for CD40. In some embodiments, the cancer cells express CD40. In some embodiments, the cancer cells are selected from the group consisting of Hodgkin lymphoma cells, cutaneous nonHodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells. In a second aspect, the disclosure features a method of producing an antibody or antigenbinding fragment thereof disclosed herein by expressing a polynucleotide encoding the antibody or antigen-binding fragment thereof in a host cell and recovering the antibody or antigen-binding fragment thereof from host cell medium.

[0058] In a third aspect, the disclosure features a construct comprising a first polypeptide domain and a second polypeptide domain, in which the first polypeptide domain and the second polypeptide domain are each, independently, an antigen-binding fragment described herein.

[0059] In some embodiments, the first polypeptide domain and the second polypeptide domain are bound by a covalent linker. In some embodiments, the covalent linker comprises an amide bond. In some embodiments, the covalent linker comprises a disulfide bond.

[0060] In a fourth aspect, the disclosure features a polynucleotide encoding an antibody or antigenbinding fragment thereof disclosed herein.

[0061] In some embodiments, the polynucleotide comprises a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to any one of SEQ ID NOs: 39-43, and a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to any one of SEQ ID NOs: 44-48.

[0062] In some embodiments, the polynucleotide comprises a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 39 and a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 44.

[0063] In some embodiments, the polynucleotide comprises a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 40 and a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 45.

[0064] In some embodiments, the polynucleotide comprises a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 41 and a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 46.

[0065] In some embodiments, the polynucleotide comprises a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 42 and a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 47.

[0066] In some embodiments, the polynucleotide comprises a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 43 and a sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to SEQ ID NO: 48.

[0067] In a fifth aspect, the disclosure features a polynucleotide encoding a construct disclosed herein.

[0068] In a sixth aspect, the disclosure features a vector comprising a polynucleotide disclosed herein.

[0069] In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a eukaryotic expression vector.

[0070] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenovirus (Ad), retrovirus, poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, and a vaccinia virus. In some embodiments, the adenovirus is a serotype 1 -60 adenovirus. In some embodiments, the adenovirus is a serotype 5, 26, 35, or 48 adenovirus. In some embodiments, the retrovirus is a y-retrovirus or a lentivirus. In some embodiments, the vaccinia virus is a modified vaccinia Ankara (MVA).

[0071] In a seventh aspect, the disclosure features an isolated host cell comprising a vector disclosed herein.

[0072] In some embodiments, the host cell is a prokaryotic cell.

[0073] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a CHO cell.

[0074] In an eighth aspect, the disclosure features a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed in the first aspect, a construct disclosed in the third aspect, a polynucleotide disclosed in the fourth or the fifth aspect, a vector disclosed in the sixth aspect, or a host cell disclosed in the seventh aspect, and a pharmaceutically acceptable carrier or excipient.

[0075] In some embodiments, the pharmaceutical composition comprises the antibody or antigen biding fragment thereof disclosed herein.

[0076] In some embodiments, at least 50% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, at least 75% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, at least 85% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, at least 90% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform. In some embodiments, at least 95% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

[0077] In some embodiments, from about 75% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, from about 80% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, from about 85% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, from about 90% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform. In some embodiments, about 95% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof yields a single detectable band upon gel electrophoresis analysis performed under non-reducing conditions.

[0079] In some embodiments, the single disulfide-bonded isoform is lgG2-A. In some embodiments, the antibody or antigen-binding fragment thereof is present in the pharmaceutical composition in an amount of from about 0.001 mg / ml to about 100 mg / ml. In some embodiments, the pharmaceutical composition is in an amount of from about 0.001 ml to about 1000 ml (e.g., about 1 -10 mL, such as about 2 mL).

[0080] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunotherapy agent described herein. In some embodiments, the additional therapeutic agent is a chemotherapy agent described herein.

[0081] In a ninth aspect, the disclosure features a method of modulating an immune response in a human subject by administering an antibody or antigen-binding fragment thereof disclosed in the first aspect, a construct disclosed in the third aspect, a polynucleotide disclosed in the fourth or the fifth aspect, a vector disclosed in the sixth aspect, a host cell disclosed in the seventh aspect, or a pharmaceutical composition disclosed in the eighth aspect, in which the antibody or antigen-binding fragment thereof is an antagonist of CD40.

[0082] In some embodiments, the method inhibits an immune response mediated by a B cell or CD8+ T cell in the human subject.

[0083] In some embodiments, the method treats an autoimmune disease in the human subject.

[0084] In some embodiments, the autoimmune disease is selected from the group consisting of Type I diabetes, alopecia areata, ankylosing spondylitis, anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behget’s disease, bullous pemphigoid, cardiomyopathy, celiac spruedermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, limited scleroderma (CREST syndrome), cold agglutinin disease, Crohn’s disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hypothyroidism, inflammatory bowel disease, autoimmune lymphoproliferative syndrome (ALPS), idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy (Berger’s Disease), juvenile arthritis, lichen planus, lupus, lupus nephritis, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud’s phenomenon, Reiter’s syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, Stiff-Man syndrome, Takayasu’s arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener’s granulomatosis, Goodpasture syndrome, transplant rejection, celiac disease, esophagitis, and antibody-mediated inflammatory central nervous system disorders.

[0085] In some embodiments, the method inhibits an immune response mediated by a T-reg cell in the human subject, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a reference level of the immune response mediated by the T-reg cell in the human subject. In some embodiments, the immune response mediated by the T-reg cell in the human subject is selected from the group consisting of secretion of immune inhibitory cytokines (e.g., IL-10, IL-35, and TGF-p), inhibition of T effector cells, inhibition of natural killer (NK) cells, inhibition of B cells, inhibition of monocyte differentiation into M1 -like phenotype, and promotion of monocyte differentiation into M2- like phenotype.

[0086] In some embodiments, the method treats an infectious disease in the human subject. In some embodiments, the infectious disease is caused by one or more agents selected from the group consisting of a virus, a bacterium, a fungus, and a parasite.

[0087] In some embodiments, the infectious disease is caused by a virus selected from the group consisting of hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, llheus virus, Israel turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus, Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, Lujo virus, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O’nyong’nyong virus, chikungunya virus, smallpox virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster virus, Kaposi’s sarcoma associated-herpesvirus (KSHV), influenza virus, severe acute respiratory syndrome (SARS) virus, rabies virus, vesicular stomatitis virus (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza virus (e.g., 1 , 2, 3, and 4), rhinovirus, mumps virus, poliovirus, human enterovirus (e.g., A, B, C, and D), hepatitis A virus, coxsackievirus, hepatitis B virus, human papilloma virus, adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus, Norwalk virus, rotavirus, human immunodeficiency virus (HIV), and human T-lymphotropic virus Types I and II.

[0088] In some embodiments, the infectious disease is caused by a bacterium belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

[0089] In some embodiments, the infectious disease is caused by a fungus belonging to a genus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia.

[0090] In some embodiments, the infectious disease is caused by a parasite selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Exemplary helminthic parasites include richuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, and Dracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes, Paragonimus westermani, Taenia solium, Taenia saginata, Hymenolepis nana, and Echinococcus granulosus.

[0091] In some embodiments, the method treats an inflammatory disease in the human subject.

[0092] In some embodiments, the inflammatory disease is selected from the group consisting of acute or chronic inflammation, cardiac fibrosis, lung fibrosis, osteoarthritis, rheumatoid arthritis, atherosclerosis, type I diabetes, type II diabetes, graft-versus-host disease, multiple sclerosis, osteomyelitis, psoriasis, Crohn’s disease, Sjogren’s syndrome, lupus erythematosus, and ulcerative colitis.

[0093] In some embodiments, the method treats a neurological disease or disorder in the human subject.

[0094] In some embodiments, the neurological disease or disorder is selected from the group consisting of a brain tumor, a brain metastasis, a brain injury, a spinal cord injury, a nerve injury, schizophrenia, epilepsy, Parkinson’s disease, autism, Huntington’s disease, stroke, Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and myasthenia gravis.

[0095] In some embodiments, prior to the administration of the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, the human subject is treated with an immunomodulatory medication, a non-steroidal anti-inflammatory drug (NSAID), acetaminophen, and / or kidney dialysis. In some embodiments, the immunomodulatory medication is a steroid. In some embodiments, the NSAID is aspirin.

[0096] In some embodiments, one or more doses of the antibody or antigen-binding fragment thereof is administered to the human subject in one or more treatment periods. In some embodiments, each dose of the antibody or antigen-binding fragment thereof comprises 10 mg, 20 mg, 40 mg, 80 mg, or 160 mg of the antibody or antigen-binding fragment thereof. In some embodiments, each treatment period lasts one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or more. In some embodiments, within each treatment period, the one or more doses of the antibody or antigen-binding fragment thereof is administered to the human subject once every week or once every two weeks.

[0097] In some embodiments, the method further comprises:

[0098] (a) determining (e.g., measuring) a level of soluble CD40 in the serum of the human subject before the human subject is administered the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, and

[0099] (b) determining (e.g., measuring) a reference level of soluble CD40 in the serum of a healthy human or comparing the level of soluble CD40 in the serum of the human subject to a predetermined reference level, in which the human subject has a level of soluble CD40 that is higher relative to a reference level of soluble CD40.

[0100] In some embodiments, the method further comprises determining (e.g., measuring) a level of IgG in the serum of the human subject before the human subject is administered the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition. In some embodiments, the human subject has a level of IgG that is higher than 16 g / L and the method comprises administering the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition to the subject. In other embodiments, the level of IgG is determined (e.g., measured) prior to administration of the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition to the subject.

[0101] In some embodiments, the method further comprises determining (e.g., measuring) a level of IgM in the serum of the human subject before the human subject is administered the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, in which the human subject has a level of IgG that is higher than 2.5 g / L.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition is administered to the human subject intravenously or subcutaneously.

[0103] In a tenth aspect, the disclosure features a kit comprising an agent selected from the group consisting of an antibody or antigen-binding fragment thereof disclosed herein, a construct disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0104] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof disclosed herein.

[0105] In some embodiments, the kit comprises a construct disclosed herein.

[0106] In some embodiments, the kit comprises a polynucleotide disclosed herein.

[0107] In some embodiments, the kit comprises a vector disclosed herein. In some embodiments, the kit further comprises instructions for transfecting the vector into a host cell. In some embodiments, the kit further comprises instructions for expressing the antibody, antigen-binding fragment thereof, or construct in the host cell. In some embodiments, the kit comprises a host cell disclosed herein. In some embodiments, the kit further comprises a reagent that can be used to express the antibody, antigen-binding fragment thereof, or construct in the host cell.

[0108] In some embodiments, the kit comprises a pharmaceutical composition disclosed herein.

[0109] In some embodiments, the kit further comprises instructions for administering the agent to a human subject.

[0110] In some embodiments, the kit further comprises instructions for making or using the agent.

[0111] In some embodiments, the kit further comprises instructions for measuring a level of soluble CD40 in a subject.

[0112] Definitions

[0113] As used herein, the term “about” refers to a value that is no more than 10% above or below the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.

[0114] As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, primatized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including e.g., Fab’, F(ab’)2, Fab, Fv, IgG, and scFv fragments. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (such as, for example, Fab and F(ab’)2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab’)2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med. 24:316, 1983; incorporated herein by reference).

[0115] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab’)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341 :544-546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. An “antigen-binding fragment” may also refer to a linear or non-linear peptide, which may be cyclic, bicylic, and / or conformationally biased. It will be appreciated by one of skill in the art that a conformationally biased antigen-binding fragment will entail a structure-based design such that the peptide includes an antigen-binding site mimic based on the 3D structure of the peptide-antigen complex. Such structural information enables the design and generation of mimics of continuous, as well as of sequentially discontinuous antigen-binding sites, which are composed of two or more protein segments that are distant in protein sequence but brought into spatial proximity through protein folding. Mimicking such discontinuous antigen-binding sites by synthetic peptides often involves splicing and / or molecular scaffolds (e.g., disulfide bonds) to enable conformation bias. See e.g., Gro3 et al., Front. Bioeng. Biotechnol. 4(39), 2016. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

[0116] As used herein, the terms “anti-CD40 antibody portion,” “anti-CD40 antibody fragment,” and the like include any protein or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule, such as, but not limited, to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, or any portion thereof, that is capable of specifically binding to CD40. In some embodiments, the portion of an immunoglobulin molecule includes all three CDRs of the heavy chain and / or all three CDRs of the light chain. For instance, two or more portions of an immunoglobulin molecule may be covalently bound to one another, e.g., via an amide bond, a thioether bond, a carbon-carbon bond, a disulfide bridge, or by a linker, such as a linker described herein or known in the art. CD40 antibodies also include antibody-like protein scaffolds, such as the tenth fibronectin type III domain (10Fn3), which contains BC, DE, and FG structural loops similar in structure and solvent accessibility to antibody CDRs. The tertiary structure of the10Fn3 domain resembles that of the variable region of the IgG heavy chain, and one of skill in the art can graft, e.g., the CDRs of a CD40 monoclonal antibody onto the fibronectin scaffold by replacing residues of the BC, DE, and FG loops of10Fn3 with residues from the CDR-H1 , CDR-H2, or CDR-H3 regions of a CD40 monoclonal antibody.

[0117] As used herein, the terms “antagonist CD40 antibody” and “CD40 antagonist antibody” refer to CD40 antibodies that are capable of inhibiting or reducing activation of CD40, attenuating one or more signal transduction pathways mediated by CD40, and / or reducing or inhibiting at least one activity mediated by activation of CD40. For example, antagonistic CD40 antibodies may inhibit or reduce the growth and proliferation of regulatory T cells. Antagonistic CD40 antibodies may inhibit or reduce CD40 activation by blocking CD40 from binding one or more ligands (e.g., CD40L). In this way, antagonistic CD40 antibodies may block the trimerization of CD40 that would otherwise be induced by interacting with one or more ligands (e.g., CD40L), thus resulting in suppression of CD40 activity.

[0118] As used herein, the terms “CD40 extracellular domain,” “CD40 ECD,” “native CD40 ECD,” and “CD40 native ECD” refer to the domain of CD40 protein that, in its natural conformation, extends into the space outside a cell. The extracellular domain of CD40 can bind to its ligands (e.g., CD40L) and initiate downstream signaling, which triggers the biological activities related to CD40. Due to its exposure, the extracellular domain of CD40 can be targeted by peptides that bind to CD40 (e.g., antibodies or antigen-binding fragments thereof). It is known to a person skilled in the art that the extracellular domain of CD40 can be expressed as a truncated polypeptide of full-length CD40, under the guidance of biochemical characterizations of CD40 (e.g., crystal structure).

[0119] As used herein, the term “bispecific antibodies” refers to antibodies (e.g., monoclonal, often human or humanized antibodies) that have binding specificities for at least two different antigens. For example, one of the binding specificities can be directed towards CD40, and the other can be for any other antigen, e.g., for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.

[0120] As used herein, the phrase “chemotherapeutic agent” refers to any chemical agent with therapeutic usefulness in the treatment of cancer, such as a cancer described herein. Chemotherapeutic agents encompass both chemical and biological agents. These agents can function to inhibit a cellular activity upon which a cancer cell depends for continued survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones, hormone analogs, and antineoplastic drugs. Exemplary chemotherapeutic agents suitable for use in conjunction with the compositions and methods described herein include, without limitation, those set forth in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison’ s Principles of Internal medicine, 14thedition; Perry et al., Chemotherapeutic, Chapter 17 in Abeloff, Clinical Oncology 2nded., 2000; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nded. St. Louis, Mosby-Year Book, 1995; Fischer D. S., Knobf M. F., Durivage H.J. (eds): The Cancer Chemotherapeutic Handbook, 4thed. St. Louis, Mosby-Year Handbook, the disclosures of each of which are incorporated herein by reference as they pertain to chemotherapeutic agents.

[0121] As used herein, the term “chimeric” antibody refers to an antibody having variable domain sequences (e.g., CDR sequences) derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human, a non-human primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, a member of the Bo vidae family (such as cattle, cow, bison, buffalo, elk, and yaks, among others), sheep, or horse, among others). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202-7, 1985; Oi et al., BioTechniques 4:214-221 , 1986; Gillies et al., J. Immunol. Methods 125:191 -202, 1985; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397; incorporated herein by reference.

[0122] As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable domains of an antibody. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The antibodies described herein may comprise modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a p-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the p-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987); incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated. The CDRs can be identified using sequence or structure-based methods that have been described by Kabat et al., Chothia et al. (J. Mol. Biol. 196:901 -917, 1987), and MacCallum et al. (J. Mol. Biol. 262:732-745, 1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. The term “CDR” may be, for example, a CDR as defined by Kabat or Chothia based on sequence comparisons.

[0123] As used herein, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and / or steric volume. These properties are summarized for each of the twenty naturally occurring amino acids in Table 1 below.

[0124] Table 1. Representative physicochemical properties of naturally occurring amino acids.

[0125] From this table, it is appreciated that the conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (Hi) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg). As is appreciated in the art, a polypeptide may retain its biological properties or functions after the introduction of one or more conservative substitutions.

[0126] Amino acid substitutions may be represented herein using the convention (AA1 )(N)(AA2), where “AA1 ” represents the amino acid normally present at particular site within an amino acid sequence, “N” represents the residue number within the amino acid sequence at which the substitution occurs, and “AA2” represents the amino acid present in the amino acid sequence after the substitution is effectuated. For example, the notation “C232S” in the context of an antibody hinge region, such as an lgG2 antibody hinge region, refers to a substitution of the naturally occurring cysteine residue for a serine residue at amino acid residue 232 of the indicated hinge amino acid sequence. Likewise, the notation “C233S” in the context of an antibody hinge region, such as an lgG2 antibody hinge region, refers to a substitution of the naturally occurring cysteine residue for a serine residue at amino acid residue 233 of the indicated hinge amino acid sequence.

[0127] As used herein, the term “conjugate” refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with an appropriately reactive functional group of another molecule.

[0128] As used herein in the context of a CD40 antagonist, the term “construct” refers to a fusion protein containing a first polypeptide domain bound to a second polypeptide domain. The polypeptide domains may each independently be antagonistic CD40 single chain polypeptides, for instance, as described herein. The first polypeptide domain may be covalently bound to the second polypeptide domain, for instance, by way of a linker, such as a peptide linker or a disulfide bridge, among others. Exemplary linkers that may be used to join the polypeptide domains of an antagonistic CD40 construct include, without limitation, those that are described in Leriche et al., Bioorg. Med. Chem. 20:571 -582, 2012, the disclosure of which is incorporated herein by reference in its entirety.

[0129] As used herein, the term “derivatized antibodies” refers to antibodies that are modified by a chemical reaction to cleave residues or add chemical moieties not native to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, addition of known chemical protecting / blocking groups, proteolytic cleavage, and / or linkage to a cellular ligand or other protein. Any of a variety of chemical modifications can be carried out by known techniques, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc., using established procedures. Additionally, the derivative can contain one or more non-natural amino acids, e.g., using amber suppression technology (see, e.g., U.S. Patent No. 6,964,859; incorporated herein by reference).

[0130] As used herein, the term “diabodies” refers to bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Accordingly, the term “triabodies” refers to trivalent antibodies comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain. In order to fold into their native structure, peptides configured in this way typically trimerize to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993; incorporated herein by reference).

[0131] As used herein, a “disulfide-bonded isoform” of an antibody or antigen-binding fragment thereof is a form of the antibody or antigen-binding fragment thereof having a particular internal disulfide bonding pattern. Disulfide-bonded isoforms are structural isomers of a given antibody or antigen-binding fragment thereof that do not differ from one another in amino acid sequence but exhibit different disulfide bond connectivity. For example, in the context of a human lgG2 antibody or variant thereof, the antibody may exist in one of four possible disulfide-bonded isoforms, represented herein as isoforms lgG2-A, lgG2-B, lgG2-A / Bi, and lgG2-A / B2.

[0132] As used herein, a “dominant antagonist” of CD40 is an antagonist (e.g., an antagonistic polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof) that is capable of inhibiting CD40 activation even in the presence of a CD40 ligand, such as CD40L. For example, a CD40 antagonist is a dominant antagonist if the IC50 of the antagonist increases by less than 200% (e.g., less than 200%, 100%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less) in the presence of a CD40 ligand (e.g., CD40L) relative to the ICso of the antagonist as measured in the same assay in the absence of the CD40 ligand. Inhibition of CD40 activation can be assessed, for instance, by measuring the inhibition of proliferation of CD40-expressing cells, such as T-reg cells, cancer cells that express CD40, or myeloid-derived suppressor cells, as well as by measuring the inhibition of NFKB signaling (e.g., by monitoring the reduction in expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11 , TRAF2, TRAF3, relB, and clAP2 / BIRC3 in a conventional gene expression assay).

[0133] As used herein, a “dual variable domain immunoglobulin” (“DVD-lg”) refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via linkers to create a tetravalent, dual-targeting single agent (Gu et al., Meth. Enzymol. 502:25-41 , 2012; incorporated by reference herein).

[0134] As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell).

[0135] As used herein, the term “epitope” refers to a portion of an antigen that is recognized and bound by a polypeptide, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct as described herein. In the context of a protein antigen (such as human CD40 or CD40 of a non-human mammal, such as a non-human mammal described herein), an epitope may be a continuous epitope, which is a single, uninterrupted segment of one or more amino acids covalently linked to one another by peptide bonds in which all of the component amino acids bind the polypeptide (e.g., antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct thereof). Continuous epitopes may be composed, for instance, of 1 , 5, 10, 15, 20, or more amino acids within an antigen, such as CD40. For example, a continuous epitope may be composed of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more amino acids within an antigen. Examples of continuous epitopes on CD40 that are bound by antagonistic polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein include two or more continuous residues of, or all residues of, the SCSPGFGVK motif (SEQ ID NO: 49) on CD40, two or more continuous residues of, or all residues of, the CEPCPVGFFS motif (SEQ ID NO: 50) on CD40, as well as corresponding regions on CD40 of non-human mammals (e.g., bison, cattle, and others described herein). In some embodiments, an epitope may be a discontinuous epitope, which contains two or more amino acids each separated from one another in the amino acid sequence of an antigen by one or more intervening amino acid residues. Discontinuous epitopes may be composed, for instance, of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such segments of amino acid residues, such as one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) segments containing amino acids from within one or more of the SCSPGFGVK motif (SEQ ID NO: 49) and / or the CEPCPVGFFS motif (SEQ ID NO: 50) within human CD40, as well as corresponding regions on CD40 of non-human mammals (e.g., bison, cattle, and others described herein). Despite this separation by intervening amino acids, the segments that compose a discontinuous epitope may be, for instance, spatially proximal to one another in the three-dimensional conformation of the antigen.

[0136] As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or cultured matter.

[0137] As used herein, the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs. FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.

