Anti-CD40 antibody constructs with high intrinsic agonism

Abstract

The present invention relates to oligomeric and oligovalent anti-CD40 antibody variants that exhibit high intrinsic, i.e., FcγR-dependent, agonistic activity. More specifically, the present invention relates to tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, decavalent, and eleven or more multivalent constructs with FcγR-independent activity, which are composed of CD40-specific Fab and / or scFv domains and / or sdAb domains and a dimeric, trimeric, or tetrameric oligomerization scaffold, preferably a TNC Fc, IgG, or trimerization domain. The present invention also relates to the forced oligomerization of multiple anti-CD40 IgG molecules, which can be achieved in a stoichiometric manner by genetic engineering, conferring potent FcγR-independent agonism, and the fact that the idiotype of the anti-CD40 antibody does not significantly affect the autonomous agonism of oligovalent anti-CD40 variants. The present invention also relates to methods for producing such oligomers and oligovalent anti-CD40 antibody constructs, pharmaceutical compositions containing them, and their use for the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to oligomeric and oligovalent anti-CD40 antibody variants that exhibit high intrinsic and therefore FcγR-dependent agonistic activity. More specifically, the present invention relates to tetravalent, hexavalent, and octavalent constructs composed of CD40-specific Fab and scFv domains with FcγR-independent activity. The present invention also relates to the forced oligomerization of multiple anti-CD40 IgG molecules, which can be achieved in a stoichiometric manner by genetic engineering, conferring potent FcγR-independent agonism, and the fact that the idiotype of the anti-CD40 antibody does not significantly affect the autonomous agonism of oligovalent anti-CD40 variants. The present invention also relates to methods for producing such oligomeric and oligovalent anti-CD40 antibody constructs, pharmaceutical compositions containing them, and their use for the treatment of cancer. [Background technology]

[0002] The overwhelming majority of preclinical and clinical studies aimed at activating CD40 in tumor therapy or as an adjuvant in pathogen vaccination have used or have used agonistic anti-CD40 antibodies (Non-Patent Documents 1-3). However, with regard to anti-CD40 antibody agonism, it is crucial to distinguish between antibody-intrinsic autonomous agonism, which is manifested by the absence of antibody binding to Fcγ receptors (FcγRs), and conditional CD40 agonism, which is only evident upon antibody binding to cell-expressed FcγR molecules. Thus, strictly and precisely, anti-CD40 antibodies with FcγR-dependent agonism are not agonists, since their binding to CD40 alone has no receptor-stimulating effect. Rather, complexes of these non-agonistic antibodies with other molecules that are themselves non-agonistic (e.g., FcγRs, cross-linking antibodies) exhibit agonistic effects. Unfortunately, this distinction has not been made in many published studies, and therefore in the literature the term "agonist" is used for both anti-CD40 antibodies with FcγR-dependent and FcγR-independent agonism, although this aspect may be absolutely crucial for the interpretation of data obtained with anti-CD40 antibodies. The majority of anti-CD40 IgG antibodies considered agonistic in the literature actually lack associated autonomous agonism and act as agonists only upon binding to FcγR or upon cross-linking with a secondary antibody or protein G (NPL 4). For example, a variant of the "agonistic" anti-IgG1 ADC-1013 (Mitazalimab) with an N297Q mutation in the Fc domain, which reduces glycosylation and FcγR binding, lacks dendritic cell binding, but agonism is restored upon cross-linking with anti-human IgG (NPL 5). Similarly, a variant of the "agonistic" anti-APX005M (Sotigalimab) lacks agonism upon removal of the Fc domain, but introduction of the S267E mutation, which improves affinity for FcγRIIb, enhances agonism toward B cells (NPL 6). Similar results have been reported for several other anti-CD40 antibodies, including SEA-CD40 (Non-Patent Document 7), G28.5 (Non-Patent Document 8), and ChiLob-7 / 4 (Non-Patent Document 9).