[0138] As used herein, the term “fusion protein” refers to a protein that is joined via a covalent bond to another molecule. A fusion protein can be chemically synthesized by, e.g., an amide-bond forming reaction between the N-terminus of one protein to the C-terminus of another protein. Alternatively, a fusion protein containing one protein covalently bound to another protein can be expressed recombinantly in a cell (e.g., a eukaryotic cell or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein, for example, from a vector or the genome of the cell. A fusion protein may contain one protein that is covalently bound to a linker, which in turn is covalently bound to another molecule. Examples of linkers that can be used for the formation of a fusion protein include peptide-containing linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally occurring proteins and are thus more resistant to degradation by endogenous proteases. Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem. 20:571 -582, 2012).

[0139] As used herein, the term “heterospecific antibodies” refers to monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Traditionally, the recombinant production of heterospecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1983). Similar procedures are disclosed, e.g., in WO 93 / 08829; U.S. Pat. Nos. 6,210,668; 6,193,967; 6,132,992; 6,106,833; 6,060,285; 6,037,453; 6,010,902; 5,989,530; 5,959,084; 5,959,083; 5,932,448; 5,833,985; 5,821 ,333; 5,807,706; 5,643,759; 5,601 ,819; 5,582,996; 5,496,549; and 4,676,980; WO 91 / 00360; WO 92 / 00373; EP 03089;

[0140] Traunecker et al., EMBO J. 10:3655, 1991 ; and Suresh et al., Methods in Enzymology 121 :210, 1986; incorporated herein by reference. Heterospecific antibodies can include Fc mutations that enforce correct chain association in multi-specific antibodies, as described by Klein et al., mAbs 4:653-663, 2012; incorporated herein by reference.

[0141] As used herein, the term “hinge region” refers to the domain of an antibody or antigen-binding fragment thereof (e.g., an lgG2 antibody or antigen-binding fragment thereof) located between the antigen-binding portion(s) of the antibody or antigen-binding fragment thereof, such as the Fab region of the antibody or antigen-binding fragment thereof, and the portion of the antibody or antigen-binding fragment thereof that dictates the isotype of the antibody or antigen-binding fragment thereof, such as the Fc region of the antibody or antigen-binding fragment thereof. For example, in the context of a monoclonal antibody, the hinge region is the polypeptide situated approximately in the center of each heavy chain, connecting the CH1 domain to the CH2 and CH3 domains. The hinge region of an antibody or antigen-binding fragment thereof may provide a chemical linkage between chains of the antibody or antigen-binding fragment thereof. For instance, in a monoclonal antibody, the cysteine residues within the hinge region form inter-chain disulfide bonds, thereby providing explicit covalent bonds between heavy chains. The amino acid sequence of wild-type human lgG2 is ERKCCVECPPCP (SEQ ID NO: 51 ). As used herein, antibody hinge regions are numbered according to the numbering system of Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987), the disclosure of which is incorporated herein by reference. For example, using the numbering scheme of Kabat et al., the wild-type human lgG2 hinge region set forth in SEQ ID NO: 51 is numbered from residues 226 to 243, such that the N- terminal glutamate residue of SEQ ID NO: 51 is residue 226 and the C-terminal proline residue of SEQ ID NO: 51 is residue 243. Throughout the present disclosure, variant lgG2 hinge regions, such as the variant set forth in SEQ ID NO: 52 (ERKCCVESPPCP), are numbered according to the convention of Kabat et al. unless explicitly stated to the contrary.

[0142] As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1 , CH2, CH3), hinge, VL, and VH) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell that can express functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when a human antibody is a single-chain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered of human origin. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111 ; and PCT publications WO 1998 / 46645; WO 1998 / 50433; WO 1998 / 24893; WO 1998 / 16654; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741 ; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patent Nos. 5,413,923; 5,625, 126; 5,633,425; 5,569,825; 5,661 ,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 ; and 5,939,598; incorporated by reference herein.

[0143] As used herein, the term “humanized” antibody refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other target-binding subdomains of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All, or substantially all, of the FR regions may also be those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7, 1988; U.S. Patent Nos 5,225,539; 5,530,101 ; 5,585,089; 5,693,761 ; 5,693,762; and 6,180,370; EP239400; PCT publication WO 91 / 09967; EP592106; and EP519596; incorporated herein by reference.

[0144] As used herein, the term “hydrophobic side chain” refers to an amino acid side chain that exhibits relatively low solubility in water due to, e.g., the steric or electronic properties of the chemical moieties present within the side chain. Examples of amino acids containing hydrophobic side chains include those containing aliphatic hydrocarbons, such as alanine, valine, leucine, isoleucine, proline, and methionine, as well as amino acids containing aromatic ring systems that are electrostatically neutral at physiological pH, such as tryptophan, phenylalanine, and tyrosine.

[0145] As used herein, the term “immunotherapy agent” refers to a compound, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct as described herein, that specifically binds an immune checkpoint protein (e.g., immune checkpoint receptor or ligand) and exerts an antagonistic effect on the receptor or ligand, thereby reducing or inhibiting the signal transduction of the receptor or ligand that would otherwise lead to a downregulation of the immune response. Immunotherapy agents include compounds, such as antibodies, antigen-binding fragments, single-chain polypeptides, and constructs, capable of specifically binding receptors expressed on the surfaces of hematopoietic cells, such as lymphocytes (e.g., T cells), and suppressing the signaling induced by the receptor or ligand that would otherwise lead to tolerance towards an endogenous (“self”) antigen, such as a tumor-associated antigen. Immunotherapy agents may reduce the signaling induced by the receptor or ligand by, for example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% relative to the signaling induced by the receptor or ligand exhibited in the absence of the immunotherapy agent. Exemplary assays that can be used to measure the extent of receptor or ligand signaling include, for example, enzyme-linked immunosorbent assay (ELISA) techniques to measure protein expression alterations that are associated with a particular signal transduction pathway, as well as polymerase chain reaction (PCR)-based techniques, such as quantitative PCR, reverse-transcription PCR, and real-time PCR experiments useful for determining changes in gene expression associated with a particular signal transduction pathway, among others. Exemplary methods that can be used to determine whether an agent is an “immunotherapy agent” include the assays described in Mahoney et al., Cancer Immunotherapy 14:561 -584, 2015, the disclosure of which is incorporated herein by reference in its entirety. Examples of immunotherapy agents include, e.g., antibodies or antigen-binding fragments thereof that specifically bind one or more of TL1 A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3, B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D, NKG2A, MICA, MICB, CD244, CSF1 R, IDO, TGFp, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, and neuropilin. Additional examples of immunotherapy agents include Targretin, Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti- chemokines, Vorinostat, gabapentin, antibodies to lymphoid cell surface receptors and / or lymphokines, antibodies to surface cancer proteins, and / or small molecular therapies such as Vorinostat. Particular examples of immunotherapy agents that may be used in or in conjunction with the compositions and methods described herein include anti-PD-1 antibodies and antigen-binding fragments thereof, such as nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab, as well as anti-PD-L1 antibodies and antigen-binding fragments thereof, such as atezolizumab and avelumab, and anti-CTLA-4 antibodies and antigen-binding fragments thereof, such as ipilimumab or tremelimumab.

[0146] As used herein, the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0147] As used herein, the term “multispecific antibodies” refers to antibodies that exhibit affinity for more than one target antigen. Multispecific antibodies can have structures similar to full immunoglobulin molecules and include Fc regions, for example, IgG Fc regions. Such structures can include, but are not limited to, IgG-Fv, lgG-(scFv)2, DVD-lg, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc- (SCFV)2. In case of lgG-(scFv)2, the scFv can be attached to either the N-terminal or the C- terminal end of either the heavy chain or the light chain. Exemplary multi-specific molecules that include Fc regions and into which anti-CD40 antibodies or antigen-binding fragments thereof can be incorporated have been reviewed by Kontermann, mAbs 4:182-197, 2012; Yazaki et al., Protein Engineering, Design & Selection 26:187-193, 2013; and Grote et al., 2012, in Proetzel & Ebersbach (eds.), Antibody Methods and Protocols, Methods in Molecular Biology vol. 901 , chapter 16:247-263; incorporated herein by reference. In some embodiments, antibody fragments can be components of multi-specific molecules without Fc regions, based on fragments of IgG or DVD or scFv. Exemplary multi-specific molecules that lack Fc regions and into which antibodies or antibody fragments can be incorporated include scFv dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies), Fab dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated), are described by Hudson and Souriau, Nature Medicine 9:129-134, 2003; incorporated herein by reference.

[0148] As used herein, the term “myeloid-derived suppressor cell” or “MDSC” refers to a cell of the immune system that modulates the activity of a variety of effector cells and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others. Myeloid derived suppressor cells are distinguished by their gene expression profile, and express all or a subset of proteins and small molecules selected from the group consisting of B7-1 (CD80), B7-H1 (PD-L1 ), CCR2, CD1 d, CD1 d1 , CD2, CD31 (PECAM-1 ), CD43, CD44, complement component C5a R1 , F4 / 80 (EMR1 ), Fey RIH (CD16), Fey RII (CD32), Fey RIIA (CD32a), Fey RUB (CD32b), Fey RIIB / C (CD32b / c), Fey RIIC (CD32c), Fey RIIIA (CD16A), Fey RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly- 6G), ICAM-1 (CD54), IL-1 Rl, IL-4Ra, IL-6Ra, integrin a4 (CD49d), integrin aL (CD1 1 a), integrin aM (CD1 1 b), M-CSFR, MGL1 (CD301 a), MGL1 / 2 (CD301 a / b), MGL2 (CD301 b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), siglec-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1 ), and VEGFR2 (KDR or Flk-1 ). Particularly, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD1 1 c, CD14, CD21 , CD23 (FCERII), CD34, CD35, CD40 (TNFRSF5), CD1 17 (c-kit), HLA-DR, and Sca-1 (Ly6).

[0149] As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30%, (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid residue as the corresponding position in the reference sequence, the molecules are identical at that position.

[0150] As used herein, the term “primatized antibody” refers to an antibody comprising framework regions from primate-derived antibodies and other regions, such as CDRs and / or constant regions, from antibodies of a non-primate source. Methods for producing primatized antibodies are known in the art. See e.g., U.S. Patent Nos. 5,658,570; 5,681 ,722; and 5,693,780; incorporated herein by reference. For instance, a primatized antibody or antigen-binding fragment thereof described herein can be produced by inserting the CDRs of a non-primate antibody or antigen-binding fragment thereof into an antibody or antigen-binding fragment thereof that contains one or more framework regions of a primate.

[0151] As used herein, the term “proliferation” in the context of a population of cells, such as a population of CD40-expressing cells (e.g., T-reg cells, MDSCs, or CD40-expressing cancer cells) refers to mitotic and cytokinetic division of a cell to produce a plurality of cells. Cell proliferation may be evidenced, for example, by a finding that the quantity of cells (e.g., CD40-expressing cells) in a subject or sample of cells has increased over a given time period, such as over the course of one or more hours, days, or weeks. One of skill in the art may monitor cell proliferation using a variety of known techniques, such as by way of visual microscopy, hemocytometry, flow cytometry, fluorescence activated cell sorting, and other assays known in the art. In the present disclosure, cell proliferation is considered “inhibited” when the rate of proliferation of a population of cells, such as a population of CD40-expressing cells contacted with an antagonistic CD40 polypeptide described herein, is decreased relative to the rate of proliferation of a population of control cells, such as a population of CD40-expressing cells not contacted with the antagonistic CD40 polypeptide. A decrease in the rate of proliferation may manifest, for example, as a reduction in the quantity of cells of interest in a subject or sample over a given time period, such as a reduction in the quantity of cells of interest in a subject or sample of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, over a given time period. Additionally or alternatively, inhibition of cell proliferation may be evidenced by a finding that the rate at which cells of interest (e.g., CD40-expressing cells contacted with an antagonistic CD40 polypeptide described herein) are dividing is reduced, e.g., by %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, relative to the rate at which control cells (e.g., CD40-expressing cells not contacted with the antagonistic CD40 polypeptide) are dividing.

[0152] As used herein, the term “operatively linked” in the context of a polynucleotide fragment is intended to mean that the two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame.

[0153] As used herein, the term “pharmacokinetic profile” refers to the absorption, distribution, metabolism, and clearance of a drug over time following administration of the drug to a subject.

[0154] As used herein, a “recessive antagonist” of CD40 is an antagonist (e.g., an antagonistic polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof) that inhibits CD40 activation to a significantly lesser extent in the presence of a CD40 ligand, such as CD40L among others, relative to the extent of inhibition of the same antagonist as measured in the absence of the CD40 ligand. For example, a CD40 antagonist is a recessive antagonist if the IC50 of the antagonist increases by, e.g., 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more in the presence of a CD40 ligand (e.g., CD40L among others) relative to the IC50 of the antagonist as measured in the same assay the absence of the CD40 ligand. Inhibition of CD40 activation can be assessed, for instance, by measuring the inhibition of proliferation of CD40- expressing cells, such as T-reg cells, cancer cells that express CD40, or myeloid-derived suppressor cells, as well as by measuring the inhibition of NFKB signaling (e.g., by monitoring the reduction in expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11 , TRAF2, TRAF3, relB, and clAP2 / BIRC3 in a conventional gene expression assay).

[0155] As used herein, the terms “CD40 natural ligand,” “endogenous CD40 ligand,” and “CD40 endogenous ligand” refer to an endogenous ligand of CD40 that may form a CD40 ligand-CD40 protein complex and induce the formation of the active, homotrimeric conformation of CD40. Exemplary CD40 ligand-CD40 protein complexes may include, but are not limited to, the CD40L- CD40 complex.

[0156] As used herein, the term “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990); incorporated herein by reference.

[0157] As used herein, the term “scFv” refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1 , CDR-L2, and / or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1 , CDR-H2, and / or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers, such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). scFv molecules are known in the art and are described, e.g., in U.S. Patent No. 5,892,019; Flo et al., Gene 77:51 , 1989; Bird et al., Science 242:423, 1988; Pantoliano et al., Biochemistry 30:10117, 1991 ; Milenic et al., Cancer Research 51 :6363, 1991 ; and Takkinen et al., Protein Engineering 4:837, 1991 . The VL and VH domains of a scFv molecule can be derived from one or more antibody molecules. It is also understood by one of ordinary skill in the art that the variable regions of the scFv molecules described herein can be modified, such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, in some embodiments, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and / or framework residues). Alternatively or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. scFv fragments are described, for example, in WO 2011 / 084714; incorporated herein by reference.

[0158] As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a Kd of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a Kd of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a Kd of greater than 100 nM (e.g., greater than 500 nm, 1 pM, 100 pM, 500 pM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0159] As used herein, the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as cancer or an infectious disease). Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bo vidae family (such as cattle, cow, bison, buffalo, elk, and yaks, among others), sheep, and horses, among others, receiving treatment for diseases or conditions, for example, cell proliferation disorders, such as cancer or infectious diseases.

[0160] As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of an exogenous polynucleotide into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium phosphate precipitation, diethylaminoethyl (DEAE)-dextran transfection, and the like.

[0161] As used herein, the terms “treat” or “treatment” refer to therapeutic treatment, in which the objective is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a cell proliferation disorder, such as cancer, an autoimmune disease, or an infectious disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable (e.g., in particular in a subject that is not treated with a composition described herein). Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in which the condition or disorder is to be prevented.

[0162] As used herein, the term “tumor microenvironment” refers to cancer cells that form a tumor and the population of non-cancer cells, molecules, and / or blood vessels within the tumor or that border or surround the cancer cells.

[0163] As used herein, the terms “tumor necrosis factor receptor superfamily,” “TNFR superfamily,” “TNFRS,” “TNFRSF,” or “TNFRSF members” refer to a group of type I transmembrane proteins with a carboxy-terminal intracellular domain and an amino-terminal extracellular domain characterized by a common cysteine-rich domain (CRD). The TNFR superfamily includes receptors that mediate cellular signaling due to binding by one or more ligands in the TNF superfamily. The TNFR superfamily can be divided into two subgroups: receptors containing the intracellular death domain and those lacking this domain. The death domain is an 80 amino acid motif that propagates apoptotic signal transduction cascades following receptor activation. Exemplary TNFR superfamily members that contain the intracellular death domain include TNFR1 , while TNFR2 represents a TNFR superfamily protein that does not contain this domain. Members of the TNFR superfamily include CD40, TNFR1 , TNFR2, RANK, CD30, lymphotoxin beta receptor (LT-p receptor or LT-pR), 0X40, Fas receptor, decoy receptor 3 (DCR3), CD27, 4-1 BB, death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DCR1 ), decoy receptor 2 (DCR2), osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, Herpesvirus entry mediator, nerve growth factor receptor, B cell maturation antigen, glucocorticoid-induced TNFR-related protein, TROY, death receptor 6 (DR6), death receptor 3 (DR3), and ectodysplasin A2 receptor.

[0164] As used herein, the terms “CD40 signaling” and “CD40 signal transduction,” and the like, are used interchangeably and refer to the cellular events that normally occur upon activation of CD40 on the surface of a CD40-expressing cell, such as T-reg cell, MDSC, or CD40-expressing cancer cell, by an endogenous CD40 ligand, such as CD40L among others. CD40 signaling may be evidenced by a finding that expression is increased for one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11 , TRAF2, TRAF3, relB, and clAP2 / BIRC3. CD40 signaling is considered “inhibited,” as used herein, when the expression (and / or post-translational modification in the event that such a modification is required for activity of the encoded protein) of one or more, or all, of the foregoing genes is decreased in a CD40-expressing cell upon contacting the cell with an agent, such as a CD40 antagonist polypeptide described herein, relative to a CD40-expressing cell that is not contacted with the agent. CD40 signaling is considered “inhibited,” for example, when the expression or post-translational modification (e.g., phosphorylation) of one or more of CHUK, NFKBIE, NFKBIA, MAP3K11 , TRAF2, TRAF3, relB, or clAP2 / BIRC3, in a CD40-expressing cell contacted with an antagonistic CD40 polypeptide is decreased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the expression or post-translational modification (e.g., phosphorylation) of one or more of these genes in a CD40-expressing cell not contacted with the antagonistic CD40 polypeptide. Exemplary assays that can be used to determine expression level and phosphorylation state are known in the art and include, e.g., western blot assays to determine protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments to determine mRNA content.

[0165] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, and a virus or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 11026; incorporated herein by reference. Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of antibodies and antibody fragments described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, internal ribosomal entry site (IRES), and polyadenylation signal site to direct efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0166] As used herein, the term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus.

[0167] As used herein, the term “stabilize” refers to the effect of a molecule (e.g., a polypeptide, such as the anti-GD40 polypeptides disclosed herein) to maintain a particular state or conformation of a protein (e.g., CD40). In some embodiments, the molecule stabilizes the particular state or conformation of the protein by binding to the protein.

[0168] As used herein, the term “destabilize” refers to the effect of a molecule (e.g., a polypeptide, such as the anti-CD40 polypeptides disclosed herein) to disrupt a particular state or conformation of a protein (e.g., CD40). in some embodiments, the molecule destabilizes the particular state or conformation of the protein by binding to the protein. In some embodiments, the molecule facilitates the conversion of the protein into a different state or conformation. As used herein, the term “reference level” refers to a threshold level or a level in a control subject or control patient population. A reference level depends on the assay performed and can be determined by one of ordinary skill in the art. A reference level can be a baseline level or a level in the same subject measured at an earlier or later point in time. In some cases, a reference level is determined in a subject prior to or after the administration of an antibody or antigen-binding fragment thereof disclosed herein, a construct disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein. For example, some non-limiting examples of reference levels of human soluble CD40 include the level of human soluble CD40 in a subject that: has not been diagnosed as having a disease; does not present with at least two or more symptoms of a disease; or has not been administered with the anti-CD40 polypeptides disclosed herein.

[0169] Brief Description of the Drawings

[0170] FIG. 1 is a line graph showing binding affinity of anti-CD40 mAbs (clonal deconvoluted or not deconvoluted) to human CD40 extracellular domain (ECD) as measured by enzyme-linked immunosorbent assay (ELISA). Binding of the mAbs to CD40 ECD, as indicated by the level of optical density (O.D.) at 405 nm (in absorbance unit), is plotted against the concentration (cone.) of purified mAbs.

[0171] FIG. 2A is a line graph showing antagonist activity of anti-CD40 mAbs (clonal deconvoluted or not deconvoluted) in the presence of human CD40L as measured using HEK-BLUE™ reporter cells. Reporter activity, as indicated by the level of O.D. at 650 nm (in absorbance unit), is plotted against the concentration of purified mAbs.

[0172] FIG. 2B is a line graph showing agonist activity of anti-CD40 mAbs (clonal deconvoluted or not deconvoluted) as measured using HEK-BLUE™ reporter cells. Reporter activity, as indicated by the level of O.D. at 650 nm (in absorbance unit), is plotted against the concentration of purified mAbs.

[0173] FIGS. 3A and 3B are line graphs showing binding affinity of anti-CD40 mAbs to nucleic acid- captured human CD40 peptide (FIG. 3A) or human CD40 ECD (FIG. 3B) as measured by ELISA. Binding of the mAbs to CD40 peptide or ECD, as indicated by the level of O.D. at 405 nm (in absorbance unit), is plotted against the concentration of mAbs.

[0174] FIGS. 4A-4C are a series of BIACORE™ sensorgrams showing binding affinity of anti-CD40 mAbs ABBV-323 (KD = 6.7 nM; FIG. 4A; positive control), 4F10 (KD = 2.5 nM; FIG. 4B; also known as CD40AB1 ), and 10D11 (KD = 6.3 nM; FIG. 4C; also known as CD40AB2) to human CD40 ECD.

[0175] FIG. 5A is a line graph showing binding affinity of anti-CD40 mAbs to human CD40 ECD as measured by antigen capture format ELISA. Binding of the mAbs to CD40 ECD, as indicated by the level of O.D. at 650 nm (in absorbance unit), is plotted against the concentration of biotinylated CD40 ECD.

[0176] FIG. 5B is a line graph showing binding affinity of anti-CD40 mAbs to Cynomolgus monkey (Macaca fascicularis, cyno) CD40 ECD as measured by antigen down ELISA. Binding of the mAbs to cyno CD40 ECD, as indicated by the level of O.D. at 405 nm (in absorbance unit), is plotted against the concentration of purified mAbs.

[0177] FIG. 6 is a set of line graphs showing antagonist activity of anti-CD40 mAbs as measured by CD40L-induced primary B cell proliferation assay performed using primary B cells from two human donors (left and right panels, respectively). Antagonist activity of anti-CD40 mAbs was compared to that of ABBV-323 (“Abbvie”; positive control) or a human lgG2 (hlgG2) isotype control (negative control). The IC50 is 0.1385, 0.05379, 0.5737, 5.220, and 0.03094 nM for antibodies 10D11 , 4F10, 6A11 , 12C2, and ABBV-323, respectively, in Donor 1 . The IC50 is 0.1821 , 0.05531 , 0.6709, 5.850, and 0.06289 nM for antibodies 10D11 , 4F10, 6A11 , 12C2, and ABBV-323, respectively, in Donor 2. Ab, antibody; hlL4, human IL-4.

[0178] FIGS. 7A-7D are a series of line graphs showing body weight (FIGS. 7A and 7B) and percent body weight change (FIGS. 7C and 7D) of humanized NSG™ mice treated with anti-CD40 mAbs 10D11 (CD40AB2) or 4F10 (CD40AB1 ), or controls (no peripheral blood mononuclear cell (PBMC) control, vehicle control, and positive control (ABBV-323)). Body weight or percent body weight change of the mice is plotted against the duration of the treatment.

[0179] FIGS. 8A-8H are a series of box plots showing serum concentrations of human IgG or IgM in mice measured at different dilutions, different study days, and from different blood sources. FIG. 8A shows human IgG concentration in retro-orbital blood of mice on study day 7 with 1 :100,000 dilution. FIG. 8B shows human IgM concentration in retro-orbital blood of mice on study day 7 with 1 :100,000 dilution. FIG. 8C shows human IgG concentration in retro-orbital blood of mice on study day 7 with 1 :50,000 dilution. FIG. 8D shows human IgM concentration in retro-orbital blood of mice on study day 7 with 1 :200 dilution. FIG. 8E shows human IgG concentration in cardiac blood of mice on study day 14 with 1 :1 ,000,000 dilution. FIG. 8F shows human IgM concentration in cardiac blood of mice on study day 14 with 1 :100,000 dilution. FIG. 8G shows human IgG concentration in cardiac blood of mice on study day 14 with 1 :50,000 dilution. FIG. 8H shows human IgM concentration in cardiac blood of mice on study day 14 with 1 :200 dilution. Small circles indicate individual mouse samples. Large circles indicate the mean value of a group. Dark grey boxes indicate the first quartile. Light grey boxes indicate the third quartile. The border between the 1st and 3rd quartiles indicates the median. Whiskers indicate the value of one standard deviation. 1 K = 1000.

[0180] FIGS. 9A-9T are a series of box plots showing flow cytometry quantification of human CD45 (huCD45), CD3, CD86, and / or CD19 in blood or spleens of mice collected at study day 14 (study terminus) represented by cells / pl flow beads or percentage of parent / grandparent population. FIG. 9A shows the population of huCD45+ live (7-AAD+) cells in spleens of the mice quantified by cells / pl flow beads. FIG. 9B shows the population of huCD45+ live cells in cardiac blood of the mice quantified by cells / pl flow beads. FIG. 9C shows the population of huCD45+ live cells in spleens of the mice quantified by percentage of parent (live cells). FIG. 9D shows the population of huCD45+ live cells in cardiac blood of the mice quantified by percentage of parent (live cells). FIG. 9E shows the population of CD3+ live cells in spleens of the mice quantified by cells / pl flow beads. FIG. 9F shows the population of CD3+ live cells in cardiac blood of the mice quantified by cells / pl flow beads. FIG. 9G shows the population of CD3+ huCD45+ live cells in spleens of the mice quantified by percentage of parent (huCD45+ cells). FIG. 9H shows the population of CD3+ live cells in cardiac blood of the mice quantified by percentage of parent (huCD45+ cells). FIG. 9I shows the population of CD86+ CD3+ live cells in spleens of the mice quantified by cells / pl flow beads. FIG. 9J shows the population of CD86+ CD3+ live cells in cardiac blood of the mice quantified by cells / pl flow beads. FIG. 9K shows the population of CD86+ CD3+ live cells in spleens of the mice quantified by percentage of parent (CD3+ cells). FIG. 9L shows the population of CD86+ CD3+ live cells in cardiac blood of the mice quantified by percentage of parent (CD3+ cells). FIG. 9M shows the population of CD19+ live cells in spleens of the mice quantified by cells / pl flow beads. FIG. 9N shows the population of CD19+ live cells in cardiac blood of the mice quantified by cells / pl flow beads. FIG. 90 shows the population of CD19+ huCD45+ live cells in spleens of the mice quantified by percentage of parent (huCD45+ cells). FIG. 9P shows the population of CD19+ huCD45+ live cells in cardiac blood of the mice quantified by percentage of parent (huCD45+ cells). FIG. 9Q shows the population of CD86+ CD19+ live cells in spleens of the mice quantified by cells / pl flow beads. FIG. 9R shows the population of CD86+ CD19+ live cells in cardiac blood of the mice quantified by cells / pl flow beads. FIG. 9S shows the population of CD86+ CD19+ live cells in spleens of the mice quantified by percentage of parent (CD19+ cells). FIG. 9T shows the population of CD86+ CD19+ live cells in cardiac blood of the mice quantified by percentage of parent (CD19+ cells). Small circles indicate individual mouse samples. Large circles indicate the mean value of a group. Dark grey boxes indicate the first quartile. Light grey boxes indicate the third quartile. The border between the 1st and 3rd quartiles indicates the median. Whiskers indicate the value of one standard deviation.

[0181] Detailed Description

[0182] Antagonistic polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that are specific for human CD40 inhibit the activation of human CD40 by binding CD40 (e.g., on the exterior surface of a cell, such as T-reg cell, a cancer cell that expresses CD40, a myeloid-derived suppressor cell (MDSC), a T cell, a B cell, a monocyte, a neutrophil, a platelet, a granulocyte, a bone marrow derived lymphoid cells, or a parenchymal cell), thereby preventing CD40 from recruiting its cognate or natural ligand.

[0183] Inactive Conformation of CD40: Anti-Parallel Dimer

[0184] Antagonistic polypeptides (e.g., antagonistic CD40 single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure may be used to inhibit the activity of CD40 by, for example, binding CD40 in an anti-parallel dimer conformation. By binding CD40 in an anti-parallel dimer structure, antibodies or antigen-binding fragments thereof form a complex with CD40 in which CD40 residues that bind a cognate or natural ligand (such as CD40 ligand) are sequestered within the interior of the complex. Thus, antibodies or antigen-binding fragments thereof of the disclosure may prevent or substantially inhibit or reduce ligand-mediated trimerization, and hence activation, of CD40 by forming a complex with CD40 that sterically precludes the endogenous ligand from accessing its cognate binding sites within CD40. CD40 is known to naturally adopt an anti-parallel dimer conformation.

[0185] Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure may be used to bind CD40 in an anti-parallel dimer conformation in order to inhibit the activity of CD40 in a target cell, such as, e.g., a T-reg cell, a CD8+ cytotoxic T cell, a CD40- expressing cancer cell, a myeloid-derived suppressor cell, a B cell, a monocyte, a neutrophil, a platelet, a granulocyte, a bone marrow-derived lymphoid cell, or a parenchymal cell. Polypeptides described herein are capable of binding CD40 and epitopes therein, such as epitopes containing two or more continuous or discontinuous residues within CRD3 and / or CRD4 of CD40. For example, antagonistic CD40 antibodies or antigen-binding fragments thereof of the disclosure may bind an epitope of, within, or including one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). In some embodiments, the epitope bound by the antibody or antigenbinding fragment thereof is or includes one or more of amino acids 124-132 of SEQ ID NO: 37. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 143-152 of SEQ ID NO: 37. Antagonistic CD40 antibodies or antigen-binding fragments thereof may also bind an epitope(s) that exhibits at least 80% sequence identity (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to one or both of these sequences and an epitope(s) that contains conservative amino acid substitutions relative to one or both of these sequences. Anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may also specifically bind an epitope within human CD40 that includes at least five continuous or discontinuous amino acid residues of amino acids 104-172 of SEQ ID NO: 37 (e.g., at least five continuous or discontinuous amino acid residues of amino acids 114-142 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 133-162 of SEQ ID NO: 37, or at least five continuous or discontinuous amino acid residues of amino acids 124-132 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 143-152 of SEQ ID NO: 37). Furthermore, anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may also specifically bind an epitope within human CD40 that includes at least five continuous or discontinuous amino acid residues of amino acids 104-172 of SEQ ID NO: 37 (e.g., at least five continuous or discontinuous amino acid residues of amino acids 114-142 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 123-172 of SEQ ID NO: 37, or at least five continuous or discontinuous amino acid residues of amino acids 124-132 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 133-162 of SEQ ID NO: 37), as well as an epitope(s) that exhibits at least 80% sequence identity (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to one or both of these sequences and an epitope(s) that contains conservative amino acid substitutions relative to one or both of these sequences. Antagonistic CD40 antibodies or antigen-binding fragments thereof can target cells that express CD40, such as, e.g., T cells, B cells, platelets, macrophages, dendritic cells, epithelial cells, endothelial cells, and mesenchymal cells.

[0186] Antagonistic CD40 Polypeptides

[0187] The anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can interact with and inhibit the activity of CD40. Thus, the anti-CD40 antibodies of the disclosure can selectively antagonize the CD40 ligand-CD40 interaction rather than promote CD40 signaling.

[0188] The CD40 polypeptides of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may bind CD40 with high affinity and may sterically sequester the receptor from a CD40 ligand (e.g., CD40L, among others) rather than to allow CD40 ligand binding to CD40 to initiate CD40 signaling, e.g., by binding CD40 in an anti-parallel dimer conformation in which the cognate CD40 ligand binding sites are sterically inaccessible.

[0189] The Antagonistic CD40 Antibody CD40AB1

[0190] An antagonistic CD40 antibody or antibody fragment of the disclosure may contain one or more CDR sequences of CD40AB1 , also referred to herein as anti-CD40 mAb 4F10, an antibody that selectively binds and inhibits CD40 by virtue of specifically binding various epitopes within this receptor. For instance, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may exhibit binding properties that are the same as or similar to those of CD40AB1 as shown in Example 5 below. Antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB1 ) may bind to an epitope containing one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 124-132 of SEQ ID NO: 37. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 143-152 of SEQ ID NO: 37. Included in the disclosure are CD40 antibodies and antibody fragments that specifically bind this epitope.

[0191] Antibodies and antibody fragments of the disclosure can be designed and identified using the knowledge of the epitopes specifically bound by CD40AB1 . For instance, one can use any of a variety of in vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in order to screen for antibodies capable of binding these epitopes with high affinity and selectivity.

[0192] The heavy chain and light chain CDRs of CD40AB1 are shown below:

[0193] CD40AB1 CDR-H1 : GFTLSNY (SEQ ID NO: 1 ) CD40AB1 CDR-H2: WYDGNI (SEQ ID NO: 6) CD40AB1 CDR-H3: DYGGNFWFDN (SEQ ID NO: 10) CD40AB1 CDR-L1 : RSSQSLLNSNGYNYLD (SEQ ID NO: 14) CD40AB1 CDR-L2: LGSNRAS (SEQ ID NO: 19) CD40AB1 CDR-L3: MQALQTPLT (SEQ ID NO: 23)

[0194] Notably, the CDR-L2 of CD40AB1 is flanked by the N-terminal framework residues LLIY (SEQ ID NO: 38). Accordingly, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure include those that contain one or more of the above CDRs of CD40AB1 , as well as N-terminal LLIY (SEQ ID NO: 38) residues that flank the CDR-L2 sequence of the antagonistic CD40 antibody or antigen-binding fragment thereof.

[0195] The antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the disclosure may also include a heavy chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the heavy chain of CD40AB1 (SEQ ID NO: 27), and a light chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the light chain of CD40AB1 (SEQ ID NO: 28).

[0196] The Antagonistic CD40 Antibody CD40AB2

[0197] An antagonistic CD40 antibody or antibody fragment of the disclosure may contain one or more CDR sequences of CD40AB2, also referred to herein as anti-CD40 mAb 10D11 , an antibody that selectively binds and inhibits CD40 by virtue of specifically binding various epitopes within this receptor. For instance, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may exhibit binding properties that are the same as or similar to those of CD40AB2 as shown in Example 5 below. Antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB2) may bind to an epitope containing one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 124-132 of SEQ ID NO: 37. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 143-152 of SEQ ID NO: 37. Included in the disclosure are CD40 antibodies and antibody fragments that specifically bind this epitope.

[0198] Antibodies and antibody fragments of the disclosure can be designed and identified using the knowledge of the epitopes specifically bound by CD40AB2. For instance, one can use any of a variety of in vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in order to screen for antibodies capable of binding these epitopes with high affinity and selectivity.

[0199] The heavy chain and light chain CDRs of CD40AB2 are shown below:

[0200] CD40AB2 CDR-H1 : GGSISSY (SEQ ID NO: 2) CD40AB2 CDR-H2: YYSGST (SEQ ID NO: 7) CD40AB2 CDR-H3: SPWIQLGWFAP (SEQ ID NO: 11 ) CD40AB2 CDR-L1 : RASQSVSSSYLA (SEQ ID NO: 15) CD40AB2 CDR-L2: GASSRAT (SEQ ID NO: 20) CD40AB2 CDR-L3: QQYGSSPWT (SEQ ID NO: 24)

[0201] Notably, the CDR-L2 of CD40AB2 is flanked by the N-terminal framework residues LLIY (SEQ ID NO: 38). Accordingly, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure include those that contain one or more of the above CDRs of CD40AB2, as well as N-terminal LLIY (SEQ ID NO: 38) residues that flank the CDR-L2 sequence of the antagonistic CD40 antibody or antigen-binding fragment thereof.

[0202] The antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the disclosure may also include a heavy chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the heavy chain of CD40AB2 (SEQ ID NO: 29), and a light chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the light chain of CD40AB2 (SEQ ID NO: 30).

[0203] The Antagonistic CD40 Antibody CD40AB3

[0204] An antagonistic CD40 antibody or antibody fragment of the disclosure may contain one or more CDR sequences of CD40AB3, also referred to herein as anti-CD40 mAb 8C4, an antibody that selectively binds and inhibits CD40 by virtue of specifically binding various epitopes within this receptor. For instance, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may exhibit binding properties that are the same as or similar to those of CD40AB3 as shown in Example 5 below. Antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB3) may bind to an epitope containing one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 124-132 of SEQ ID NO: 37. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 143-152 of SEQ ID NO: 37. Included in the disclosure are CD40 antibodies and antibody fragments that specifically bind this epitope.

[0205] Antibodies and antibody fragments of the disclosure can be designed and identified using the knowledge of the epitopes specifically bound by CD40AB3. For instance, one can use any of a variety of in vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in order to screen for antibodies capable of binding these epitopes with high affinity and selectivity.

[0206] The heavy chain and light chain CDRs of CD40AB3 are shown below:

[0207] CD40AB3 CDR-H1 : GFTFSTY (SEQ ID NO: 3) CD40AB3 CDR-H2: KQDGGE (SEQ ID NO: 8) CD40AB3 CDR-H3: EFSVGRGYFGMDV (SEQ ID NO: 12) CD40AB3 CDR-L1 : RSSQSLLYSNGHNYLD (SEQ ID NO: 16) CD40AB3 CDR-L2: LGSNRAS (SEQ ID NO: 21 ) CD40AB3 CDR-L3: MQALRTPPT (SEQ ID NO: 25)

[0208] Notably, the CDR-L2 of CD40AB3 is flanked by the N-terminal framework residues LLIY (SEQ ID NO: 38). Accordingly, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure include those that contain one or more of the above CDRs of CD40AB3, as well as N-terminal LLIY (SEQ ID NO: 38) residues that flank the CDR-L2 sequence of the antagonistic CD40 antibody or antigen-binding fragment thereof.

[0209] The antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the disclosure may also include a heavy chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the heavy chain of CD40AB3 (SEQ ID NO: 31 ), and a light chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the light chain of CD40AB3 (SEQ ID NO: 32). The Antagonistic CD40 Antibody CD40AB4 and Derivatives

[0210] An antagonistic CD40 antibody or antibody fragment of the disclosure may contain one or more CDR sequences of CD40AB4, also referred to herein as anti-CD40 mAb 7F12, an antibody that selectively binds and inhibits CD40 by virtue of specifically binding various epitopes within this receptor. For instance, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may exhibit binding properties that are the same as or similar to those of CD40AB4 as shown in Example 5 below. Antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB4) may bind to an epitope containing one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 124-132 of SEQ ID NO: 37. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 143-152 of SEQ ID NO: 37. Included in the disclosure are CD40 antibodies and antibody fragments that specifically bind this epitope.

[0211] Antibodies and antibody fragments of the disclosure can be designed and identified using the knowledge of the epitopes specifically bound by CD40AB4. For instance, one can use any of a variety of in vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in order to screen for antibodies capable of binding these epitopes with high affinity and selectivity.

[0212] Two variants of CD40AB4, CD40AB4.1 (also referred to herein as anti-CD40 mAb 7F12_C1 .5.4) and CD40AB4.2 (also referred to herein as anti-CD40 mAb 7F12_C1 .5-10), have been identified and characterized in this disclosure. The heavy chain and light chain CDRs of CD40AB4.1 are shown below:

[0213] CD40AB4.1 CDR-H1 : GFTFGDY (SEQ ID NO: 4)

[0214] CD40AB4.1 CDR-H2: SWDGRT (SEQ ID NO: 9)

[0215] CD40AB4.1 CDR-H3: DGVGNLFDY (SEQ ID NO: 13)

[0216] CD40AB4.1 CDR-L1 : RASQSVSNNLA (SEQ ID NO: 17)

[0217] CD40AB4.1 CDR-L2: GASTRNT (SEQ ID NO: 22)

[0218] CD40AB4.1 CDR-L3: QQYNNWPPIT (SEQ ID NO: 26)

[0219] The heavy chain and light chain CDRs of CD40AB4.2 are shown below:

[0220] CD40AB4.2 CDR-H1 : GFTFADY (SEQ ID NO: 5)

[0221] CD40AB4.2 CDR-H2: SWDGRT (SEQ ID NO: 9)

[0222] CD40AB4.2 CDR-H3: DGVGNLFDY (SEQ ID NO: 13)

[0223] CD40AB4.2 CDR-L1 : RASQSVSFNLA (SEQ ID NO: 18)

[0224] CD40AB4.2 CDR-L2: GASTRNT (SEQ ID NO: 22)

[0225] CD40AB4.2 CDR-L3: QQYNNWPPIT (SEQ ID NO: 26) Notably, the CDR-L2 of CD40AB4.1 and CD40AB4.2 is flanked by the N-terminal framework residues LLIY (SEQ ID NO: 38). Accordingly, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure include those that contain one or more of the above CDRs of CD40AB4.1 or CD40AB4.2, as well as N-terminal LLIY (SEQ ID NO: 38) residues that flank the CDR-L2 sequence of the antagonistic CD40 antibody or antigenbinding fragment thereof.

[0226] The antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the disclosure may also include a heavy chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the heavy chain of CD40AB4.1 (SEQ ID NO: 33), and a light chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the light chain of CD40AB4.1 (SEQ ID NO: 34). Alternatively, the antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure may also include a heavy chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the heavy chain of CD40AB4.2 (SEQ ID NO: 35), and a light chain having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, 95%, 97%, 99%, or 100% identical) to the light chain of CD40AB4.2 (SEQ ID NO: 36).

[0227] The Antagonistic CD40 Antibody ABBV-323

[0228] An antagonistic CD40 antibody or antibody fragment of the disclosure may contain one or more CDR sequences of ABBV-323, an antibody that selectively binds and inhibits CD40. The crystal structure of ABBV-323, its Fab heavy chain sequence (SEQ ID NO: 61 ), and its Fab light chain sequence (SEQ ID NO: 62) are known in the art and can be accessed by UniProt Accession No. 6PE7. Structural studies of ABBV-323 Fab in complex with CD40 (UniProt Accession No. 6PE8) revealed that conformational changes of the antibody in the CDRs upon binding to CD40 are crucial for antagonism. In particular, the CD40 antagonism of ABBV-323 may be achieved by the stabilization of the proposed functional antiparallel dimer for CD40 receptor via contact to CDR-L1 of ABBV-323 (Argiriadi et al. BMC Mol. Cell. Biol. 20: 29, 2019; incorporated by reference herein).

[0229] The heavy chain and light chain CDRs of ABBV-323 are shown below:

[0230] ABBV-323 CDR-H1 : GFTFSDY (SEQ ID NO: 55)

[0231] ABBV-323 CDR-H2: SSGRGN (SEQ ID NO: 56)

[0232] ABBV-323 CDR-H3: SWGYFDV (SEQ ID NO: 57)

[0233] ABBV-323 CDR-L1 : KSSQSLLNRGNQKNYLT (SEQ ID NO: 58)

[0234] ABBV-323 CDR-L2: WASTRES (SEQ ID NO: 59)

[0235] ABBV-323 CDR-L3: QNDYTYPLT (SEQ ID NO: 60) Framework Regions of Antagonistic CD40 Polypeptides

[0236] Antagonistic CD40 polypeptides of the disclosure (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may contain a framework (FW) region between each of the CDR sequences of the polypeptides described herein. The amino acid sequences of the FW regions can be natural sequences, such as those present in an antibody that is produced by immunization of an animal (e.g., a non-human animal) with an antigen sequence, such as one or more of those described herein. Native FW region sequence(s) or human FW sequence(s) can be used in the polypeptides of the present disclosure. The FW sequence(s) can also be prepared by using human FW sequence(s) that are modified to include one or more amino acids of a native FW region sequence.

[0237] The FW region amino acid sequences recognize, and are bound by, MHC class II proteins, including human leukocyte antigens (HLA) DR and DQ, among others. Typically, antibodies that are found to contain amino acid sequences that bind MHC proteins are engineered to remove such motifs, since antibodies that bind MHC proteins are susceptible to being degraded upon administration to a subject (e.g., a mammalian subject, such as a human subject) and positioned on the exterior of an antigen-presenting cell of the immune system, thereby triggering an inappropriate immune response against the administered antibody. Methods of determining whether a particular amino acid sequence is prone to bind MHC molecules are known in the art, and are described, e.g., in Wang et al., BMC Bioinformatics 11 :568, 2010; Nielsen et al., BMC Bioinformatics 8:238, 2007; Gonzalez-Galarza et al., Nucleic Acid Research 39:D913-D919, 2011 ; and Greenbaum et al., Immunogenetics 63:325, 2011 , the disclosures of each of which are incorporated herein by reference in their entirety.

[0238] The FW regions described above are not immunogenic peptides, despite their propensity to bind MHC class II molecules. Antagonistic CD40 polypeptides of the disclosure (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) that contain FW region sequences exhibit the unexpected and beneficial property of CD40 binding affinity without inducing an immunogenic response against the polypeptide upon administration of the polypeptide to a subject (e.g., a mammalian subject, such as a human).