[0003] Significant FcγR-independent agonism has been reported for a few anti-CD40 IgGs. For example, the Fab2 fragment of CP-870,893 (selicrelumab) exhibited similar B cell activation as the corresponding intact antigen, and secondary cross-linking of the latter did not further enhance the associated activity (Non-Patent Document 10). Similar findings have been obtained for the anti-CD40 antibody CDX-1140 (Non-Patent Document 11). There is evidence that the isotype of an anti-CD40 antibody is particularly related to its agonistic activity. Thus, both CP-870,893 and CDX-1140 are IgG2 isotypes, and conversion of the anti-CD40 IgG1 ChiLob-4 / 7 to the IgG2 isotype resulted in significant FcγR-independent agonism (Non-Patent Document 12). Interestingly, the agonistic activity of anti-CD40 hIgG2 antibodies can be assigned to isoform B of the hIgG2 isotype, which differs from isoform A of the hIgG2 molecule in the formation of disulfide bridges between the CH1 and CL domains, and therefore the arrangement of the two Fab domains of this molecule is less flexible. Thus, anti-CD40 hIgG2 antibodies with mutations that direct the formation of only isoform A (e.g., HC-C233S) or isoform B (e.g., HC-C127S or LC-C214S / HC-C233S) exhibit no or enhanced FcγR-independent agonism compared to the parent hIgG2 molecule (Non-Patent Documents 12 and 13). However, the general relevance of the IgG2 and IgG2B isotypes to FcγR-independent CD40 agonism has been verified by studies showing i) the loss of agonism of deglycosylated CP-870,893-IgG2, which has reduced affinity for FcγRIIb, and ii) similar agonism of the two IgG2 isoforms (Non-Patent Document 14). Furthermore, the FcγR-independent agonism of anti-CD40-hIgG2 or anti-CD40-hIgG2A antibodies (sometimes referred to as "superagonists") is still significantly lower than that of FcγR-binding anti-CD40 antibodies (Non-Patent Documents 14 and 15).Thus, in the presence of transfectants expressing large amounts of FcγRIIb, conditions that provide the best chance for all CD40-occupied antibody molecules to actually also bind to FcγR molecules, CD40 agonism is uniformly and strongly enhanced (Non-Patent Document 15).

[0004] Over the past decade, extensive in vitro and in vivo studies have revealed that virtually all CD40-specific antigens exhibit agonistic activity when bound to Fcγ receptors (Non-Patent Documents 14-18). In this context, the specific FcγR type and its signaling properties appear to be irrelevant to the agonism of Fcγ-binding anti-CD40 antibodies; rather, the intact plasma membrane-bound form of expression appears to be required for an Fcγ-binding anti-CD40 antibody molecule to acquire agonism. Accordingly, bispecific mutants recognizing CD40 and a second membrane-associated antibody have been found to exhibit up to a 1000-fold increase in CD40 stimulatory activity after binding to this second antibody (Non-Patent Documents 8, 19-24). Improving FcγR binding of antibodies through genetic engineering is currently the state of the art, and while achieving potent CD40 agonism in vivo with the aid of an anti-CD40 antibody with potent, conditional FcγR-dependent agonism appears trivial, the requirement for FcγR binding entails several limitations. CD40 expression levels are usually very high, potentially limiting the availability of immune cells expressing FcγR. Therefore, it must be considered that in vivo, only a portion of the CD40 molecule can be targeted by an FcγR-binding anti-CD40 antibody, resulting in submaximal stimulation of CD40 activity. For example, an anti-CD40 mouse IgG1 antibody significantly stimulates proliferation of B cells from wild-type mice but not FcγRIIB-deficient B cells. However, when an FcγR-expressing transfectant is added to such experiments, proliferation can be further increased by one to two orders of magnitude (Non-Patent Document 15). Clearly, physiological levels of FcγR expressed by B cells are insufficient for an FcγR-binding anti-CD40 antibody to reach all CD40 molecules present. Another aspect that limits the usefulness of FcγR-dependent agonism of conventional anti-CD40 antibodies for immune stimulation is their binding to activating FcγRs, such as FcγRIIIa. In such cases, FcγR binding not only results in CD40 agonism but can also trigger killing of CD40-expressing target cells by effector functions of FcγR-expressing cells (e.g., ADCC, CDC). [Prior art documents] [Non-patent literature]

[0005] [Non-licensed document 1] Vonderheide RH. CD40 Agonist Antibodies in Cancer Immunotherapy. Annu Rev Med. 2020 Jan 27;71:47-58. [Non-licensed document 2] Li DK, Wang W. Characteristics and clinical trial results of agonistic anti-CD40 antibodies in the treatment of malignancies. Oncol Lett. 2020 Nov;20(5):176 [Non-licensed document 3] Bullock TNJ. CD40 stimulation as a molecular adjuvant for cancer vaccines and other immunotherapies. Cell Mol Immunol. 2022 Jan;19(1):14-22.

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[0006] The characteristics of agonistic anti-CD40 antibodies vary widely. Some anti-CD40 antibodies exhibit agonism solely in association with binding to FcγR+ immune cells, whereas others have been found to exhibit intrinsic agonism that is idio- and / or isotype-dependent. However, it is worth noting that all of these antibodies exhibit increased activity when cells overexpressing FcγRs are available, indicating that i) idio- or isotype-based selection of agonistic anti-FcγRs is not sufficient to obtain antibodies that maximally potentiate FcγR activation, and ii) the availability of endogenously expressed FcγRs may be limited, limiting the extent of FcγR activation. Given the well-documented fact that oligomerization of soluble CD40L trimers by genetic engineering strongly enhances their CD40-stimulatory function, we here substantiate the hypothesis that genetically engineered oligomeric and oligovalent anti-CD40 antibody variants exhibit enhanced intrinsic and therefore FcγR-dependent agonistic activity. We generated tetravalent, hexavalent, and dodecavalent variants of six anti-CD40 antibodies with significantly different characteristics. All of these variants, including those derived from antagonistic anti-CD40 antibodies, exhibited strongly enhanced CD40 agonism compared with their conventional counterparts. In most cases, CD40 agonism reached the maximal response induced by FcγR-binding anti-CD40 antibodies or membrane CD40L (the natural engager of CD40). In summary, the present data demonstrate that increasing anti-CD40 antibody titers by genetic engineering regularly yields molecules with high autonomous agonism, overcoming the specific limitations of the parent antibody.

[0007] The present inventors have identified antibody constructs, including, inter alia, oligomeric and oligovalent anti-CD40 antibody variants, that exhibit high intrinsic and therefore FcγR-independent agonistic activity. The present inventors have found that i) forced oligomerization of certain anti-CD40 molecules, which can be achieved in a stoichiometric manner by genetic engineering, confers potent FcγR-independent agonism, and ii) the idiotype of the anti-CD40 antibody has no significant effect on the autonomous agonism of oligovalent anti-CD40 variants. According to the present invention, these novel antibody-based CD40 agonists stimulate CD40 in an FcγR-independent manner, and this advantage is expected to lead to the development of new application fields, including clinical applications such as cancer treatment or vaccination against infectious diseases. [Means for solving the problem]

[0008] Accordingly, the present invention relates to the following preferred embodiments. 1. A multivalent anti-CD40 antibody construct comprising at least four antigen binding sites for CD40, which are not naturally occurring IgA and IgM molecules. 2. The multivalent anti-CD40 antibody construct according to item 1, comprising at least one IgG molecule comprising two of the antigen-binding sites for CD40. 3. The multivalent anti-CD40 antibody construct according to item 1, comprising a modified IgG molecule in which each of the two variable domains of the heavy chain has been modified by replacing with at least one single domain antibody (sdAb) comprising one of the antigen-binding sites for CD40, and in which each of the two variable domains of the light chain has been modified by replacing with at least one single domain antibody (sdAb) comprising one of said antigen-binding sites for CD40. 4. The multivalent anti-CD40 antibody construct according to item 2 or 3, further comprising an scFv or single domain antibody (sdAb) covalently attached to the C-terminus of one of the two heavy chains of the IgG molecule or modified IgG molecule, said scFv or single domain antibody (sdAb) comprising one of the antigen-binding sites for CD40. 5. The multivalent anti-CD40 antibody construct according to item 4, further comprising an scFv or single domain antibody (sdAb) covalently linked to the C-terminus of the other of the two heavy chains of the IgG molecule or modified IgG molecule, wherein the scFv or single domain antibody (sdAb) comprises one of the antigen-binding sites for CD40.