[0239] Molecular Determinants of CD40 Affinity and Antagonism

[0240] Notably, there are distinct sequence similarities between the CDR-L1 regions of the antagonistic CD40 antibodies CD40AB1 , CD40AB2, CD40AB3, CD40AB4 (including CD40AB4.1 and CD40AB4.2). An analysis of the residues common to the CDR-L1 sequences of these antibodies provides insight into the molecular features of antibodies that bind CD40 and exhibit an antagonistic effect, such as a dominant antagonistic effect. The antagonistic CD40 antibodies may bind to an epitope containing one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). The structural similarities between corresponding CDR-L1 regions provide a basis for predicting residue substitutions that may preserve or enhance CD40 affinity and antagonism (e.g., dominant antagonism). Inspection of the CDR-L1 sequences of these antibodies demonstrates that several residues and physicochemical characteristics are conserved throughout this region, while other positions within this CDR can be varied significantly without loss of affinity and antagonistic function. The CDR-L1 sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 are shown below:

[0241] RSSQSLLNSNGYNYLD (CD40AB1 CDR-L1 , SEQ ID NO: 14)

[0242] RASQSVSSSY - LA (CD40AB2 CDR-L1 , SEQ ID NO: 15)

[0243] RSSQSLLYSNGHNYLD (CD40AB3 CDR-L1 , SEQ ID NO: 16)

[0244] RASQSVSNNL (CD40AB4.1 CDR-L1 , SEQ ID NO: 17) R SQSVSFNL (CD40AB4.2 CDR-L1 , SEQ ID NO: 18) R-SQS - (Consensus sequence)

[0245] Inspection of the CDR-L1 sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 reveals conserved arginine, serine, glutamine, and serine residues at the first, third, fourth, and fifth positions within this CDR, respectively. Additionally, the second position in the above CDR-L1 sequences features a sterically small serine in CD40AB1 and CD40AB3, and a similarly small alanine in CD40AB2, CD40AB4.1 , and CD40AB4.2. The fifth position in the above CDR-L1 sequences features a hydrophobic leucine in CD40AB1 and CD40AB3, and a similarly hydrophobic valine in CD40AB2, CD40AB4.1 , and CD40AB4.2. Notably, residues of varying steric and electrostatic properties are tolerated in the remaining positions. For instance, the sixth position of the CDR-L1 sequence tolerates amino acid residues of contrasting polarity, as said position of CDR-L1 in CD40AB2, CD40AB4.1 , and CD40AB4.2 features a polar serine residue, while a nonpolar, hydrophobic leucine residue is found at the corresponding position in CD40AB1 and CD40AB3.

[0246] Collectively, the shared structural features of the above CDR-L1 sequences provide insight into those residues that are important for selectively binding residues within CD40 that promote receptor antagonism (e.g., dominant receptor antagonism), such as residues 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO: 37) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37) and demonstrate that other amino acids can be varied while retaining affinity and dominant antagonistic activity. For instance, antagonistic CD40 polypeptides, such a dominant antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the disclosure may contain a CDR-L1 represented by the consensus sequence RZ1SQSZ2(J)2Z3(J)2-7, wherein each J is independently a naturally occurring amino acid, each Z1is independently a naturally occurring amino acid containing a sterically small side chain (e.g., glycine, serine, and alanine), each Z2is independently a naturally occurring amino acid containing a hydrophobic side chain (e.g., leucine, valine, and isoleucine), and each Z3is independently a naturally occurring amino acid containing a uncharged, polar side chain at physiological pH (e.g., asparagine, serine, glutamine, and threonine). For instance, the CDR-L1 may be derived from CD40AB1 and have the amino acid sequence RSSQSLLNSNGYNYLD (SEQ ID NO: 14). The CDR-L1 may be derived from CD40AB2 and have the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 15). The CDR-L1 may be derived from CD40AB3 and have the amino acid sequence RSSQSLLYSNGHNYLD (SEQ ID NO: 16). The CDR-L1 may be derived from CD40AB4.1 and have the amino acid sequence RASQSVSNNLA (SEQ ID NO: 17). The CDR-L1 may be derived from CD40AB4.2 and have the amino acid sequence RASQSVSFNLA (SEQ ID NO: 18).

[0247] In addition to the above CDR-L1 sequences, other CDR sequences can be included in antagonistic CD40 polypeptides, such as dominant antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the disclosure. Additional CDR sequences that promote dominant CD40 antagonism can be determined, for instance, by alignment of the CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2. For example, the CDR-H1 sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 are shown below:

[0248] GFTLSNY (CD40AB1 CDR-H1 , SEQ ID NO: 1 )

[0249] GGSISSY (CD40AB2 CDR-H1 , SEQ ID NO: 2)

[0250] GFTFSTY (CD40AB3 CDR-H1 , SEQ ID NO: 3)

[0251] GFTFGDY (CD40AB4.1 CDR-H1 , SEQ ID NO: 4)

[0252] GFTFADY (CD40AB4.2 CDR-H1 , SEQ ID NO: 5)

[0253] G - Y (Consensus sequence)

[0254] Alignment of the sequences reveals a shared GXXXXXY motif, wherein “X” designates any amino acid. These CDR-H1 sequences feature a conserved glycine residue at the first position and a conserved tyrosine residue at the seventh position. Additionally, the third position in the above CDR- H1 sequences features a hydroxyl group-containing serine in CD40AB2, and a similarly hydroxyl group-containing threonine in CD40AB1 , CD40AB3, CD40AB4.1 , and CD40AB4.2. Similarly, the fourth position in the above CDR-H1 sequences features a hydrophobic leucine in CD40AB1 , a similarly hydrophobic isoleucine in CD40AB2, and a similarly hydrophobic phenylalanine in CD40AB3, CD40AB4.1 , and CD40AB4.2. Notably, inspection of these sequences demonstrates that the CDR-H1 region is tolerant of substitutions at the remaining positions. Side chains of size are tolerated at the second position, for example, as both glycine, the smallest amino acid, and phenylalanine, containing a bulky aromatic side chain, are found in this position in the CDR-H1 region of the CD40 antibodies. Additionally, while the sixth position is occupied by charged aspartate residue in the CDR-H1 of CD40AB4.1 and CD40AB4.2, this position features a polar, uncharged asparagine, serine, or threonine residue in the CDR-H1 of CD40AB1 , CD40AB2, and CD40AB3, respectively. This diversity demonstrates that these positions can be substituted with amino acids of diverse steric and electrostatic properties without loss of CD40 affinity and antagonism. Sequence analysis of the CDR-H3 regions of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 similarly reveals a conserved amino acid at the ninth position throughout these regions:

[0255] — DYG— GNFWFDN (CD40AB1 CDR-H3, SEQ ID NO: 10) SPWIQLGWFA — P (CD40AB2 CDR-H3, SEQ ID NO: 11 ) EFSVGRGYFGMDV (CD40AB3 CDR-H3, SEQ ID NO: 12) -DGVG-NLFDY — (CD40AB4.1 and CD40AB4.2 CDR-H3, SEQ ID NO: 13) -F- (Consensus sequence)

[0256] Analysis of this sequence alignment demonstrates that the CDR-H3 sequences feature a conserved phenylalanine at the ninth position, with side chains of variable molecular size, polarity, and electrostatic charge tolerated at the remaining positions.

[0257] Sequence analysis of the CDR-L2 regions of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 similarly reveals a set of conserved amino acids at various positions throughout these regions:

[0258] LGSN-RAS (CD40AB1 CDR-L2, SEQ ID NO: 19)

[0259] - GASSRAT (CD40AB2 CDR-L2, SEQ ID NO: 20)

[0260] LGSN-RAS (CD40AB3 CDR-L2, SEQ ID NO: 21 )

[0261] - GASTRNT (CD40AB4.1 and CD40AB4.2 CDR-L2, SEQ ID NO: 22)

[0262] -G - R — (Consensus sequence)

[0263] Inspection of the CDR-L2 sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 reveals conserved glycine and arginine residues within this CDR. Additionally, the third position in the above CDR-L2 sequences features a sterically small serine in CD40AB1 and CD40AB3, and a similarly small alanine in CD40AB2, CD40AB4.1 , and CD40AB4.2. The eighth position in the above CDR-L2 sequences features a hydroxyl group-containing serine in CD40AB1 and CD40AB3, and a similarly hydroxyl group-containing threonine in CD40AB2, CD40AB4.1 , and CD40AB4.2. Notably, residues of varying steric and electrostatic properties are tolerated in the remaining positions. For instance, the seventh position of the CDR-L2 sequence tolerates amino acid residues of contrasting size and hydrogen bond-forming tendencies, as said position of CDR-L2 in CD40AB4.1 and CD40AB4.2 features a polar asparagine residue containing a carboxamide side chain with hydrogen bond donor and acceptor moieties, while an alanine residue bearing an unfunctionalized methyl side chain is found at the corresponding position in CD40AB1 , CD40AB2, and CD40AB3. Similarly, analysis of the CDR-L3 regions of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , and CD40AB4.2 reveals a set of conserved amino acids at various positions throughout these regions: MQALQTPLT- (CD40AB1 CDR-L3, SEQ ID NO: 23) QQYGSSPWT- (CD40AB2 CDR-L3, SEQ ID NO: 24)

[0264] MQALRTPPT- (CD40AB3 CDR-L3, SEQ ID NO: 25)

[0265] QQYNNWPPIT (CD40AB4.1 and CD40AB4.2 CDR-L3, SEQ ID NO: 26)

[0266] -Q - P - (Consensus sequence)

[0267] Analysis of the above sequence alignment demonstrates that conserved glutamine residues are featured at the second position of these CDR-L3 sequences and conserved proline residues are featured at the seventh position of these CDR-L3 sequences, while substitutions are widely tolerated at the remaining residues.

[0268] Collectively, the shared structural features of the above CDR-H and CDR-L sequences provide insight into those residues that are important for selectively binding one or more epitopes of CD40 (e.g., SEQ ID NO: 49 or 50) in an anti-parallel dimer configuration and demonstrate that certain amino acids can be varied while retaining affinity and dominant antagonistic activity.

[0269] In some embodiments, antagonistic TNFR2 polypeptides of the disclosure, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1 -5 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 6-9 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 10-13 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-18 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 19-22 or a variant thereof with up to two conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-26 or a variant thereof with up to two conservative amino acid substitutions.

[0270] In some embodiments, antagonistic TNFR2 polypeptides of the disclosure, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof with up to two conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof with up to two conservative amino acid substitutions.

[0271] In some embodiments, antagonistic TNFR2 polypeptides of the disclosure, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof with up to two conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof with up to two conservative amino acid substitutions.

[0272] In some embodiments, antagonistic TNFR2 polypeptides of the disclosure, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof with up to two conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof with up to two conservative amino acid substitutions.

[0273] In some embodiments, antagonistic TNFR2 polypeptides of the disclosure, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof with up to two conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof with up to two conservative amino acid substitutions.

[0274] In some embodiments, antagonistic TNFR2 polypeptides of the disclosure, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof with up to two conservative amino acid substitutions, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof with up to two conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof with up to two conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof with up to two conservative amino acid substitutions.

[0275] Humanized, Primatized, and Chimeric Antibodies

[0276] Antibodies of the disclosure include human, humanized, primatized, and chimeric antibodies that contain one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, or one or more CDR sequences that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR sequences or sequences that contain conservative mutations relative to these CDR sequences. For instance, antibodies of the disclosure also include human, humanized, primatized, and chimeric antibodies that contain one or more CDR sequences that are identical to the one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, except for conservative amino acid substitutions. In some embodiments, a humanized, primatized, or chimeric antibody may contain one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, or one or more CDR sequences that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR sequences or sequences that contain conservative mutations relative to these CDR sequences. For example, antagonistic CD40 antibodies of the disclosure can be generated by incorporating one or more of the above CDR sequences into the framework regions (e.g., FW1 , FW2, FW3, and FW4) of a human antibody. Exemplary framework regions that can be used for the development of a humanized anti-CD40 antibody containing one or more of the above CDRs include, without limitation, those described in U.S. Patent No. 7,732,578, U.S. Patent No. 8,093,068, and WO 2003 / 105782; incorporated herein by reference.

[0277] One strategy that can be used to design humanized antibodies of the disclosure is to align the sequences of the heavy chain variable region and light chain variable region of an antagonistic CD40 antibody, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, with the heavy chain variable region and light chain variable region of a consensus human antibody. Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991 ; Tomlinson et al., J. Mol. Biol. 227:776-98, 1992; and Cox et al, Eur. J. Immunol. 24:827-836, 1994; incorporated herein by reference). In this way, the variable domain framework residues and CDRs can be identified by sequence alignment (see Kabat, supra). One can substitute, for example, one or more CDR sequences of the consensus human antibody with the one or more CDR sequences of an antagonistic CD40 antibody, such as a CDR-L2 of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, in order to produce a humanized CD40 antagonist antibody. Exemplary variable domains of a consensus human antibody include the heavy chain variable domain:

[0278] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVA VISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDR GGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 53), and the light chain variable domain:

[0279] DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAA

[0280] SSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGT

[0281] KVEIKRT (SEQ ID NO: 54), identified in US Patent No. 6,054,297; incorporated herein by reference (CDRs shown in bold were determined according to the method of Chothia et al. J. Mol. Biol. 196:901 -917, 1987). These amino acid substitutions can be made, for example, by recombinant expression of polynucleotides encoding the heavy and light chains of a humanized antibody in a host cell using methods known in the art or described herein.

[0282] Similarly, this strategy can also be used to produce primatized anti-CD40 antibodies, as one can substitute, for example, the one or more CDRs of a primate antibody consensus sequence with, for example, the one or more CDRs of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2. Consensus primate antibody sequences known in the art (see e.g., U.S. Patent Nos. 5,658,570; 5,681 ,722; and 5,693,780; incorporated herein by reference).

[0283] In some embodiments, it may be desirable to import particular framework residues in addition to CDR sequences from a CD40 antibody, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2 into the heavy and / or light chain variable domains of a human antibody. For instance, U.S. Patent No. 6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody. In some embodiments, framework residues may engage in non- covalent interactions with the antigen and thus contribute to the affinity of the antibody for the target antigen. In other cases, individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody with the antigen. Alternatively, certain framework residues may form the interface between VH and VL domains and may therefore contribute to the global antibody structure. In other cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser / Thr) which may dictate antibody structure and antigen affinity upon attachment to carbohydrate moieties. In cases such as those described above, it may be beneficial to retain certain framework residues of a CD40 antagonist antibody (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2) in the antagonistic antibodies and antigen-binding fragments thereof of the disclosure, such as humanized antibodies, as various framework residues may promote high epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment thereof.

[0284] Antibodies of the disclosure also include antibody fragments, Fab domains, F(ab’) molecules, F(ab’)2 molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi-specific antibodies, bispecific antibodies, and heterospecific antibodies that contain the one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2 or one or more CDR sequences that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of CDR sequences. Antibodies and antigen-binding fragments thereof of the disclosure include those that also contain one or more CDR sequences having between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2. These molecules can be expressed recombinantly, e.g., by incorporating polynucleotides encoding these proteins into expression vectors for transfection in a eukaryotic or prokaryotic cell using techniques described herein or known in the art, or synthesized chemically, e.g., by solid phase peptide synthesis methods described herein or known in the art.

[0285] Polypeptides of the disclosure additionally include antibody-like scaffolds that contain, for example, the one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, or one or more CDR sequences that exhibit at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to these CDR sequences or sequences that contain between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2. Examples of antibody-like scaffolds include proteins that contain a tenth fibronectin type III domain (10Fn3), which contains BC, DE, and FG structural loops analogous to canonical antibodies. It has been shown that the tertiary structure of the10Fn3 domain resembles that of the variable region of the IgG heavy chain, and one of skill in the art can graft, e.g., the one or more CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2, or sequences having at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to one or more of these CDR sequences or sequences containing conserved amino acid substitutions relative to these CDR sequences onto the fibronectin scaffold by replacing residues of the BC, DE, and FG loops of10Fn3 with residues of the CDR sequences of CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , or CD40AB4.2. This can be achieved by recombinant expression of a modified10Fn3 domain in a prokaryotic or eukaryotic cell (e.g., using the vectors and techniques described herein). Examples of using the10Fn3 domain as an antibody-like scaffold for the grafting of CDRs from antibodies onto the BC, DE, and FG structural loops are reported in WO 2000 / 034784, WO 2009 / 142773, WO 2012 / 088006, and U.S. Patent No. 8,278,419; incorporated herein by reference.

[0286] Antagonists Targeting Cell Proliferation Disorders and Infectious Diseases

[0287] Antagonistic polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) of the disclosure that bind CD40 can be used to suppress T-reg cell growth and proliferation and can be administered to a mammalian subject, such as a human subject with a cell proliferation disorder or an infectious disease, in order to enhance the effectiveness of an immune response (e.g., an immune response against cancerous cells or pathogenic organisms) in the subject. This is particularly important for therapeutic applications, e.g., cancer immunotherapy, as TRAF-binding TNFRSF member (e.g., CD40) activation upon association with ligand (e.g., CD40L for CD40) leads to propagation of the MAPK and TRAF2 / 3 signal cascade and activation of NFKB-mediated transcription of genes involved in T-reg cell growth and escape from apoptosis (Faustman, et al., Nat. Rev. Drug Disc. 9:482-493, 2010).

[0288] Antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) of the disclosure may demonstrate the ability to attenuate T-reg and / or cancer cell proliferation even in the presence of a CD40 agonist (such as TL1 A, TRAIL, LT beta, LTa, CD153, N-APP, or RANKL, among others) or an agonistic CD40 antibody, or growth-promoting molecules, such as IL-2. Without being limited by mechanism, antagonistic CD40 single-chain polypeptides, antibodies, or antigenbinding fragments thereof of the disclosure may exhibit this property due to the ability of these antibodies or antigen-binding fragments thereof to bind a specific CD40 and stabilize the dimeric, antiparallel dimer conformation of this receptor. This structural configuration is not capable of potentiating NFKB signaling. By maintaining CD40 in an inactive structural state, antagonistic CD40 single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the disclosure may prevent CD40 agonists from restoring cell growth.

[0289] Another property that may be exhibited by antagonistic TRAF-binding TNFRSF member (e.g., CD40) polypeptides is the ability to not only reduce proliferation of a T-reg cell, a CD40-expressing cancer cell, an MDSC, a T cell, a B cell, a monocyte, a neutrophil, a platelet, a granulocyte, a bone marrow-derived lymphoid cell, and / or a parenchymal cell, but also the ability to reduce the total quantity of these cells within a subject or sample (e.g., within a subject, such as a human subject who was administered the antagonist). Antagonistic CD40 single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may be capable of reducing the total quantity of T-reg cells, cancer cells (such as cutaneous T cell lymphoma cells, ovarian cancer cells, colon cancer cells, renal cell carcinoma cells or multiple myeloma cells, among others), and / or MDSCs in a subject or in a sample treated with an antagonist CD40 polypeptide (such as a sample isolated from a human subject undergoing treatment for cancer or an infectious disease as described herein) by, e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to a subject or sample not treated with an antagonist CD40 antibody or antigen-binding fragment thereof.

[0290] The ability of antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) to attenuate T-reg and / or cancer cell growth may be due to the ability of these polypeptides to diminish the quantity of soluble CD40 within a subject or sample (e.g., a sample isolated from a human subject undergoing treatment for cancer, an infectious disease, or an autoimmune disease as described herein). Soluble CD40 can be secreted by, e.g., T-reg cells and can interfere with the ability of CD40 antagonists to localize to CD40 at the surface of a T-reg cell, CD40-expressing cancer cell, or MDSC by binding and sequestering such antagonists in the extracellular environment. By reducing CD40 secretion, antagonistic CD40 singlechain polypeptides, antibodies, or antigen-binding fragments thereof of the disclosure may render T- reg cells, CD40-expressing cancer cells, MDSCs, T cells, B cells, monocytes, neutrophils, platelets, granulocytes, bone marrow-derived lymphoid cells, and / or parenchymal cells increasingly susceptible to therapeutic molecules, such as an antagonistic CD40 antibody or antigen-binding fragment thereof, and / or additional anti-cancer agents described herein or known in the art that may be used in conjunction with the compositions and methods of the disclosure.

[0291] Antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may be capable of inhibiting the proliferation or reducing the total quantity of a population of T-reg cells in a subject or sample (e.g., a sample isolated from a human subject undergoing treatment for cancer or an infectious disease as described herein) and may act selectively on T-reg cells in an actively dividing state. Antagonistic CD40 single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the disclosure may selectively target active T-reg cells that express CD25Hiand CD45RALow, e.g., over resting T-reg cells that express CD25Medand CD45RAHi. For instance, antagonistic CD40 polypeptides of the disclosure may be capable of reducing the proliferation of a population of T-reg cells expressing CD25Hiand CD45RALowby, e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more relative to a population of T-reg cells that does not express the CD25Hiand CD45RALowproteins, such as a population of T-reg cells that expresses CD25Medand CD45RAHiproteins.

[0292] Antagonistic CD40 antibodies of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may inhibit growth of cells that express CD40, such as, e.g., a T-reg, a cancer cell, a MDSC, a T cell, a B cell, a monocyte, a neutrophil, a platelet, a granulocyte, a bone marrow-derived lymphoid cell, and / or a parenchymal cell with a similar potency as that exhibited by antigen-binding fragments of such antibodies. For instance, removal of the Fc region of an antagonistic CD40 antibody of the disclosure may not alter the ability of the molecule to attenuate the proliferation or reduce the total quantity of T-reg cells and / or cancer cells in a subject or sample (e.g., a sample isolated from a human subject undergoing treatment for cancer or an infectious disease as described herein). Antagonistic CD40 antibodies and antigen-binding fragments thereof of the disclosure may function by a pathway distinct from antibody-dependent cellular cytotoxicity (ADCC), in which an Fc region is required to recruit effector proteins to induce cell death. Additionally, antagonistic CD40 antibodies or antigen-binding fragments thereof may not be susceptible to a loss of inhibitory capacity in the presence of cross-linking agents.

[0293] Antagonistic CD40 antibodies or antigen-binding fragments thereof of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may therefore exhibit therapeutic activity in a variety of isotypes, such as IgG, IgA, IgM, IgD, or IgE, or in a variety of forms, such as a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigenbinding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigenbinding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem scFv (taFv). Antagonistic polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure that stabilize the anti-parallel dimer conformation of CD40 can interrupt cell growth and thus be used to treat diseases such as cancer and infectious diseases. For example, anti-CD40 antibodies of the disclosure can be administered to a subject to treat cancer (such as, e.g., breast cancer, pancreatic cancer, and adenocarcinoma) or infectious diseases by inhibiting the proliferation of, e.g., T-reg cells, cancer cells expressing CD40, MDSCs, T cells, B cells, monocytes, neutrophils, platelets, and / or granulocytes.

[0294] Specific Binding Properties of Antagonistic CD40 Polypeptides

[0295] The specific binding of a single-chain polypeptide, antibody or antibody fragment of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) to human CD40 can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody needed to achieve half-maximal inhibition of the CD40 ligand-CD40 interaction in vitro (IC50) and the equilibrium constant (Kd) of the polypeptide-CD40 complex dissociation. The equilibrium constant, Kd, that describes the interaction of CD40 with a polypeptide of the disclosure is the chemical equilibrium constant for the dissociation reaction of a CD40-antibody complex into solvent-separated CD40 protein and antibody molecules that do not interact with one another.

[0296] CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure include those that specifically bind to CD40 with a Kd value of less than 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In some embodiments, antibodies of the disclosure are those that specifically bind to CD40 with a Kd value of less than 1 nM (e.g., (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM,

[0297] 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM,

[0298] 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM,

[0299] 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 1 10 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).

[0300] CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the Kd or IC50 of a CD40 single-chain polypeptide, antibody, or fragment thereof include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays, among others. ELISA represents a particularly useful method for analyzing antibody activity, as such assays typically require minimal concentrations of antibodies. A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a CD40 antibody of the disclosure). Polypeptides of the disclosure are capable of binding CD40 and epitopes derived thereof. For instance, polypeptides of the disclosure may bind peptides containing the amino acid sequence of SEQ ID NOs: 49 or 50, or a variant thereof with up to 80% or greater sequence identity thereto. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of a horseradish peroxidase (HRP) substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate (ABTS)) with an antigen-antibody complex bound to an HRP-conjugated secondary antibody. For instance, polypeptides of the disclosure may induce a luminescence response of about 400 absorbance units or more when incubated with surface-immobilized antigen and an HRP-conjugated secondary antibody in the presence of an HRP substrate. In some embodiments, the luminescence observed can be from about 400 to about 900 absorbance units (e.g., 400-900 absorbance units, 500-800 absorbance units, or 600-700 absorbance units). In particular embodiments, the luminescence observed can be from about 600 to about 900 absorbance units (e.g., 600-900 absorbance units or 700-800 absorbance units).

[0301] Kinetic Properties of Antagonistic CD40 Polypeptides

[0302] In addition to the thermodynamic parameters of a CD40-polypeptide interaction, it is also possible to quantitatively characterize the kinetic association and dissociation of a single-chain polypeptide, antibody, or antibody fragment of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) with CD40. This can be done, e.g., by monitoring the rate of antibody-antigen complex formation according to established procedures. For example, one can use surface plasmon resonance (SPR) to determine the rate constants for the formation (kon) and dissociation (kotf) of an antibody-CD40 complex. These data also enable calculation of the equilibrium constant of (Kd) of antibody-CD40 protein complex dissociation, since the equilibrium constant of this unimolecular dissociation can be expressed as the ratio of the kOff to kOn values. SPR is a technique that is particularly advantageous for determining kinetic and thermodynamic parameters of receptorantibody interactions since the experiment does not require that one component be modified by attachment of a chemical label. Rather, the receptor is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of antibody. Antibodyreceptor binding induces distortion in the angle of reflection of incident light at the metallic surface, and this change in refractive index over time as antibody is introduced to the system can be fit to established regression models to calculate the association and dissociation rate constants of an antibody-receptor interaction.

[0303] CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure exhibit high kon and low kotf values upon interaction with CD40, consistent with high- affinity receptor binding. Polypeptides of the disclosure may exhibit kOn values in the presence of CD40 of greater than 104M’1s’1(e.g., 1.0 x 104M’1s’1, 1.5 x 104M’1s’1, 2.0 x 104M’1s’1, 2.5 x 104M’1s’1, 3.0 x 104M-1s1,3.5x 104M’1S’1, 4.0 x 104M’1S’1, 4.5 x 104M’1S’1, 5.0 x 104M’1S’1, 5.5 x 104M’1S’1, 6.0 x 104M-1s1, 6.5x 104M’1S’1, 7.0 x 104M’1S’1, 7.5 x 104M’1S’1, 8.0 x 104M’1S’1, 8.5 x 104M’1S’1, 9.0 x 104M-1s1, 9.5 x 104M-1s1, 1.0 x 105M’1S’1, 1.5 x 105M’1S’1, 2.0 x 105M’1S’1, 2.5 x 105M’1S’1, 3.0 x 105M-1s1, 3.5 x 105M-1s1, 4.0 x 105M’1S’1, 4.5 x 105M’1S’1, 5.0 x 105M’1S’1, 5.5 x 105M’1S’1, 6.0 x 105M-1s1, 6.5 x 105M-1s1, 7.0 x 105M’1S’1, 7.5 x 105M’1S’1, 8.0 x 105M’1S’1, 8.5 x 105M’1S’1, 9.0 x 105M’1s-1, 9.5 x 105or 1.0 x 106M’1s-1). Polypeptides of the disclosure exhibit low kotr values when bound to CD40, since antibodies can interact with distinct CD40 epitopes with a high affinity. Residues within these epitopes form strong intermolecular contacts with CD40, which serves to slow the dissociation of the polypeptide-CD40 complex. This high receptor affinity is manifested in low kotr values. Antibodies of the disclosure may exhibit kotr values of less than 10-3s-1when complexed to CD40 (e.g., 1.0x 1 O’3s1, 9.5x 1 O’4s1, 9.0x 1 O’4s1, 8.5x 1 O’4s1, 8.0 x 104s1, 7.5 x 1 O’4s1, 7.0 x 1 O’4s-1, 6.5 x 1 O’4s-1, 6.0 x 1 O’4s1, 5.5 x 1 O’4s1, 5.0 x 1 O’4s1, 4.5 x 1 O’4s1, 4.0 x 1 O’4s1, 3.5 x 1 O’4s-1,3.0x IO4s1, 2.5x IO4s1,2.0x IO4S’1, 1.5 x 10’4S’1,1.0x 10’4S’1, 9.5 x 10’5S’1, 9.0 x 10’5S’1, 8.5 x 10-5s-1, 8.0 x 10-5s-1, 7.5 x 10-5S’1, 7.0 x 1 O’5S’1, 6.5 x 1 O’5S’1, 6.0 x 1 O’5S’1, 5.5 x 1 O’5S’1, 5.0 x 1 O’5S’1, 4.5 x 1 O’5s-1, 4.0 x 1 O’5s-1, 3.5 x 1 O’5s’1, 3.0 x 1 O’5s’1, 2.5 x 1 O’5s’1, 2.0 x 1 O’5s’1, 1.5 x 1 O’5s’1, or 1.Ox 105s-1).

[0304] Epitopes within CD40 Bound by Antagonistic CD40 Polypeptides

[0305] The affinities (e.g., Kd < 1 pM) of polypeptides (e.g., antagonistic CD40 single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure for CD40 coupled with the rapid onset of polypeptide-CD40 complex formation and the slow dissociation of these complexes render these polypeptides well-suited for therapeutic applications as modulators (e.g., suppressors or enhancers) of, e.g., T-reg and CD8+ cytotoxic cell growth and proliferation via antagonism of CD40. The high kOn values, for instance, indicate that polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the disclosure are capable of localizing to the surface of a CD40-expressing cell (e.g., a T-reg cell or a CD8+ cytotoxic cell) and rapidly associating with CD40, thereby preventing receptor activation that may otherwise be induced by a cognate or natural CD40 ligand (such as CD40L, among others), e.g., by inhibiting the trimerization of CD40 by its cognate or natural ligand. Moreover, the slow dissociation of the polypeptide-CD40 complex can be indicative of a long half-life of the complex in vivo, which results in stable, sustained modulation (e.g., downregulation or up-regulation) of the growth of the CD40-expressing cell (e.g., T-reg or CD8+ cytotoxic cell growth). These ideal thermodynamic and kinetic parameters of CD40 binding are consistent with the strong intermolecular contacts that are established upon association of polypeptides of the disclosure with CD40.

[0306] Among the difficulties in developing anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) that are capable of antagonizing CD40 has been the elucidation of epitopes within CD40 that participate in antagonistic complex formation rather than epitopes that promote signal transduction. Various discrete peptide fragments found within the CD40 primary structure bind antagonistic antibodies of the disclosure by virtue of the spatial orientation of these residues in the native conformation of the receptor. Significantly, these residues have been difficult to identify, as many isolated linear CD40-derived peptides do not appear to interact with antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antibody fragments) due to the different conformations these peptides exhibit when structurally pre-organized within the full-length protein and when isolated in solution. Epitope mapping analysis using constrained cyclic and bicyclic peptides derived from various regions of CD40 indicates that antagonistic CD40 antibodies of the disclosure bind epitopes from distinct regions of the CD40 amino acid sequence in a conformationdependent manner.

[0307] The present disclosure also features anti-CD40 polypeptides in the form of an lgG2 isotype, which demonstrate substantially improved CD40 antagonist effects. It has been discovered that this class of CD40 polypeptides exhibits a surprisingly superior ability to disrupt CD40 signaling, modulate (e.g., down-regulate or up-regulate) T-reg cell and CD8+ cytotoxic cell growth, and / or augment the proliferation of effector T cells relative to CD40-binding polypeptides of other isotypes. lgG2 Isotype Antibodies Promotes Optimal CD40 Antagonism

[0308] Antagonistic CD40 polypeptides of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can adopt a single disulfide-bonded isoform. For example, pharmaceutical compositions of the disclosure include those containing an antagonist CD40 polypeptide in which, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more, of the polypeptide in the pharmaceutical composition is present in a single disulfide-bonded isoform. Antagonistic CD40 polypeptides of the disclosure may advantageously adopt an lgG2-A disulfide-bonded isoform, which has surprisingly been found to promote a substantially more robust level of CD40 antagonism relative to other lgG2 disulfide-bonded isoforms, such as the lgG2-B, lgG2-A / Bi, and lgG2-A / B2 isoforms. Polypeptides of the disclosure may be engineered to predominantly adopt an lgG2-A isoform, for example, by introducing mutations into the lgG2 hinge region that prohibit the formation of other disulfide-bonded isoforms. Exemplary mutations in the amino acid sequence of a human lgG2 hinge region that promote the formation of the lgG2-A isoform at the exclusion of the remaining isoforms described above include the deletions and / or substitutions of the cysteine residues at positions 232 and 233 of the wild-type human lgG2 hinge amino acid sequence. By removing one or both residues and optionally replacing these residues with amino acids that are incapable of forming disulfide bonds, one can bias the disulfide bonding pattern in a population of lgG2 isoforms towards the lgG2-A isoform. Examples of amino acid substitutions that can be used to obtain a population of lgG2-A isoform antibodies include conservative amino acid substitutions, such as the C232S and C233S amino acid substitutions. Due to the similar molecular volume and polarity of cysteine and serine, the C232S and C233S substitutions feature the beneficial effect of preserving the steric and electronegativity properties of the naturally occurring cysteine residue while prohibiting the formation of a disulfide bond at position 232 and / or 233 of the lgG2 hinge region. By incorporating C232S and / or C233S substitutions into a CD40 antibody or fragment thereof, a population of CD40 antagonist antibodies or fragments having an lgG2-A isoform can be obtained. Methods of effectuating amino acid substitutions and deletions into an antibody or antigen-binding fragment thereof include mutagenesis techniques described herein and known in the art.

[0309] Spacing between Antigen-Binding Sites

[0310] Antagonistic CD40 polypeptide (e.g., single-chain polypeptides, antibody, antigen-binding fragment thereof, or construct thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) described herein may contain antigen-binding sites (i.e., antigenbinding arms) that are separated from one another by a distance of at least about 133 A, which is the spacing observed between antigen-binding arms in human lgG2 isotype antibodies. It has been discovered that this spacing gives rise to antibodies having optimal CD40 antagonistic properties. In contrast, polypeptides that contain antigen-binding sites separated from one another by fewer than about 133 A, such as IgG 1 antibodies and antigen-binding fragments thereof that contain antigenbinding sites separated from one another by about 117 A and lgG3 antibodies and antigen-binding fragments thereof that contain antigen-binding sites separated from one another by 125 A, do not express optimal CD40 antagonistic properties. CD40 antagonist polypeptides of the disclosure include those containing antigen-binding arms separated by, e.g., a distance of from about 133 A to about 160 A, such as a distance of about 133 A, 134 A, 135 A, 136 A, 137 A, 138 A, 139 A, 140 A, 141 A, 142 A, 143 A, 144 A, 145 A, 146 A, 147 A, 148 A, 149 A, 150 A, 151 A, 152 A, 153 A, 154 A, 155 A, 156 A, 157 A, 158 A, 159 A, or 160 A). For example, the polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) may contain antigenbinding sites that are separated from one another by a distance of from about 133 A to about 150 A, such as by a distance of about 133 A, 134 A, 135 A, 136 A, 137 A, 138 A, 139 A, 140 A, 141 A, 142 A, 143 A, 144 A, 145 A, 146 A, 147 A, 148 A, 149 A, or 150 A. In some embodiments, the antigenbinding sites are separated from one another by a distance of from about 133 A to about 145 A, such as by a distance of about 133 A, 134 A, 135 A, 136 A, 137 A, 138 A, 139 A, 140 A, 141 A, 142 A, 143 A, 144 A, or 145 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of from about 133 A to about 139 A, such as by a distance of about 133 A, 134 A, 135 A, 136 A, 137 A, 138 A, or 139 A. In some embodiments, the antigen-binding sites are separated from one another by a distance of from about 134 A to about 139 A, such as by a distance of about 134 A, 135 A, 136 A, 137 A, 138 A, or 139 A.

[0311] The CD40 antagonist polypeptides described herein (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may have, e.g., two, three, four, five, or more, antigenbinding arms separated by a distance specified above. Examples of antibody fragments that have two or more antigen-binding arms include, without limitation, diabodies, triabodies, F(ab’)2 molecules, and tandem scFv (taFv) molecules, among others. Methods of generating these antibody fragments include peptide synthesis and recombinant protein expression techniques described herein and known in the art. There exist a variety of methods for measuring the distance between antigen-binding arms of an antibody or antibody fragment. For example, distances between antigen-binding arms of an antibody can be made by analyzing the three-dimensional structure of an antibody or antibody fragment using computer software, such as by PYMOL® and other molecular imaging software. Three-dimensional structures of polypeptides, such as antibodies and antibody fragments, can be calculated using the data obtained from X-ray crystallography experiments and nuclear magnetic resonance (NMR) techniques known in the art. Examples of X-ray crystallography and NMR methods that can be used to obtain three-dimensional polypeptide structures are described, e.g., in Eigenbrot et al., Journal of Molecular Biology 229:969-995, 1993; and Huang et al., Science 317:1930-1934, 2007, the disclosures of each of which are incorporated herein by reference in their entirety.

[0312] Uniformity of Populations of CD40 Antagonist Polypeptides

[0313] Pharmaceutical compositions can be generated in which the CD40 antagonist polypeptide (e.g., antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) described herein is present as a single disulfide-bonded isoform. For example, at least 10%, or more, of the polypeptide in the pharmaceutical composition may be present as a single disulfide-bonded isoform (e.g., the lgG2-A isoform). This may be achieved, for example, by way of amino acid substitutions or deletions at one or both of cysteine residues 232 and 233 of the wild-type human lgG2 hinge region, thereby preventing or reducing the occurrence of disulfide bonding that could give rise to an lgG2 isoform other than lgG2-A. The pharmaceutical compositions of the disclosure include those in which, for example, about 10% to about 99.999% of the antagonist CD40 polypeptide in the pharmaceutical composition is present in a single disulfide-bonded isoform, such as the lgG2-A isoform. For example, pharmaceutical compositions of the disclosure include those containing an antagonist CD40 polypeptide in which, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more, of the polypeptide in the pharmaceutical composition is present in a single disulfide-bonded isoform.

[0314] Techniques for measuring the relative quantities of various disulfide-bonded isoforms present in a subject or sample of an antagonist CD40 polypeptide include liquid chromatography techniques known in the art and described herein, such as those exemplified in Wypych et al., The Journal of Biological Chemistry 283:16194-16205, 2008, the disclosure of which is incorporated herein by reference in its entirety.

[0315] Effects on T-reg Cell Proliferation Using Antagonistic CD40 Polypeptides for Treating Cancer and Infectious Disease

[0316] Antagonistic CD40 polypeptides described herein (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can be used to attenuate the activity of T-reg cells that typically accompanies T cell-mediated cytotoxicity against “self” cells, such as the attack of a tumor cell by a T lymphocyte. This can be achieved, for instance, due to the ability of antagonistic CD40 polypeptides described herein to inhibit the proliferation of, and / or to directly kill, T-reg cells. Antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can, thus, be administered (e.g., by any of a variety of routes of administration described herein) to a mammalian subject, such as a human, to prolong the duration of an adaptive immune response, such as a response against a cancer cell or a pathogenic organism. In this way, for example, antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof described herein, may synergize with existing techniques to enhance T lymphocyte-based therapy for cancer and for infectious diseases. For instance, CD40 antagonists described herein may be administered to suppress T-reg cell activity, thereby enhancing the cytotoxic effect of tumor reactive T cells. CD40 antagonists may also synergize with existing strategies to promote tumor-reactive T cell survival, such as lymphodepletion and growth factor therapy, and in turn prolong the duration of anti-tumor reactivity in vivo.

[0317] Antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, and antigenbinding fragments thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can also be used to treat a broad array of infectious diseases in a mammalian subject (e.g., a human), as inhibition of T-reg proliferation promotes the activity of CD8+ T lymphocytes capable of mounting an attack on pathogenic organisms. Additionally, antagonistic CD40 antibodies and antigen-binding fragments thereof described herein can be used to treat a wide variety of infectious diseases, such as Mycobacterium tuberculosis, in a human or an agricultural farm animal (e.g., a bovine mammal, pig, cow, horse, sheep, goat, cat, dog, rabbit, hamster, guinea pig, or other non-human mammal).

[0318] Direct Effects on CD40-Expressing Cancer Cells

[0319] Antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, or antigenbinding fragments thereof described herein (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may bind and inactivate CD40 on the surface of a cancer cell, such as a CD40-expressing tumor cell. For instance, antagonistic CD40 antibodies and antigen-binding fragments thereof described herein may bind CD40 on the surface a T cell lymphoma cell (e.g., a Hodgkin or cutaneous non-Hodgkin lymphoma cell), ovarian cancer cell, colon cancer cell, multiple myeloma cell, or renal cell carcinoma cell, among others. The ability of antagonistic CD40 antibodies and antigen-binding fragments thereof described herein to bind CD40 directly on a cancer cell provides another pathway by which these molecules may attenuate cancer cell survival and proliferation. For instance, an antagonistic CD40 polypeptide described herein, such as an antagonistic CD40 single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct, may bind CD40 directly on the surface of a cancer cell (e.g., a cutaneous T cell lymphoma cell, ovarian cancer cell, colon cancer cell, or multiple myeloma cell, such as an ovarian cancer cell) to suppress the ability of the cell to proliferate and / or to promote apoptosis of the cell. CD40 Antagonist Polypeptides Are Not Reliant on Additional CD40-Binding Agents for

[0320] Activity

[0321] Significantly, antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof described herein (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof), are capable of binding CD40, thereby suppressing CD40-mediated signaling without the need for an endogenous CD40 binding agent, such as CD40L, among others. Antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof described herein do not require CD40 ligand to suppress the natural ligand functions described herein. Without being limited by mechanism, antagonistic CD40 antibodies or antigen-binding fragments thereof described herein may exhibit this property due to the ability of these antibodies or antigen-binding fragments thereof to bind CD40 at particular epitopes that, when bound, stabilize the anti-parallel dimer conformation of CD40. This structural configuration is not capable of potentiating a signal. By maintaining CD40 in an inactive structural state, antagonistic CD40 polypeptides described herein may prevent CD40 agonists from restoring activity of a CD40-expressing cell.

[0322] For instance, antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof described herein (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof), may bind CD40 on the surface of a CD40- expressing cell, such as a T-reg cell, cancer cell, myeloid-derived suppressor cell (MDSC), T cells, B cells, monocytes, neutrophils, platelets, granulocytes, bone marrow-derived lymphoid cells, and parenchymal cells, and inhibit the proliferation of such cells in the presence or absence of a cognate or natural CD40 ligand. For example, antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof described herein, may inhibit the proliferation of such cells by, e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cells that are not treated with the CD40 antagonist polypeptide. The antagonistic CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-biding fragment thereof) may exhibit an IC50 value in such a cell proliferation assay that is largely unchanged by the presence or absence of a CD40 cognate or natural ligand (e.g., an IC50 value in the presence of TNFa that is changed by less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1 % relative to the IC50 value of the antagonistic CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) in the same cell proliferation assay in the absence of the CD40 cognate or natural ligand). Similarly, antagonistic CD40 polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof described herein, may inhibit CD40 signaling as assessed by measuring the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K1 1 , TRAF2, TRAF3, relB, and clAP2 / BIRC3 by, e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cells that are not treated with the CD40 antagonist polypeptide. The antagonistic CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-biding fragment thereof) may exhibit an IC50 value in such a gene expression assay that is largely unchanged by the presence or absence of a CD40 cognate or natural ligand (e.g., an IC50 value in the presence of CD40 cognate or natural ligand that is changed by less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1 % relative to the IC50 value of the antagonistic CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) in the same gene expression assay in the absence of CD40 cognate or natural ligand such as CD153).

[0323] Direct Killing of T-reg Cells, MDSCs, and CD40-Expressing Cancer Cells

[0324] Antagonistic CD40 polypeptides disclosed herein (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof), and constructs thereof, may, for instance, not only reduce the proliferation of T-reg cells, CD40-expressing cancer cells, and / or MDSCs, but may also induce the death of T-reg cells, CD40-expressing cancer cells, and / or MDSCs within a sample (e.g., within a subject, such as a human subject administered the antagonist). Antagonistic CD40 polypeptides described herein may be capable, for instance, of reducing the total quantity of T-reg cells, cancer cells (such as cutaneous T cell lymphoma cells, ovarian cancer cells, colon cancer cells, renal cell carcinoma cells or multiple myeloma cells, among others), and / or MDSCs in a subject or in a sample treated with an antagonist CD40 antibody or antigen-binding fragment thereof (such as a sample isolated from a human subject undergoing treatment for cancer or an infectious disease as described herein) by, e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to a subject or sample not treated with an antagonist CD40 antibody or antigen-binding fragment thereof.

[0325] The ability of antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) described herein to attenuate T-reg, MDSC, and / or cancer cell growth may be due, in part, to the ability of these polypeptides to diminish the quantity of soluble CD40 within a subject or sample (e.g., a sample isolated from a human subject undergoing treatment for cancer or an infectious disease as described herein). In the absence of this beneficial activity, soluble CD40 can be secreted by, e.g., T-reg cells, and could otherwise interfere with the ability of CD40 antagonists to localize to CD40 at the surface of a T-reg cell, CD40-expressing cancer cell, or MDSC by binding and sequestering such antagonists in the extracellular environment. By reducing CD40 secretion, antagonistic CD40 antibodies or antigen-binding fragments thereof described herein may render T-reg cells, CD40-expressing cancer cells, and / or MDSCs increasingly susceptible to therapeutic molecules, such as an antagonistic CD40 antibody or antigen-binding fragment thereof, and / or additional anti-cancer agents, such as those described herein or known in the art, that may be used in conjunction with the compositions and methods described herein.