[0009] 6. A multivalent anti-CD40 antibody construct according to claim 1, comprising an Fc fragment of an IgG molecule, wherein each of the two N-termini and / or each of the two C-termini of said Fc fragment is covalently linked to a polypeptide chain comprising one, two or three, preferably three, single domain antibodies (sdAbs), each of said one, two or three, preferably three, single domain antibodies (sdAbs) comprising one of said antigen binding sites for CD40. 7. The multivalent anti-CD40 antibody construct according to any one of items 1 to 6, comprising six antigen-binding sites for CD40. 8. The multivalent anti-CD40 antibody construct according to any one of items 1 to 7, comprising 12 antigen-binding sites for CD40. 9. The multivalent anti-CD40 antibody construct according to any one of items 1 to 8, wherein any two of the antigen-binding sites for CD40 are oriented in parallel. 10. The multivalent anti-CD40 antibody construct according to any one of paragraphs 2 to 9, comprising a trimer of three of said IgG molecules or modified IgG molecules formed by a trimerization domain fused to the C-terminus of each heavy chain of each of said IgG molecules or modified IgG molecules.

[0010] 11. The multivalent anti-CD40 antibody construct of any one of paragraphs 4 to 10, wherein the trimerization domain is the trimerization domain of tenascin-C. 12. The multivalent anti-CD40 antibody construct according to item 10, wherein the trimerization domain comprises an amino acid sequence set forth in SEQ ID NO: 60, or a sequence at least 70% identical thereto, preferably at least 80% identical thereto, more preferably at least 85% identical thereto, more preferably at least 90% identical thereto, more preferably at least 93% identical thereto, or more preferably at least 96% identical thereto. 13. The multivalent anti-CD40 antibody construct according to any one of items 2 to 12, wherein the IgG molecule or modified IgG molecule comprises an oligomerization mutation. 14. The multivalent anti-CD40 antibody construct according to item 13, wherein the oligomerization mutations are E345R / E430G / S440Y mutations. 15. The multivalent anti-CD40 antibody construct of any one of items 1 to 14, comprising one or more Fab1 domains, scFv domains, and / or sdAb domains, each comprising one of said antigen-binding sites for CD40.

[0011] 16. The multivalent anti-CD40 antibody construct according to any one of items 2 to 15, wherein the IgG molecule or modified IgG molecule is IgG1, IgG2, IgG3, or IgG4. 17. The multivalent anti-CD40 antibody construct according to any one of items 2 to 16, wherein the IgG molecule or modified IgG molecule is IgG2 or IgG4. 18. The multivalent anti-CD40 antibody construct according to any one of items 2 to 17, wherein the Fc domain of the IgG molecule or modified IgG molecule or the Fc fragment of the IgG molecule comprises a mutation that reduces FcγR binding. 19. The multivalent anti-CD40 antibody construct according to item 18, wherein the mutation is an N297A mutation. 20. A multivalent anti-CD40 antibody construct comprising at least two antigen binding sites for CD40, which are not naturally occurring IgA and IgM molecules, and wherein two of the antigen binding sites for CD40 are oriented antiparallel.