[0326] Modulation of T-reg Cells, MDSCs, and T Effector Cells in the Tumor Microenvironment

[0327] Antagonist CD40 polypeptides described herein (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof), may inhibit the proliferation of T-reg cells with a greater potency in a subject suffering from cancer relative to a subject that does not have cancer. The antagonist CD40 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may inhibit the proliferation of T-reg cells with a greater potency in the tumor microenvironment relative to a site that is free of cancer cells, such as a site distal from a tumor in a subject suffering from cancer or in a subject without cancer. This effect may be determined using, for example, a cell death assay as described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may exhibit an IC50 for reducing or inhibiting the proliferation of T-reg cells in the tumor microenvironment that is less than the IC50 of the polypeptides for reducing or inhibiting the proliferation of T-reg cells in a site that is free of cancer cells by, for example, 1 .1 -fold, 1 .2-fold, 1 .3- fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1 .7-fold, 1 .8-fold, 1 .9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1 ,000- fold, 10,000-fold, or more. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit the proliferation of T-reg cells or may promote the apoptosis of T-reg cells with a potency that is greater in the tumor microenvironment containing CD40-expressing cancer cells, such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic or lymphoid cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, stomach cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, than in a site that is free of such cancer cells, such as a site distal from a tumor in a subject suffering from one or more of the foregoing cancers or a in a subject without cancer.

[0328] The CD40 polypeptides described herein (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof), and constructs thereof, may inhibit the proliferation of MDSCs with a greater potency in a subject suffering from cancer relative to a subject that does not have cancer. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit the proliferation of MDSCs with a greater potency in the tumor microenvironment relative to a site that is free of cancer cells, such as a site distal from a tumor in a subject suffering from cancer or in a subject without cancer. This effect may be determined using, for example, a cell death assay described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may have an IC50 for reducing or inhibiting the proliferation of MDSCs in the tumor microenvironment that is less than the IC50 of the polypeptides for reducing or inhibiting the proliferation of MDSCs in a site that is free of cancer cells by, for example, 1 .1 -fold, 1 .2-fold, 1 .3-fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20- fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1 ,000-fold, 10,000-fold, or more. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit the proliferation of MDSCs or may promote the apoptosis of MDSCs with a potency that is greater in the tumor microenvironment containing CD40- expressing cancer cells, such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic or lymphoid cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, stomach cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, than in a site that is free of such cancer cells, such as a site distal from a tumor in a subject suffering from one or more of the foregoing cancers or a in a subject without cancer.

[0329] Dual Proliferative and Cell Killing Effects of CD40 Antagonist Polypeptides

[0330] The stimulation of T-reg and MDSC proliferation engenders important therapeutic benefits, as these cells suppress the activity of T effector cells, such as autoreactive CD8+ T cells that mount an inappropriate immune response against “self” tissue. Similarly, the direct killing of B cells, monocytes, neutrophils, platelets, macrophages, dendritic cells, epithelial cells, endothelial cells, granulocytes, mesenchymal cells, and T effector cells imparts therapeutic activity, as CD40 antagonist polypeptides can be used to reduce the quantity of self-reactive T cells in a subject suffering from autoimmunity, GVHD, transplant rejection, allergies, chronic inflammatory disease, asthma, or another disorder described herein. CD40 antagonist polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can be used to suppress immunological conditions in a subject, such as a mammalian subject (e.g., a human subject).

[0331] Additionally, the ability of CD40 antagonist polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) to stimulate proliferation of CD40-positive parenchymal cells provides the beneficial effect of inducing tissue and organ regeneration. Exemplary CD40-positive parenchymal cells that can be induced to proliferate using the CD40 antagonist polypeptides of the disclosure include, without limitation, cells of the pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, structures of the head, eye, thymus, tongue, bone, liver, small intestine, large intestine, gut, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryos, and testes. Thus, CD40 antagonist polypeptides described herein can be used to treat disorders in which the regeneration, protection, and / or healing of one or more of these cell types is desired. Exemplary diseases that can be treated using the CD40 antagonist polypeptides of the disclosure are described herein.

[0332] Antagonistic CD40 Polypeptides that Bind CD40 from Non-Human Animals

[0333] In addition to binding epitopes within human CD40, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure also include those that specifically bind epitopes containing the equivalent motif within CD40 derived from non-human animals, such as non-human mammals, e.g., in a cow, bison, mouse, rat, or monkey, among others.

[0334] Antagonistic CD40 Single-Chain Polypeptides

[0335] CD40 antagonists of the disclosure (e.g., antibodies or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be in the form of a single-chain polypeptide, such as a single-chain polypeptide that contains one, two, or three heavy chain CDRs of a monoclonal CD40 antagonist antibody described herein, and / or one, two, or three light chain CDRs of a monoclonal CD40 antagonist antibody described. Single-chain polypeptides may be in the form of an antibody fragment, e.g., an antibody fragment described herein or known in the art, such as a scFv fragment. Single chain polypeptides may alternatively contain one or more CDRs described herein covalently bound to one another using conventional bond-forming techniques known in the art, for instance, by an amide bond, a thioether bond, a carbon-carbon bond, or by a linker, such as a peptide linker or a multi-valent electrophile (e.g., a bis(bromomethyl) arene derivative, such as a bis(bromomethyl)benzene or bis(bromomethyl)pyridine) described herein or known in the art.

[0336] Single-chain polypeptides can be produced by a variety of recombinant and synthetic techniques, such as by recombinant gene expression or solid-phase peptide synthesis procedures described herein or known in the art. For instance, one of skill in the art can design polynucleotides encoding, e.g., two or more of the above CDRs operatively linked to one another in frame to produce a continuous, single-chain peptide containing these CDRs. Optionally, the CDRs may be separated by a spacer, such as by a framework region (e.g., a framework sequence described herein or a framework region of a germline consensus sequence of a human antibody) or a flexible linker, such as a poly-glycine or glycine / serine linker described herein or known in the art. When produced by chemical synthesis methods, native chemical ligation can optionally be used as a strategy for the synthesis of long peptides (e.g., greater than 50 amino acids). Native chemical ligation protocols are known in the art and have been described, e.g., by Dawson et al. Science, 266:776-779, 1994; incorporated herein by reference. A detailed description of techniques for the production of singlechain polypeptides, full-length antibodies, and antibody fragments is provided in the sections that follow.

[0337] Nucleic Acids and Expression Systems

[0338] Antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or antigenbinding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can be prepared by any of a variety of established techniques. For instance, an antagonistic CD40 polypeptide (e.g., single-chain polypeptides, antibodies, or antigenbinding fragments thereof) of the disclosure can be prepared by recombinant expression of one or more immunoglobulin light and heavy chain genes in a host cell. For instance, to express an antibody recombinantly, a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates, 1989), and in U.S. Patent No. 4,816,397; incorporated herein by reference.

[0339] Vectors for Expression of Antagonistic CD40 Polypeptides

[0340] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments of the disclosure include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent. No. 5,801 ,030); incorporated herein by reference.

[0341] Genome Editing Techniques

[0342] In addition to viral vectors, a variety of additional methods have been developed for the incorporation of genes, e.g., those encoding antibody light and heavy chains, single-chain polypeptides, single-chain variable fragments (scFvs), tandem scFvs, Fab domains, F(ab’)2 domains, diabodies, and triabodies, among others, into the genomes of target cells for polypeptide expression. One such method that can be used for incorporating polynucleotides encoding anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) into prokaryotic or eukaryotic cells includes transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by excision sites at the 5’ and 3’ positions. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some cases, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a prokaryotic or eukaryotic cell by transposase-catalyzed cleavage of similar excision sites that exist within nuclear genome of the cell. This allows the gene encoding, e.g., an anti-CD40 antibody or fragment or domain thereof to be inserted into the cleaved nuclear DNA at the excision sites, and subsequent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the prokaryotic or eukaryotic cell genome completes the incorporation process. In some embodiments, the transposon may be a retrotransposon, such that the gene encoding the antibody is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the prokaryotic or eukaryotic cell genome. Exemplary transposon systems include the piggyBac transposon (described in detail in WO 2010 / 085699) and the sleeping beauty transposon (described in detail in US20050112764); incorporated herein by reference.

[0343] Another useful method for the integration of nucleic acid molecules encoding anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) into the genome of a prokaryotic or eukaryotic cell is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which is a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against infection by viruses. The CRISPR / Cas system consists of palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeatspacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can theoretically design a CRISPR / Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al., Nat. Biotech., 31 :227-229, 2013) and can be used as an efficient means of site-specifically editing eukaryotic or prokaryotic genomes in order to cleave DNA prior to the incorporation of a polynucleotide encoding an anti-CD40 polypeptide of the disclosure. The use of CRISPR / Cas to modulate gene expression has been described in U.S. Patent No. 8,697,359, which is incorporated herein by reference. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a polynucleotide encoding a CD40 antibody or antibody fragment of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) include the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. Zinc finger nucleases and TALENs for use in genome editing applications are described in Urnov et al. (Nat. Rev. Genet. 11 :636-646, 2010); and in Joung et al., (Nat. Rev. Mol. Cell. Bio. 14:49-55, 2013); incorporated herein by reference. Additional genome editing techniques that can be used to incorporate polynucleotides encoding antibodies of the disclosure into the genome of a prokaryotic or eukaryotic cell include the use of ARCUS™ meganucleases that can be rationally designed to site- specifically cleave genomic DNA. The use of these enzymes for the incorporation of polynucleotides encoding antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or antibody fragments) of the disclosure into the genome of a prokaryotic or eukaryotic cell is particularly advantageous in view of the structure-activity relationships that have been established for such enzymes. Single-chain meganucleases can thus be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations. These single-chain nucleases have been described extensively, e.g., in U.S. Patent Nos. 8,021 ,867 and 8,445,251 ; incorporated herein by reference.

[0344] Polynucleotide Sequence Elements

[0345] To express antagonistic polypeptides (e.g., antagonistic CD40 single-chain polypeptides, antibodies, or antibody fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure, polynucleotides encoding partial or full-length light and heavy chains, or CDRs thereof, e.g., obtained as described above, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Polynucleotides encoding, e.g., the light chain gene and the heavy chain of a CD40 antibody can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector using established techniques described herein or known in the art.

[0346] In addition to polynucleotides encoding the heavy and light chains of an antibody (or a polynucleotide encoding a single-chain polypeptide or an antibody fragment, such as a scFv molecule), the recombinant expression vectors of the disclosure may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed or the level of expression of protein desired. For instance, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5, 168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615.

[0347] In addition to the antibody chain or CDR genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216; 4,634,665; and 5,179,017). For example, typically the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin, or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection). To express the light and heavy chains of a CD40 antibody or a CD40 antibody fragment, the expression vector(s) containing polynucleotides encoding the heavy and light chains can be transfected into a host cell by standard techniques.

[0348] Polynucleotides Encoding Modified Antagonistic CD40 Polypeptides

[0349] Antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or antibody fragments of the disclosure, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may feature differences in the sequence of one or more CDRs. In other cases, the polypeptides of the disclosure may feature differences in one or more framework regions. Exemplary framework regions include, for example, human framework regions described in U.S. Patent No. 7,829,086, and primate framework regions as described in EP 1945668; incorporated herein by reference. Alternatively, polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the disclosure may exhibit differences in the sequence of one or more CDRs and differences in one or more framework regions. To generate nucleic acids encoding such CD40 antagonist polypeptides, DNA fragments encoding, e.g., one or more CDRs, or at least one, or both, of the light chain variable regions and the heavy chain variable regions can be produced by chemical synthesis (e.g., by solid-phase polynucleotide synthesis techniques), in vitro gene amplification (e.g., by polymerase chain reaction techniques), or by replication of the polynucleotide in a host organism. For instance, nucleic acids encoding anti-CD40 polypeptides of the disclosure may be obtained by amplification and modification of germline DNA or cDNA encoding light and heavy chain variable sequences.

[0350] This can be achieved, for example, by performing site-directed mutagenesis of germline DNA or cDNA and amplifying the resulting polynucleotides using the polymerase chain reaction (PCR) according to established procedures. Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991 ; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al., Eur. J. Immunol. 24:827-836, 1994; incorporated herein by reference). Chimeric nucleic acid constructs encoding human heavy and light chain variable regions containing one or more of the CDRs of the disclosed antibody can be produced, e.g., using established cloning techniques known in the art. Additionally, a polynucleotide encoding the heavy or light chain variable region of the disclosed antibody can be synthesized and used as a template for mutagenesis to generate a variant as described herein using routine mutagenesis techniques. Alternatively, a DNA fragment encoding the variant can be directly synthesized (e.g., by established solid phase nucleic acid chemical synthesis procedures).

[0351] Once DNA fragments encoding related VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0352] The isolated DNA encoding the VH region of an anti-CD40 antibody of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can be converted to a full-length heavy chain gene (as well as a Fab heavy chain gene), e.g., by operatively linking the VH- encoding DNA to another DNA molecule encoding heavy chain constant region domains (CH1 , CH2, CH3, and, optionally, CH4). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991 ) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG 1 , lgG2, lgG3, lgG4, IgA, IgE, IgM, or IgD constant region, and in certain examples is an IgG 1 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 domain.

[0353] The isolated DNA encoding the VL region of an anti-CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof, such as such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991 )) and DNA fragments encompassing these regions can be obtained, e.g., by amplification in a prokaryotic or eukaryotic cell of a polynucleotide encoding these regions, by PCR amplification, or by chemical polynucleotide synthesis. The light chain constant region can be a kappa (K) or lambda (A) constant region, but in certain examples is a kappa constant region. To create a scFv gene, the VH and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., a polynucleotide encoding a flexible, hydrophilic amino acid sequence, such as the amino acid sequence (Gly4Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the linker (see e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990). Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to a CD40. The molecules expressed from such truncated DNA molecules are also encompassed by the polypeptides of the disclosure. Dual specific antibodies, i.e., antibodies that bind CD40 and a different antigen using the same binding site, can be produced by mutating amino acid residues in the light chain and / or heavy chain CDRs.

[0354] Dual specific antibodies, e.g., antibodies that bind a CD40 and a different antigen using the same binding site, can be produced by mutating amino acid residues in the light chain and / or heavy chain CDRs. Dual functional antibodies can be made by expressing a polynucleotide engineered to encode a dual specific antibody.

[0355] Modified antagonistic CD40 antibodies and antibody fragments of the disclosure can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984 The Pierce Chemical Co., Rockford, 111 ; incorporated herein by reference). Variant antibodies can also be generated using a cell-free synthetic platform (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals); incorporated herein by reference).

[0356] Host Cells for Expression of Antagonistic CD40 Polypeptides

[0357] It is possible to express the polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure in either prokaryotic or eukaryotic host cells. In some embodiments, expression of polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) is performed in eukaryotic cells, e.g., mammalian host cells, for optimal secretion of a properly folded and immunologically active antibody. Exemplary mammalian host cells for expressing the recombinant polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the disclosure include Chinese Hamster Ovary (CHO cells) (including DHFR- CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216- 4220, 1980, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, Mol. Biol. 159:601 -621 , 1982, NSO myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. Additional cell types that may be useful for the expression of single-chain polypeptides, antibodies, and fragments thereof include bacterial cells, such as BL-21 (DE3) E. co / / cells, which can be transformed with vectors containing foreign DNA according to established protocols. Additional eukaryotic cells that may be useful for expression of polypeptides include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art. When recombinant expression vectors encoding antibody genes (e.g., genes encoding one or more CDRs, an antibody heavy chain, or an antibody light chain) are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown.

[0358] Polypeptides of the disclosure (e.g., antagonistic CD40 single-chain polypeptides, antibodies, and antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. The disclosure also includes methods in which the above procedure is varied according to established protocols known in the art. For example, it can be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-CD40 antibody of this disclosure in order to produce an antigen-binding fragment of the antibody.

[0359] Once an anti-CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) thereof of the disclosure has been produced by recombinant expression, it can be purified by any method known in the art, such as a method useful for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a CD40 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the disclosure can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification or to produce therapeutic conjugates.

[0360] Once isolated, an anti-CD40 antibody or antigen-binding fragments thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980); incorporated herein by reference), or by gel filtration chromatography, such as on a SUPERDEX™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0361] Exemplary CD40 Antagonist Antibodies or Antigen-Binding Fragments

[0362] The disclosure features the production of antagonistic antibodies or antigen-binding fragments thereof of the disclosure (e.g., antagonistic CD40 antibodies or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof), for example, by expressing a polynucleotide engineered to encode the antagonistic antibodies or antigen-binding fragments thereof. For instance, nucleic acids encoding such antagonistic CD40 antibodies or antigen-binding fragments thereof of the disclosure may bind an epitope.

[0363] For instance, a nucleic acid molecule encoding an antagonistic CD40 antibody or antigenbinding fragments thereof that binds an epitope within amino acids 104-172 of SEQ ID NO: 37 can be delivered to and expressed in a cell to produce the antibody or antigen-binding fragment thereof. For example, the antagonistic CD40 antibodies or antigen-binding fragments thereof encoded by the nucleic acid molecule may bind an epitope of, within, or including one or more of amino acids 104-152 (e.g., amino acids 124-132, SCSPGFGVK, SEQ ID NO: 49) of the CD40 amino acid sequence (SEQ ID NO:4) and / or amino acids 123-172 (e.g., amino acids 143-152, CEPCPVGFFS, SEQ ID NO: 50) of the CD40 amino acid sequence (SEQ ID NO: 37). In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 124-132 of SEQ ID NO: 37. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof is or includes one or more of amino acids 143-152 of SEQ ID NO: 37. The antagonistic CD40 antibodies or antigen-binding fragments thereof encoded by the nucleic acid molecule may also bind an epitope(s) that exhibits at least 80% sequence identity (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to one or both of these sequences and an epitope(s) that contains conservative amino acid substitutions relative to one or both of these sequences. The anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) encoded by the nucleic acid molecule may also specifically bind an epitope within human CD40 that includes at least five continuous or discontinuous amino acid residues of amino acids 104-172 of SEQ ID NO: 37 (e.g., at least five continuous or discontinuous amino acid residues of amino acids 114-142 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 133-162 of SEQ ID NO: 37, or at least five continuous or discontinuous amino acid residues of amino acids 124-132 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 143-152 of SEQ ID NO: 37). Furthermore, anti- CD40 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) encoded by the nucleic acid molecule may also specifically bind an epitope within human CD40 that includes at least five continuous or discontinuous amino acid residues of amino acids 104-172 of SEQ ID NO: 37 (e.g., at least five continuous or discontinuous amino acid residues of amino acids 114-142 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 133-162 of SEQ ID NO: 37, or at least five continuous or discontinuous amino acid residues of amino acids 124-132 of SEQ ID NO: 37 and / or at least five continuous or discontinuous amino acid residues of amino acids 143-152 of SEQ ID NO: 37), as well as an epitope(s) that exhibits at least 80% sequence identity (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to one or both of these sequences and an epitope(s) that contains conservative amino acid substitutions relative to one or both of these sequences. A nucleic acid molecule encoding the CD40 antibodies or antigenbinding fragments thereof can be engineered to target cells that express CD40, such as, e.g., T cells, B cells, platelets, macrophages, dendritic cells, epithelial cells, endothelial cells, and mesenchymal cells.

[0364] Antagonistic CD40 Polypeptide Conjugates

[0365] Prior to administration of antagonistic CD40 polypeptides (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) of the disclosure to a mammalian subject (e.g., a human), it may be desirable to conjugate the polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) to a second molecule, e g., to modulate the activity of the polypeptide in vivo. Antagonistic CD40 single-chain polypeptides, antibodies, and fragments thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can be conjugated to other molecules at either the N-terminus or C-terminus of a light or heavy chain of the polypeptide using any one of a variety of established conjugation strategies that are well-known in the art. Examples of pairs of reactive functional groups that can be used to covalently tether an antagonistic CD40 single-chain polypeptide, antibody, or fragment thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) to another molecule include, without limitation, thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha, beta-unsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides.

[0366] Antagonistic CD40 single-chain polypeptides, antibodies, and fragments thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can be covalently appended directly to another molecule by chemical conjugation as described. Alternatively, fusion proteins containing antagonistic CD40 single-chain polypeptides, antibodies, and fragments thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell). This can be accomplished, for example, by incorporating a polynucleotide encoding the fusion protein into the nuclear genome of a cell (e.g., using techniques described herein or known in the art). Optionally, single-chain polypeptides, antibodies, and fragments thereof of the disclosure (e.g., antagonistic CD40 single-chain polypeptides, antibodies, and fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can be joined to a second molecule by forming a covalent bond between the antibody and a linker. This linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to ligation to the anti-CD40 single-chain polypeptide, antibody, or fragment thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof). Examples of linkers that can be used for the formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some cases, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally occurring proteins and are thus more resistant to degradation by endogenous proteases. Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell). Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571 -582, 2012).

[0367] Drug-Polypeptide Conjugates

[0368] An antagonistic CD40 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) of the disclosure can additionally be conjugated to, admixed with, or administered separately from a therapeutic agent, such as a cytotoxic molecule. Conjugates of the disclosure may be applicable to the treatment or prevention of a disease associated with aberrant cell proliferation, such as a cancer described herein. Exemplary cytotoxic agents that can be conjugated to, admixed with, or administered separately from an antagonistic CD40 polypeptide include, without limitation, antineoplastic agents such as: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin; aldesleukin; altretamine; ambomycin; a. metantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; camptothecin; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daca (n- [2- (dimethyl-amino) ethyl] acridine-4-carboxamide); dactinomycin; daunorubicin hydrochloride; daunomycin; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; dolasatins; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; ethiodized oil i 131 ; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198; homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-i a; interferon gamma-i b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin ; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium chloride sr 89; sulofenur; talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine; tomudex; top53; topotecan hydrochloride; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2' deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2chloro-2'-arabino- fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP- 751 ; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N-nitrosourea (MNU); N, N'-Bis (2-chloroethyl)-N-nitrosourea (BCNU); N- (2-chloroethyl)-N' cyclohexyl-N-nitrosourea (CCNU); N- (2-chloroethyl)-N'- (trans-4- methylcyclohexyl-N-nitrosourea (MeCCNU); N- (2-chloroethyl)-N'- (diethyl) ethylphosphonate-N- nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin; carboplatin; ormaplatin; oxaliplati n ;C1 -973; DWA 21 14R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-amino camptothecin; topotecan; CPT-11 ; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; N- (4- hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or 2- chlorodeoxyadenosine (2-Cda).

[0369] Other therapeutic compounds that can be conjugated to, admixed with, or administered separately from an antagonistic CD40 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the disclosure in order to treat, prevent, or study the progression of a disease associated with aberrant cell proliferation include, but are not limited to, cytotoxic agents such as 20-pi-1 ,25 dihydroxyvitamin D3; 5- ethynyluracil; abiraterone; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein-1 ; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A ; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816 ; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2'deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9- ; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epothilones (A, R = H; B, R = Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4'-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; rnerbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factorsaporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06- benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; podophyllotoxin; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1 ; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

[0370] Labeled Anti-CD40 Polypeptides

[0371] Antagonistic CD40 single-chain polypeptides, antibodies, or antigen-binding fragments thereof (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) may be conjugated to another molecule (e.g., an epitope tag) for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is a common strategy that is employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Examples of epitope tag molecules that can be conjugated to antagonistic CD40 single-chain polypeptides, antibodies, or fragments thereof for the purposes of molecular recognition include, without limitation, maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by such complementary molecules as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify an antagonistic CD40 single-chain polypeptide, antibody, or fragment thereof of the disclosure that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid-phase resin containing an complementary molecule that can selectively recognize and bind the epitope tag of the antagonistic anti- CD40 antibody or fragment thereof. Examples of solid-phase resins include agarose beads, which are compatible with purifications in aqueous solution.

[0372] An antagonistic CD40 polypeptide of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can also be covalently appended to a fluorescent molecule, e.g., to detect the antibody or antigen-binding fragment thereof by fluorimetry and / or by direct visualization using fluorescence microscopy. Exemplary fluorescent molecules that can be conjugated to polypeptides of the disclosure include green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, Hoechst, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhodamine, and cyanine. Additional examples of fluorescent molecules suitable for conjugation to polypeptides of the disclosure are well-known in the art and have been described in detail in, e.g., U.S. Patent Nos. 7,417,131 and 7,413,874, each of which is incorporated by reference herein.

[0373] Antagonistic CD40 polypeptides containing a fluorescent molecule are particularly useful for monitoring the cell-surface localization properties of polypeptides, such as single-chain polypeptides, antibodies, and fragments thereof of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof). For instance, one can expose cultured mammalian cells (e.g., T-reg cells, T cells, B cells, monocytes, neutrophils, platelets, granulocytes, bone marrow- derived lymphoid cells, and parenchymal cells) to antagonistic CD40 single-chain polypeptides, antibodies, or fragments thereof of the disclosure that have been covalently conjugated to a fluorescent molecule and subsequently analyze these cells using conventional fluorescent microscopy techniques known in the art. Confocal fluorescent microscopy is a particularly powerful method for determining cell-surface localization of antagonistic anti-CD40 single-chain polypeptides, antibodies, or fragments thereof, as individual planes of a cell can be analyzed in order to distinguish antibodies or fragments thereof that have been internalized into a cell’s interior, e.g., by receptor-mediated endocytosis, from those that are bound to the external face of the cell membrane. Additionally, cells can be treated with antagonistic CD40 antibodies conjugated to a fluorescent molecule that emits visible light of a particular wavelength (e.g., fluorescein, which fluoresces at about 535 nm) and an additional fluorescent molecule that is known to localize to a particular site on the T-reg cell surface and that fluoresces at a different wavelength (e.g., a molecule that localizes to CD25 and that fluoresces at about 599 nm). The resulting emission patterns can be visualized by confocal fluorescence microscopy and the images from these two wavelengths can be merged in order to reveal information regarding the location of the antagonistic CD40 single-chain polypeptide, antibody, or antigen-binding fragment thereof on the T-reg cell surface with respect to other receptors.

[0374] Bioluminescent proteins can also be incorporated into a fusion protein for the purposes of detection and visualization of an antagonistic anti-CD40 polypeptide of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof). Bioluminescent proteins, such as Luciferase and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine). Exemplary bioluminescent proteins suitable for use as a diagnostic sequence and methods for their use are described in, e.g., U.S. Patent Nos. 5,292,658; 5,670,356; 6,171 ,809; and 7,183,092, each of which is herein incorporated by reference. Antagonistic CD40 single-chain polypeptides, antibodies, or fragments thereof labeled with bioluminescent proteins are a useful tool for the detection of antibodies of the disclosure following an in vitro assay. For instance, the presence of an antagonistic CD40 antibody that has been conjugated to a bioluminescent protein can be detected among a complex mixture of additional proteins by separating the components of the mixture using gel electrophoresis methods known in the art (e.g., native gel analysis) and subsequently transferring the separated proteins to a membrane to perform a Western blot. Detection of the antagonistic CD40 antibody among the mixture of other proteins can be achieved by treating the membrane with an appropriate Luciferase substrate and subsequently visualizing the mixture of proteins on film using established protocols.

[0375] The polypeptides (e.g., antagonistic CD40 single-chain polypeptides, antibodies, and fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can also be conjugated to a molecule comprising a radioactive nucleus, such that an antibody or fragment thereof of the disclosure can be detected by analyzing the radioactive emission pattern of the nucleus. Alternatively, an antagonistic CD40 antibody or fragment thereof can be modified directly by incorporating a radioactive nucleus within the antibody during the preparation of the protein. Radioactive isotopes of methionine (35S), nitrogen (15N), or carbon (13C) can be incorporated into antibodies or fragments thereof of the disclosure by, e.g., culturing bacteria in media that has been supplemented with nutrients containing these isotopes. Optionally, tyrosine derivatives containing a radioactive halogen can be incorporated into an antagonistic CD40 antibody or fragment thereof by, e.g., culturing bacterial cells in media supplemented with radiolabeled tyrosine. It has been shown that tyrosine functionalized with a radioactive halogen at the C2 position of the phenol system are rapidly incorporated into elongating polypeptide chains using the endogenous translation enzymes in vivo (U.S. Patent No. 4,925,651 ; incorporated herein by reference). Halogens include fluorine, chlorine, bromine, iodine, and astatine. Additionally, antagonistic CD40 antibodies or fragments thereof can be modified following isolation and purification from cell culture by functionalizing antibodies or fragments thereof of the disclosure with a radioactive isotope. The halogens represent a class of isotopes that can be readily incorporated into a purified protein by aromatic substitution at tyrosine or tryptophan, e.g., via reaction of one or more of these residues with an electrophilic halogen species. Examples of radioactive halogen isotopes include18F,75Br, 77gr1221 1231 124| 1251 1291 131 | or211At.

[0376] Another alternative strategy for the incorporation of a radioactive isotope is the covalent attachment of a chelating group to the antagonistic anti-CD40 polypeptide (e.g., single-chain polypeptide, antibody, or fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof). Chelating groups can be covalently appended to an antagonistic CD40 polypeptide by attachment to a reactive functional group, such as a thiol, amino group, alcohol, or carboxylic acid. The chelating groups can then be modified to contain any of a variety of metallic radioisotopes, including, without limitation, such radioactive nuclides as125l,67Ga,1111n, "Tc,169Yb,186Re,123l,124l,125l,131l,99mTc,1111n,64Cu,67Cu,186Re,188Re,177Lu,90Y,77As,72As,86Y,89Zr,211At,212Bi,213Bi, or225Ac.

[0377] In some embodiments, it may be desirable to covalently conjugate the polypeptides (e.g., antagonistic CD40 single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure with a chelating group capable of binding a metal ion from heavy elements or rare earth ions, such as Gd3+, Fe3+, Mn3+, or Cr2+. Conjugates containing chelating groups that are coordinated to such paramagnetic metals are useful as in magnetic resonance imaging (MRI) applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III). In this way, antagonistic CD40 antibodies can be detected by MRI spectroscopy. For instance, one can administer antagonistic CD40 antibodies or fragments thereof conjugated to chelating groups bound to paramagnetic ions to a mammalian subject (e.g., a human subject) to monitor the distribution of the antibody following administration. This can be achieved by administration of the antibody to a subject by any of the administration routes described herein, such as intravenously, and subsequently analyzing the location of the administered antibody by recording an MRI of the subject according to established protocols.

[0378] Antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can additionally be conjugated to other molecules for the purpose of improving the solubility and stability of the protein in aqueous solution. Examples of such molecules include polyethylene glycol (PEG), PSA, bovine serum albumin (BSA), and human serum albumin (HSA), among others. For instance, one can conjugate an antagonistic CD40 antibody or fragment thereof to carbohydrate moieties to evade detection of the antibody or fragment thereof by the immune system of the subject receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B-cell receptors in circulation. Alternatively, antagonistic CD40 antibodies or fragments thereof can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of antibodies of the disclosure. Exemplary molecules that can be conjugated to or inserted within anti-CD40 antibodies or fragments thereof of the disclosure to attenuate clearance and improve the pharmacokinetic profile of these antibodies and fragments include salvage receptor binding epitopes. These epitopes are found within the Fc region of an IgG immunoglobulin and have been shown to bind Fc receptors and prolong antibody half-life in human serum. The insertion of salvage receptor binding epitopes into anti-CD40 antibodies or fragments thereof can be achieved, e.g., as described in U.S. Patent No. 5,739,277; incorporated herein by reference.

[0379] Modified Antagonistic CD40 Polypeptides

[0380] In addition to conjugation to other therapeutic agents and labels for identification or visualization, anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can also be modified so as to improve their pharmacokinetic profile, biophysical stability, or inhibitory capacity. For instance, any cysteine residue not involved in maintaining the proper conformation of the anti-CD40 antibody or fragment thereof may be substituted with an isosteric amino acid (e.g., serine) in order to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cystine bond(s) may be added to the antibody or fragment thereof to improve its stability (particularly where the antibody is an antibody fragment, such as an Fv fragment). This can be accomplished, e.g., by altering a polynucleotide encoding the antibody heavy and light chains or a polynucleotide encoding an antibody fragment so as to encode one or more additional pairs of cysteine residues that can form disulfide bonds under oxidative conditions in order to reinforce antibody tertiary structure (see, e.g., U.S. Patent No. 7,422,899; incorporated herein by reference).

[0381] Another useful modification that may be made to anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure includes altering the glycosylation profile of these antibodies and fragments thereof. This can be achieved, e.g., by substituting, inserting, or deleting amino acids in an antagonistic CD40 antibody so as to insert or remove a glycosylation site. Glycosylation of antibodies typically occurs in N-linked or O-linked fashion. N-linked glycosylation is a process whereby the attachment of a carbohydrate moiety to an antibody occurs at the side chain of an asparagine residue. Consensus amino acid sequences for N-linked glycosylation include the tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X is any amino acid except proline. The insertion of either of these tripeptide sequences in a polypeptide (e.g., an anti-CD40 antibody) creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine are also competent substrates for glycoside formation. Addition of glycosylation sites to an anti-CD40 antibody can thus be accomplished by altering the amino acid sequence of the antibody (e.g., using recombinant expression techniques as described herein) such that it contains one or more of the above-described tripeptide sequences to promote N-linked glycosylation, or one or more serine or threonine residues to the sequence of the original antibody engender O-linked glycosylation (see, e.g., U.S. Patent No. 7,422,899; incorporated herein by reference).

[0382] In alternative cases, it may be desirable to modify the antibody or fragment thereof of the disclosure (e.g., an antagonistic CD40 antibody or fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. For instance, cysteine residues may be introduced in the Fc region of an anti-CD40 antibody or fragment thereof (e.g., by recombinant expression techniques as described herein), so as to facilitate additional inter-chain disulfide bond formation in this region. The homodimeric antibody thus generated may have increased conformational constraint, which may foster improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. {Cane. Res., 53:2560-2565, 1993); incorporated herein by reference. Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities (see Stevenson et al. {Anti-Canc. Drug Des., 3:219-230, 1989); incorporated herein by reference).

[0383] The serum half-life of anti-CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can be improved in some examples by incorporating one more amino acid modifications, such as by altering the CH1 or CL region of the Fab domain to introduce a salvage receptor motif, e.g., that found in the two loops of a CH2 domain of an Fc region of an IgG. Such alterations are described, for instance, in U.S. Patent No. 5,869,046 and U.S. Patent No. 6,121 ,022; incorporated herein by reference. Additional framework modifications can also be made to reduce immunogenicity of the antibody or fragment thereof or to reduce or remove T cell epitopes that reside therein, as described for instance in US2003 / 0153043; incorporated herein by reference.

[0384] Methods of Treatment

[0385] A physician of ordinary skill in the art can readily determine an effective amount of an antagonistic CD40 polypeptide described herein (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician could start prescribing doses of an antagonistic CD40 polypeptide at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Examples of the desired effect, without limitation, includes alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). Alternatively, a physician may begin a treatment regimen by administering an antagonistic CD40 polypeptide at a high dose and subsequently administer progressively lower doses until a therapeutic effect is achieved (e.g., a reduction in the proliferation of a population of CD8+ T cells, CD4+ T cells, and / or B cells) or a decrease in the peripheral secretion of IFN-y). In general, a suitable daily dose of an antibody or antigen-binding fragment thereof of the disclosure (e.g., an antagonistic CD40 antibody or antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) will be an amount of the antibody which is the lowest dose effective to produce a therapeutic effect. An antibody or antigen-binding fragment thereof of the disclosure (e.g., an antagonistic CD40 antibody or antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be administered by injection, e.g., by intravenous, intramuscular, intraperitoneal, or subcutaneous injection, optionally proximal to the site of a target tissue. A daily dose of a therapeutic composition of an antibody or antigen-binding fragment thereof of the disclosure (e.g., an antagonistic CD40 antibody or antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for an antibody or fragment thereof of the disclosure (e.g., an antagonistic CD40 antibody or antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents.

[0386] Methods of Treating Cell Proliferation Disorders

[0387] Antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof) of the disclosure, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof are useful therapeutics for the treatment of a wide array of cancers and cell proliferation disorders. Certain polypeptides of the disclosure (e.g., an antagonistic CD40 polypeptides, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can be administered to a mammalian subject, such as a human, suffering from a cell proliferation disorder, such as cancer, e.g., to enhance the effectiveness of the adaptive immune response against the target cancer cells.

[0388] In particular, antagonistic CD40 polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) of the disclosure can be administered to a mammalian subject, such as a human, to reduce or inhibit T-reg cell growth and activation, which allows tumor-infiltrating T-lymphocytes to localize to cells presenting tumor-associated antigens and to promote cytotoxicity. In addition, polypeptides of the disclosure (e.g., an antagonistic CD40 polypeptides, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may synergize with existing adoptive T-cell therapy platforms, as one of the limitations on the effectiveness of this strategy has been the difficulty of prolonging cytotoxicity of tumor-reactive T-cells following infusion into a mammalian subject (e.g., a human).

[0389] Antagonist CD40 antibodies and antigen-binding fragments thereof of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can mitigate the T- reg-mediated depletion of tumor-reactive T-cells by suppressing the growth and proliferation of T-reg cells that typically accompanies T-cell infusion. For instance, polypeptides of the disclosure (e.g., polypeptides which antagonize CD40, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be capable of reducing the growth of a population of T-reg cells by about 50% to about 200% relative to untreated cells (e.g., 50%, 75%, 100%, 125%, 150%, 175%, or 200%). The reduction in cellular growth occurs even in the presence of a cognate or natural ligand of the receptor. In some embodiments, polypeptides of the disclosure (e.g., an antagonistic CD40 polypeptides, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be capable of restricting the growth of a population of T-reg cells in the presence of the natural ligand of the CD40 to between 90% and 150% relative to untreated cells (e.g., 90%, 100%, 110%, 120%, 130%, 140%, or 150%). Antagonistic CD40 polypeptides of the disclosure (e.g., singlechain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) are also capable of restricting the proliferation of a population of T-reg cells to less than 70% (e.g., 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of that of an untreated population of T-reg cells. Antagonistic CD40 polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) are also capable of decreasing the survival of a population of T-reg cells by about 10% (e.g., by about 20%, 30%, 40%, or 50%, or more) relative to an untreated population of T-reg cells.

[0390] Antagonist CD40 antibodies and antigen-binding fragments thereof of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can mitigate the MDSC-mediated depletion of tumor-reactive T-cells by suppressing the growth and proliferation of MDSCs that may accompany T-cell infusion. For instance, polypeptides of the disclosure (e.g., polypeptides which antagonize CD40, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be capable of reducing the growth of a population of MDSCs by about 50% to about 200% relative to untreated cells (e.g., 50%, 75%, 100%, 125%, 150%, 175%, or 200%). The reduction in cellular growth occurs even in the presence of a cognate or natural ligand of the receptor. In some embodiments, polypeptides of the disclosure (e.g., an antagonistic CD40 polypeptides, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be capable of restricting the growth of a population of MDSCs in the presence of the natural ligand of the CD40 to between 90% and 150% relative to untreated cells (e.g., 90%, 100%, 110%, 120%, 130%, 140%, or 150%). Antagonistic CD40 polypeptides of the disclosure (e.g., singlechain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) are also capable of restricting the proliferation of a population of MDSCs to less than 70% (e.g., 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of that of an untreated population of MDSCs. Antagonistic CD40 polypeptides of the disclosure (e.g., singlechain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) are also capable of decreasing the survival of a population of MDSCs by about 10% (e.g., by about 20%, 30%, 40%, or 50%, or more) relative to an untreated population of MDSCs.

[0391] Antagonist CD40 antibodies and antigen-binding fragments thereof of the disclosure (e.g., CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, or variants thereof) can directly kill cells expressing CD40, such as B cells, parenchymal cells, dendritic cells, platelets, and granulocytes. For instance, polypeptides of the disclosure (e.g., polypeptides which antagonize CD40, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be capable of reducing the growth of a population of CD40 expressing cells by about 50% to about 200% relative to untreated cells (e.g., 50%, 75%, 100%, 125%, 150%, 175%, or 200%). The reduction in cellular growth may occur even in the presence of a cognate or natural ligand of the receptor. In some embodiments, polypeptides of the disclosure (e.g., an antagonistic CD40 polypeptides, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be capable of restricting the growth of a population of CD40 expressing cells in the presence of the natural ligand of CD40 to between 90% and 150% relative to untreated cells (e.g., 90%, 100%, 110%, 120%, 130%, 140%, or 150%). Antagonistic CD40 polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) are also capable of restricting the proliferation of a population of CD40- expressing cells to less than 70% (e.g., 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of that of an untreated population of CD40-expressing cells. Antagonistic CD40 polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) are also capable of decreasing the survival of a population of CD40-expressing cells by about 10% (e.g., by about 20%, 30%, 40%, or 50%, or more) relative to an untreated population of CD40-expressing cells.

[0392] Antagonistic CD40 polypeptides of the disclosure (e.g., single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can be administered to a mammalian subject (e.g., a human) suffering from cancer in order to improve the condition of the subject by promoting the immune response against cancer cells and tumorigenic material. Antibodies of the disclosure (e.g., antagonistic CD40 antibodies or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can be administered to a subject, e.g., via any of the routes of administration described herein. Polypeptides of the disclosure (e.g., antagonistic CD40 single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can also be formulated with excipients, biologically acceptable carriers, and may be optionally conjugated to, admixed with, or co-administered separately (e.g., sequentially) with additional therapeutic agents, such as anti-cancer agents. Cancers that can be treated by administration of polypeptides of the disclosure (e.g., antagonistic CD40 single-chain polypeptides, antibodies, or fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) include such cancers as leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer. Particular cancers that can be treated by administration of antibodies or antigen-binding fragments thereof of the disclosure (e.g., antagonistic CD40 antibodies or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) include, without limitation, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, Burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, Wilms tumor and other childhood kidney tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm (e.g., multiple myeloma or refractory multiple myeloma), myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, rhabdomyosarcoma, Sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia. An anti-CD40 polypeptide of the disclosure (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) can also be co-administered with a therapeutic antibody that exhibits reactivity towards a cancer cell. In this way, antagonistic CD40 polypeptides of the disclosure (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may synergize not only with the adaptive immune response, e.g., by prolonging T-lymphocyte tumor reactivity, but also with other inhibitors of tumor cell growth. Examples of additional therapeutic antibodies that can be used to treat cancer and other cell proliferation disorders include those that exhibit reactivity with a tumor antigen or a cell-surface protein that is overexpressed on the surface of a cancer cell. Exemplary antibodies that can be admixed, co-administered, or sequentially administered with antagonistic CD40 polypeptides of the disclosure (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) include, without limitation, Trastuzumab (HERCEPTIN®), Bevacizumab (AVASTIN®), Cetuximab (ERBITUX®), Panitumumab (VECTIBIX®), Ipilimumab (YERVOY®), Rituximab (RITUXAN® and MABTHERA®), Alemtuzumab (CAMPATH®), Ofatumumab (ARZERRA®), Gemtuzumab ozogamicin (MYLOTARG®), Brentuximab vedotin (ADCETRIS®),90Y- Ibritumomab Tiuxetan (ZEVALIN®), and131l-Tositumomab (BEXXAR®), which are described in detail in Scott et al. Cancer Immun. 12:14-21 , 2012; incorporated herein by reference.

[0393] A physician having ordinary skill in the art can readily determine an effective amount of an antagonistic CD40 polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician could start prescribing doses of a polypeptide of the disclosure (e.g., an antagonistic CD40 polypeptide, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering an antagonistic CD40 antibody or antibody fragment of the disclosure (e.g., an antibody or antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) at a high dose and subsequently administer progressively lower doses until a therapeutic effect is achieved (e.g., a reduction in the volume of one or more tumors, a decrease in the population of T-reg cells and / or MDSCs, or remission of a cell proliferation disorder). In general, a suitable daily dose of an antibody or antigenbinding fragment thereof of the disclosure (e.g., an antagonistic CD40 antibodies or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) will be an amount of the antibody which is the lowest dose effective to produce a therapeutic effect. A single-chain polypeptide, antibody, or antigen-binding fragment thereof of the disclosure (e.g., an antagonistic CD40 single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be administered by injection, e.g., by intravenous, intramuscular, intraperitoneal, or subcutaneous injection, optionally proximal to the site of the target tissue (e.g., a tumor). A daily dose of a therapeutic composition of an antibody or antigen-binding fragment thereof of the disclosure (e.g., an antagonistic CD40 antibodies or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for an antibody or fragment thereof of the disclosure (e.g., an antagonistic CD40 antibodies or antigen-binding fragments thereof, such as CD40AB1 , CD40AB2, CD40AB3, CD40AB4.1 , CD40AB4.2, and variants thereof) to be administered alone, it may also be ad...

Claims

Claims1 . An antibody or antigen-binding fragment thereof capable of specifically binding human CD40, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDR) 1 (CDR-H1 ), CDR-H2, and CDR-H3, and a light chain CDR 1 (CDR-L1 ), CDR-L2, and CDR-L3, wherein:(a) the CDR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 1 -5 or a variant thereof with up to two conservative amino acid substitutions;(b) the CDR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 6-9 or a variant thereof with up to two conservative amino acid substitutions;(c) the CDR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 10-13 or a variant thereof with up to two conservative amino acid substitutions;(d) the CDR-L1 comprises the amino acid sequence of any one of SEQ ID NOs: 14-18 or a variant thereof with up to two conservative amino acid substitutions;(e) the CDR-L2 comprises the amino acid sequence of any one of SEQ ID NOs: 19-22 or a variant thereof with up to two conservative amino acid substitutions; and(f) the CDR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 23-26 or a variant thereof with up to two conservative amino acid substitutions.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof with up to two conservative amino acid substitutions;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof with up to two conservative amino acid substitutions;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof with up to two conservative amino acid substitutions;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof with up to two conservative amino acid substitutions;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof with up to two conservative amino acid substitutions; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof with up to two conservative amino acid substitutions.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1 ;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 14;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 19; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 23.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigenbinding fragment thereof comprises:(a) a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 27; and(b) a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 28.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein:(a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 27; and(b) the light chain comprises the amino acid sequence of SEQ ID NO: 28.

6. The antibody or antigen-binding fragment thereof of any one of claims 2-5, wherein the antibody or antigen-binding fragment thereof does not compete with antibody ABBV-323 for binding to CD40.

7. The antibody or antigen-binding fragment thereof of claim 1 , wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof with up to two conservative amino acid substitutions;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof with up to two conservative amino acid substitutions;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof with up to two conservative amino acid substitutions;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof with up to two conservative amino acid substitutions;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof with up to two conservative amino acid substitutions; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof with up to two conservative amino acid substitutions.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 2;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 7;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 11 ;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 15;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 20; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 24.

9. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigenbinding fragment thereof comprises:(a) a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 29; and(b) a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 30.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein:(a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 29; and(b) the light chain comprises the amino acid sequence of SEQ ID NO: 30.11 . The antibody or antigen-binding fragment thereof of any one of claims 7-10, wherein the antibody or antigen-binding fragment thereof does not compete with antibody ABBV-323 for binding to CD40.

12. The antibody or antigen-binding fragment thereof of claim 1 , wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof with up to two conservative amino acid substitutions;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof with up to two conservative amino acid substitutions;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof with up to two conservative amino acid substitutions;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof with up to two conservative amino acid substitutions;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof with up to two conservative amino acid substitutions; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof with up to two conservative amino acid substitutions.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 3;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 8;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 12;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 16);(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 21 ; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 25.

14. The antibody or antigen-binding fragment thereof of claim 12, wherein the antibody or antigen-binding fragment thereof comprises:(a) a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 31 ; and(b) a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 32.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein:(a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 31 ; and(b) the light chain comprises the amino acid sequence of SEQ ID NO: 32.

16. The antibody or antigen-binding fragment thereof of any one of claims 12-15, wherein the antibody or antigen-binding fragment thereof competes with antibody ABBV-323 for binding to CD40.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the antibody or antigen-binding fragment thereof competes with antibody ABBV-323 for binding to CD40 at an epitope within the N-terminal peptide of CD40.

18. The antibody or antigen-binding fragment thereof of claim 1 , wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof with up to two conservative amino acid substitutions;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof with up to two conservative amino acid substitutions;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof with up to two conservative amino acid substitutions;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof with up to two conservative amino acid substitutions;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof with up to two conservative amino acid substitutions; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof with up to two conservative amino acid substitutions.

19. The antibody or antigen-binding fragment thereof of claim 18, wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 17;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26.

20. The antibody or antigen-binding fragment thereof of claim 18, wherein the antibody or antigen-binding fragment thereof comprises:(a) a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 33; and(b) a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 34.21 . The antibody or antigen-binding fragment thereof of claim 20, wherein:(a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 33; and(b) the light chain comprises the amino acid sequence of SEQ ID NO: 34.

22. The antibody or antigen-binding fragment thereof of claim 1 , wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof with up to two conservative amino acid substitutions;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof with up to two conservative amino acid substitutions;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof with up to two conservative amino acid substitutions;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof with up to two conservative amino acid substitutions;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof with up to two conservative amino acid substitutions; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof with up to two conservative amino acid substitutions.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein:(a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5;(b) the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 9;(c) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 13;(d) the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 18;(e) the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22; and(f) the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 26.

24. The antibody or antigen-binding fragment thereof of claim 22, wherein the antibody or antigen-binding fragment thereof comprises:(a) a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 35; and(b) a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 36.

25. The antibody or antigen-binding fragment thereof of claim 24, wherein:(a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 35; and(b) the light chain comprises the amino acid sequence of SEQ ID NO: 36.

26. The antibody or antigen-binding fragment thereof of any one of claims 1 -25, wherein the antibody or antigen-binding fragment thereof specifically binds an epitope comprising five or more of amino acids 124-132 of SEQ ID NO: 37 and / or five or more of amino acids 143-152 of SEQ ID NO: 37, or an epitope with at least 80% or greater sequence identity thereto.

27. The antibody or antigen-binding fragment thereof of any one of claims 1 -26, wherein the antibody or antigen-binding fragment thereof comprises a framework region comprising the amino acid sequence LLIY (SEQ ID NO: 38) bound to the N-terminus of the CDR-L2.

28. The antibody or antigen-binding fragment thereof of any one of claims 1 -27, wherein the antibody or antigen-binding fragment thereof lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether.

29. The antibody or antigen-binding fragment thereof of any one of claims 1 -28, wherein the antibody or antigen-binding fragment thereof inhibits signaling associated with CD40.

30. The antibody or antigen-binding fragment thereof of any one of claims 1 -29, wherein the antibody or antigen-binding fragment thereof binds CD40 with a Kd of between about 0 and about 10 nM or with a Kd of between about 0.001 pg / ml and about 50 pg / ml.31 . The antibody or antigen-binding fragment thereof of claim 30, wherein the antibody or antigen-binding fragment thereof binds CD40 with a Kd of between about 0 and about 1 nM.

32. The antibody or antigen-binding fragment thereof of any one of claims 1 -31 , wherein the antibody or antigen-binding fragment thereof binds CD40 to form an antibody-antigen complex with a kOn of between about 104M-1s-1and about 106M-1s-1.

33. The antibody or antigen-binding fragment thereof of any one of claims 1 -32, wherein the antibody or antigen-binding fragment thereof binds CD40 to form an antibody-antigen complex, and wherein the complex dissociates with a kOff of no greater than about 10-3s-1.

34. The antibody or antigen-binding fragment thereof of any one of claims 1 -33, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigenbinding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), adiabody, a triabody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem scFv (taFv).

35. The antibody or antigen-binding fragment thereof of any one of claims 1 -34, wherein the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

36. The antibody or antigen-binding fragment thereof of claim 35, wherein the antibody or antigen-binding fragment thereof is an IgG isotype.

37. The antibody or antigen-binding fragment thereof of claim 36, wherein the antibody or antigen-binding fragment thereof is an IgG 1 , lgG2, lgG3, or lgG4 isotype.

38. The antibody or antigen-binding fragment thereof of claim 37, wherein the antibody or antigen-binding fragment thereof is an lgG2 isotype.

39. The antibody or antigen-binding fragment thereof of any one of claims 1 -38, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region that lacks a cysteine residue at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region.

40. The antibody or antigen-binding fragment thereof of claim 39, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region that lacks a cysteine residue at position 232 of the amino acid sequence of the lgG2 hinge region.41 . The antibody or antigen-binding fragment thereof of claim 39 or 40, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region that lacks a cysteine residue at position 233 of the amino acid sequence of the lgG2 hinge region.

42. The antibody or antigen-binding fragment thereof of any one of claims 39-41 , wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having an amino acid other than cysteine at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region.

43. The antibody or antigen-binding fragment thereof of claim 42, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having an amino acid other than cysteine at position 232 of the amino acid sequence of the lgG2 hinge region.

44. The antibody or antigen-binding fragment thereof of claim 42 or 43, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having an amino acid other than cysteine at position 233 of the amino acid sequence of the lgG2 hinge region.

45. The antibody or antigen-binding fragment thereof of any one of claims 39-44, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having a serine residue at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region.

46. The antibody or antigen-binding fragment thereof of claim 45, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having a serine residue at position 232 of the amino acid sequence of the lgG2 hinge region.

47. The antibody or antigen-binding fragment thereof of claim 45 or 46, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region having a serine residue at position 233 of the amino acid sequence of the lgG2 hinge region.

48. The antibody or antigen-binding fragment thereof of any one of claims 39-47, wherein the antibody or antigen-binding fragment thereof comprises a human lgG2 hinge region comprising an amino acid substitution or deletion at one or both of cysteine residues 232 and 233.

49. The antibody or antigen-binding fragment thereof of claim 48, wherein the lgG2 hinge region comprises an amino acid substitution at one or both of cysteine residues 232 and 233.

50. The antibody or antigen-binding fragment thereof of claim 49, wherein the lgG2 hinge region comprises an amino acid substitution at cysteine residue 232.51 . The antibody or antigen-binding fragment thereof of claim 49 or 50, wherein the lgG2 hinge region comprises an amino acid substitution at cysteine residue 233.

52. The antibody or antigen-binding fragment thereof of any one of claims 48-51 , wherein the amino acid substitution is a conservative amino acid substitution.

53. The antibody or antigen-binding fragment thereof of claim 52, wherein the lgG2 hinge region comprises a C232S substitution.

54. The antibody or antigen-binding fragment thereof of claim 52 or 53, wherein the lgG2 hinge region comprises a C233S substitution.

55. The antibody or antigen-binding fragment thereof of any one of claims 1 -54, wherein the antibody or antigen-binding fragment thereof comprises antigen-binding sites separated from one another by a distance of at least about 133 A.

56. The antibody or antigen-binding fragment thereof of claim 55, wherein the antigen-binding sites are separated from one another by a distance of at least about 134 A.

57. The antibody or antigen-binding fragment thereof of claim 56, wherein the antigen-binding sites are separated from one another by a distance of at least about 139 A.

58. The antibody or antigen-binding fragment thereof of claim 57, wherein the antigen-binding sites are separated from one another by a distance of at least about 150 A.

59. The antibody or antigen-binding fragment thereof of any one of claims 1 -54, wherein the antigen-binding sites are separated from one another by a distance of from about 133 A to about 150 A.

60. The antibody or antigen-binding fragment thereof of claim 59, wherein the antigen-binding sites are separated from one another by a distance of from about 133 A to about 145 A.61 . The antibody or antigen-binding fragment thereof of claim 60, wherein the antigen-binding sites are separated from one another by a distance of from about 133 A to about 139 A.

62. The antibody or antigen-binding fragment thereof of claim 61 , wherein the antigen-binding sites are separated from one another by a distance of from about 134 A to about 139 A.

63. The antibody or antigen-binding fragment thereof of any one of claims 1 -62, wherein the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent.

64. The antibody or antigen-binding fragment thereof of claim 63, wherein the therapeutic agent is a cytotoxic agent.

65. The antibody or antigen-binding fragment thereof of any one of claims 1 -64, wherein the antibody or antigen-binding fragment thereof comprises a framework region from a human antibody or chimeric antibody.

66. The antibody or antigen-binding fragment thereof of any one of claims 1 -65, wherein the antibody or antigen-binding fragment thereof stabilizes an anti-parallel dimer conformation of CD40.

67. The antibody or antigen-binding fragment thereof of any one of claims 1 -66, wherein the antibody or antigen-binding fragment thereof destabilizes a trimeric conformation of CD40.

68. The antibody or antigen-binding fragment thereof of any one of claims 1 -67, wherein the antibody or antigen-binding fragment thereof reduces secretion of a soluble version of CD40.

69. The antibody or antigen-binding fragment thereof of any one of claims 1 -68, wherein the antibody or antigen-binding fragment thereof inhibits expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K1 1 , TRAF2, TRAF3, relB, and clAP2 / BIRC3.

70. The antibody or antigen-binding fragment thereof of any one of claims 1 -69, wherein the antibody or antigen-binding fragment thereof:(a) inhibits NFKB activation;(b) reduces or inhibits the proliferation of a population of T-reg cells;(c) induces the proliferation of a population of CD8+ effector T cells;(d) reduces or inhibits the proliferation of a population of cancer cells;(e) inhibits CD40 signaling in proliferating cells;(f) does not inhibit CD40 signaling in resting cells;(g) reduces or inhibits the proliferation of a population of myeloid-derived suppressor cells (MDSCs);(h) selectively reduces or inhibits the proliferation of a population of T-reg cells expressing CD25Hi; and / or(i) reduces or inhibits the proliferation of a population of T-reg cells in the presence of a ligand for CD40.71 . The antibody or antigen-binding fragment thereof of claim 70, wherein the cancer cells express CD40.

72. The antibody or antigen-binding fragment thereof of claim 70 or 71 , wherein the cancer cells are selected from the group consisting of Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells.

73. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1 -72, the method comprising expressing a polynucleotide encoding the antibody or antigen-binding fragment thereof in a host cell and recovering the antibody or antigen-binding fragment thereof from host cell medium.

74. A construct comprising a first polypeptide domain and a second polypeptide domain, wherein the first polypeptide domain and the second polypeptide domain are each, independently, an antigenbinding fragment of any one of claims 1 -72.

75. The construct of claim 74, wherein the first polypeptide domain and the second polypeptide domain are bound by a covalent linker.

76. The construct of claim 75, wherein the covalent linker comprises an amide bond.

77. The construct of claim 75, wherein the covalent linker comprises a disulfide bond.

78. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 -72.

79. The polynucleotide of claim 78, wherein the polynucleotide comprises:(a) a sequence of any one of SEQ ID NOs: 39-43; and(b) a sequence of any one of SEQ ID NOs: 44-48.

80. The polynucleotide of claim 78 or 79, wherein the polynucleotide comprises the sequence of SEQ ID NO: 39 and the sequence of SEQ ID NO: 44.81 . The polynucleotide of claim 78 or 79, wherein the polynucleotide comprises the sequence of SEQ ID NO: 40 and the sequence of SEQ ID NO: 45.

82. The polynucleotide of claim 78 or 79, wherein the polynucleotide comprises the sequence of SEQ ID NO: 41 and the sequence of SEQ ID NO: 46.

83. The polynucleotide of claim 78 or 79, wherein the polynucleotide comprises the sequence of SEQ ID NO: 42 and the sequence of SEQ ID NO: 47.

84. The polynucleotide of claim 78 or 79, wherein the polynucleotide comprises the sequence of SEQ ID NO: 43 and the sequence of SEQ ID NO: 48.

85. A polynucleotide encoding the construct of any one of claims 74-77.

86. A vector comprising the polynucleotide of any one of claims 78-85.

87. The vector of claim 86, wherein the vector is an expression vector.

88. The vector of claim 87, wherein the expression vector is a eukaryotic expression vector.

89. The vector of claim 86, wherein the vector is a viral vector.

90. The vector of claim 89, wherein the viral vector is selected from the group consisting of adenovirus (Ad), retrovirus, poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, and a vaccinia virus.91 . The vector of claim 90, wherein the adenovirus is a serotype 1 -60 adenovirus.

92. The vector of claim 91 , wherein the adenovirus is a serotype 5, 26, 35, or 48 adenovirus.

93. The vector of claim 90, wherein the retrovirus is a y-retrovirus or a lentivirus.

94. The vector of claim 90, wherein the vaccinia virus is a modified vaccinia Ankara (MVA).

95. An isolated host cell comprising the vector of any one of claims 86-94.

96. The host cell of claim 95, wherein the host cell is a prokaryotic cell.

97. The host cell of claim 95, wherein the host cell is a eukaryotic cell.

98. The host cell of claim 97, wherein the eukaryotic cell is a mammalian cell.

99. The host cell of claim 98, wherein the mammalian cell is a CHO cell.

100. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 -72, the construct of any one of claims 74-77, the polynucleotide of any one of claims 78-85, the vector of any one of claims 86-94, or the host cell of any one of claims 95 and 97- 99, and a pharmaceutically acceptable carrier or excipient.101 . The pharmaceutical composition of claim 100, wherein the pharmaceutical composition comprises the antibody or antigen biding fragment thereof of any one of claims 1 -72.

102. The pharmaceutical composition of claim 101 , wherein at least 50% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform.

103. The pharmaceutical composition of claim 102, wherein at least 75% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform.

104. The pharmaceutical composition of claim 103, wherein at least 80% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform.

105. The pharmaceutical composition of claim 104, wherein at least 85% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform.

106. The pharmaceutical composition of claim 105, wherein at least 90% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform.

107. The pharmaceutical composition of claim 106, wherein at least 95% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide- bonded isoform.

108. The pharmaceutical composition of claim 103, wherein from about 75% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

109. The pharmaceutical composition of claim 108, wherein from about 80% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

110. The pharmaceutical composition of claim 109, wherein from about 85% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

111. The pharmaceutical composition of claim 110, wherein from about 90% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

112. The pharmaceutical composition of claim 111 , wherein from about 95% to about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform.

113. The pharmaceutical composition of any one of claims 100-112, wherein the antibody or antigen-binding fragment thereof yields a single detectable band upon gel electrophoresis analysis performed under non-reducing conditions.

114. The pharmaceutical composition of any one of claims 102-113, wherein the single disulfide- bonded isoform is lgG2-A.

115. The pharmaceutical composition of any one of claims 100-114, wherein the antibody or antigen-binding fragment thereof is present in the pharmaceutical composition in an amount of from about 0.001 mg / ml to about 100 mg / ml.

116. The pharmaceutical composition of any one of claims 100-115, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

117. A method of modulating an immune response in a human subject, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1 - 72, the construct of any one of claims 74-77, the polynucleotide of any one of claims 78-85, the vector of any one of claims 86-94, the host cell of any one of claims 95 and 97-99, or the pharmaceutical composition of any one of claims 100-116, wherein the antibody or antigen-binding fragment thereof is an antagonist of CD40.

118. The method of claim 117, wherein the method inhibits an immune response mediated by a B cell or CD8+ T cell in the human subject.

119. The method of claim 117, wherein the method treats an autoimmune disease in the human subject.

120. The method of claim 119, wherein the autoimmune disease is selected from the group consisting of Type I diabetes, alopecia areata, ankylosing spondylitis, anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behget’s disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, limited scleroderma (CREST syndrome), cold agglutinin disease, Crohn’s disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hypothyroidism, inflammatory bowel disease, autoimmune lymphoproliferative syndrome (ALPS), idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, lupus nephritis, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud’s phenomenon, Reiter’s syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, Stiff-Man syndrome, Takayasu’s arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener’s granulomatosis, Goodpasture syndrome, transplant rejection, celiac disease, esophagitis, and antibody-mediated inflammatory central nervous system disorders.121 . The method of claim 117, wherein the method inhibits an immune response mediated by a T- reg cell in the human subject.

122. The method of claim 117, wherein the method treats an infectious disease in the human subject.

123. The method of claim 122, wherein the infectious disease is caused by one or more agents selected from the group consisting of a virus, a bacterium, a fungus, and a parasite.

124. The method of claim 123, wherein the infectious disease is caused by a virus selected from the group consisting of hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, llheus virus, Israel turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus, Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, Lujo virus, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O’nyong’nyong virus, chikungunya virus, smallpox virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster virus, Kaposi’s sarcoma associated-herpesvirus (KSHV), influenza virus, severe acute respiratory syndrome (SARS) virus, rabies virus, vesicular stomatitis virus (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza virus (e.g., 1 , 2, 3, and 4), rhinovirus, mumps virus, poliovirus, human enterovirus (e.g., A, B, C, and D), hepatitis A virus, coxsackievirus, hepatitis B virus, human papilloma virus, adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus, Norwalk virus, rotavirus, human immunodeficiency virus (HIV), and human T- lymphotropic virus Types I and II.

125. The method of claim 123, wherein the infectious disease is caused by a bacterium belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

126. The method of claim 123, wherein the infectious disease is caused by a fungus belonging to a genus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia.

127. The method of claim 123, wherein the infectious disease is caused by a parasite selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Exemplary helminthic parasites include richuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, and Dracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes, Paragonimus westermani, Taenia solium, Taenia saginata, Hymenolepis nana, and Echinococcus granulosus.

128. The method of claim 117, wherein the method treats an inflammatory disease in the human subject.

129. The method of claim 128, wherein the inflammatory disease is selected from the group consisting of acute or chronic inflammation, cardiac fibrosis, lung fibrosis, osteoarthritis, rheumatoid arthritis, atherosclerosis, type I diabetes, type II diabetes, graft-versus-host disease, multiple sclerosis, osteomyelitis, psoriasis, Crohn’s disease, Sjogren’s syndrome, lupus erythematosus, and ulcerative colitis.

130. The method of claim 117, wherein the method treats a neurological disease or disorder in the human subject.131 . The method of claim 130, wherein the neurological disease or disorder is selected from the group consisting of a brain tumor, a brain metastasis, a brain injury, a spinal cord injury, a nerve injury, schizophrenia, epilepsy, Parkinson’s disease, autism, Huntington’s disease, stroke, Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and myasthenia gravis.

132. The method of any one of claims 117-131 , wherein, prior to the administration of the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, the human subject is treated with:(a) an immunomodulatory medication, optionally wherein the immunomodulatory medication is a steroid;(b) a non-steroidal anti-inflammatory drug (NSAID), optionally wherein the NSAID is aspirin;(c) acetaminophen; and / or(d) kidney dialysis.

133. The method of any one of claims 117-132, wherein one or more doses of the antibody or antigen-binding fragment thereof is administered to the human subject in one or more treatment periods.

134. The method of claim 133, wherein each dose of the antibody or antigen-binding fragment thereof comprises 10 mg, 20 mg, 40 mg, 80 mg, or 160 mg of the antibody or antigen-binding fragment thereof.

135. The method of claim 133 or 134, wherein each treatment period lasts one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or more.

136. The method of any one of claims 133-135, wherein within each treatment period, the one or more doses of the antibody or antigen-binding fragment thereof is administered to the human subject once every week or once every two weeks.

137. The method of any one of claims 117-136, wherein the method further comprises:(a) measuring a level of soluble CD40 in the serum of the human subject before the human subject is administered the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, and(b) measuring a reference level of soluble CD40 in the serum of a healthy human, wherein the human subject has a level of soluble CD40 that is higher relative to a reference level of soluble CD40.

138. The method of any one of claims 117-137, wherein the method further comprises measuring a level of IgG in the serum of the human subject before the human subject is administered the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, wherein the human subject has a level of IgG that is higher than 16 g / L.

139. The method of any one of claims 117-138, wherein the method further comprises measuring a level of IgM in the serum of the human subject before the human subject is administered the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition, wherein the human subject has a level of IgG that is higher than 2.5 g / L.

140. The method of any one of claims 117-139, wherein the antibody or antigen-binding fragment thereof, the construct, the polynucleotide, the vector, the host cell, or the pharmaceutical composition is administered to the human subject intravenously or subcutaneously.

141. A kit comprising an agent selected from the group consisting of the antibody or antigenbinding fragment thereof of any one of claims 1 -72, the construct of any one of claims 74-77, the polynucleotide of any one of claims 78-85, the vector of any one of claims 86-94, the host cell of any one of claims 95-99, or the pharmaceutical composition of any one of claims 100-116.

142. The kit of claim 141 , wherein the kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1 -72.

143. The kit of claim 141 , wherein the kit comprises the construct of any of claims 74-77.

144. The kit of claim 141 , wherein the kit comprises the polynucleotide of any one of claims 78-85.

145. The kit of claim 141 , wherein the kit comprises the vector of any one of claims 86-94.

146. The kit of claim 145, wherein the kit further comprises instructions for transfecting the vector into a host cell.

147. The kit of claim 146, wherein the kit further comprises instructions for expressing the antibody, antigen-binding fragment thereof, or construct in the host cell.

148. The kit of claim 141 , wherein the kit comprises the host cell of any one of claims 95-99.

149. The kit of claim 148, wherein the kit further comprises a reagent that can be used to express the antibody, antigen-binding fragment thereof, or construct in the host cell.

150. The kit of claim 141 , wherein the kit comprises the pharmaceutical composition of any one of claims 100-116.151 . The kit of claim 141 , further comprising instructions for administering the agent to a human subject.

152. The kit of claim 141 , further comprising instructions for making or using the agent.

153. The kit of any one of claims 141 -152, further comprising instructions for measuring a level of soluble CD40 in a subject.

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