[0012] 21. The multivalent anti-CD40 antibody construct according to item 20, wherein the domains comprising the binding sites are linked by a linker sequence or a peptide linker sequence. 22. The multivalent anti-CD40 antibody construct according to any one of items 1 to 21, which is an Fc-independent CD40 agonist. 23. The following: a) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 2, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, or most preferably an amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence set forth in SEQ ID NO: 2; b) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 3, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3, or most preferably an amino acid sequence that is set forth in SEQ ID NO: 1 and an amino acid sequence that is set forth in SEQ ID NO: 3; c) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 4, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 4, or most preferably an amino acid sequence set forth in SEQ ID NO: 1 and an amino acid sequence set forth in SEQ ID NO: 4: d) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 6, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, or most preferably an amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence set forth in SEQ ID NO: 6; e) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 7, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, or most preferably an amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence set forth in SEQ ID NO: 7; f) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 8, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 8, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 8, or most preferably an amino acid sequence set forth in SEQ ID NO: 5 and an amino acid sequence set forth in SEQ ID NO: 8; g) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 10, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 10, or most preferably an amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence set forth in SEQ ID NO: 10; h) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11, or most preferably an amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence set forth in SEQ ID NO: 11; i) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 9 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 12, or most preferably an amino acid sequence set forth in SEQ ID NO: 9 and an amino acid sequence set forth in SEQ ID NO: 12; j) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 14, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 14, or most preferably an amino acid sequence set forth in SEQ ID NO: 13 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 14; k) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 15, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, or most preferably an amino acid sequence set forth in SEQ ID NO: 13 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 15; l) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 16, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 16, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 16, or most preferably an amino acid sequence set forth in SEQ ID NO: 13 and an amino acid sequence set forth in SEQ ID NO: 16; m) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 18, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 18, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 18, or most preferably an amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence set forth in SEQ ID NO: 18; n) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 19, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 19, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, or most preferably an amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence set forth in SEQ ID NO: 19; o) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 20, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 20, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, or most preferably an amino acid sequence set forth in SEQ ID NO: 17 and an amino acid sequence set forth in SEQ ID NO: 20; p) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 24, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 24, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 24, or most preferably an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 24; q) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 25, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 25, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 25, or most preferably an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 25; r) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 21 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 26, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 21 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 26, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 21 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 26, or most preferably an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 26; s) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 24, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 24, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 24, or most preferably an amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence set forth in SEQ ID NO: 24; t) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 25, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 25, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 25, or most preferably an amino acid sequence represented by SEQ ID NO: 22 and an amino acid sequence represented by SEQ ID NO: 25; u) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 26, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 26, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 26, or most preferably an amino acid sequence represented by SEQ ID NO: 22 and an amino acid sequence represented by SEQ ID NO: 26; v) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 24, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 24, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 24, or most preferably an amino acid sequence set forth in SEQ ID NO: 23 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 24; w) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 25, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 25, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 25, or most preferably an amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence represented by SEQ ID NO: 25; x) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 26, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 26, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 26, or most preferably an amino acid sequence set forth in SEQ ID NO: 23 and an amino acid sequence set forth in SEQ ID NO: 26; y) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 27, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 27, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 27, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 27, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 27, or most preferably an amino acid sequence set forth in SEQ ID NO: 27; z) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 28, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 28, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 28, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 28, or most preferably an amino acid sequence set forth in SEQ ID NO: 28; aa) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 29, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 29, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 29, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 29, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 29, or most preferably the amino acid sequence set forth in SEQ ID NO: 29; bb) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 31, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 31, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 31, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 31, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 31, or most preferably the amino acid sequence set forth in SEQ ID NO: 31; cc) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 32, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 32, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 32, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 32, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 32, or most preferably an amino acid sequence set forth in SEQ ID NO: 32; dd) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 33, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 33, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 33, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 33, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 33, or most preferably the amino acid sequence set forth in SEQ ID NO: 33; ee) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 34, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 34, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 34, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 34, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 34, or most preferably an amino acid sequence set forth in SEQ ID NO: 34; ff) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 35, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 35, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 35, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 35, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 35, or most preferably an amino acid sequence represented by SEQ ID NO: 35; gg) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 36, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 36, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 36, or most preferably an amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence set forth in SEQ ID NO: 36; hh) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 36, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 36, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 36, or most preferably an amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence set forth in SEQ ID NO: 36; ii) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 36, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 36, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 23 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 36, or most preferably an amino acid sequence set forth in SEQ ID NO: 23 and an amino acid sequence set forth in SEQ ID NO: 36; jj) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 39, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 39, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 39, or most preferably an amino acid sequence that is set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 39; kk) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 39, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 39, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 39, or most preferably an amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence set forth in SEQ ID NO: 39; ll) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 39, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 39, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 39, or most preferably an amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence represented by SEQ ID NO: 39; mm) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 40, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 40, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 40, or most preferably an amino acid sequence set forth in SEQ ID NO: 21 and an amino acid sequence set forth in SEQ ID NO: 40; nn) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 40, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 40, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 40, or most preferably an amino acid sequence represented by SEQ ID NO: 22 and an amino acid sequence represented by SEQ ID NO: 40; oo) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 40, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 40, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 40, or most preferably an amino acid sequence represented by SEQ ID NO: 23 and an amino acid sequence represented by SEQ ID NO: 40; pp) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 41, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 41, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 41, or most preferably an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 41; qq) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 41, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 41, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 41, or most preferably an amino acid sequence set forth in SEQ ID NO: 22 and an amino acid sequence set forth in SEQ ID NO: 41; rr) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 23 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 41, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 41, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 23 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 41, or most preferably an amino acid sequence set forth in SEQ ID NO: 23 and an amino acid sequence set forth in SEQ ID NO: 41; ss) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 42, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 42, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 42, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 42, or most preferably the amino acid sequence set forth in SEQ ID NO: 42; tt) an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 39, preferably an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 39, more preferably an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 39, or most preferably an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence set forth in SEQ ID NO: 39; uu) an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence represented by SEQ ID NO: 41 that is at least 85% identical, preferably an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence represented by SEQ ID NO: 41 that is at least 90% identical, more preferably an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence represented by SEQ ID NO: 41 that is at least 99% identical, or most preferably an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence represented by SEQ ID NO: 41; vv) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 44, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 44, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 44, or most preferably an amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence set forth in SEQ ID NO: 44; ww) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 45, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 45, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 45, or most preferably an amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence set forth in SEQ ID NO: 45; xx) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 43 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 46, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 43 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 46, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 43 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 46, or most preferably an amino acid sequence set forth in SEQ ID NO: 43 and an amino acid sequence set forth in SEQ ID NO: 46; yy) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 47 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 48, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 47 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 48, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 47 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 48, or most preferably an amino acid sequence represented by SEQ ID NO: 47 and an amino acid sequence represented by SEQ ID NO: 48; zz) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 49, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 49, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 49, or most preferably an amino acid sequence that is set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 49; aaa) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 50, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 50, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 50, or most preferably an amino acid sequence that is set forth in SEQ ID NO: 47 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 50; bbb) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 52, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 52, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 52, or most preferably an amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence set forth in SEQ ID NO: 52; ccc) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 53, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 53, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 53, or most preferably an amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence set forth in SEQ ID NO: 53; ddd) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 54, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 54, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 54, or most preferably an amino acid sequence that is set forth in SEQ ID NO: 51 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 54; eee) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 55 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 56, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 55 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 56, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 55 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 56, or most preferably an amino acid sequence set forth in SEQ ID NO: 55 and an amino acid sequence set forth in SEQ ID NO: 56; fff) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 57, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 57, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 57, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 57, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 57, or most preferably an amino acid sequence set forth in SEQ ID NO: 57; or ggg) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 58, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 58, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 58, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 58, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 58, or most preferably the amino acid sequence set forth in SEQ ID NO: 58; 23. The multivalent anti-CD40 antibody construct of any one of items 1 to 22, comprising: 24. The multivalent anti-CD40 antibody construct according to any one of items 1 to 23, further comprising an N-terminal amino acid sequence comprising one or two of the following (a) and (b): (a) a leader sequence comprising the amino acid sequence set forth in SEQ ID NO: 30, optionally followed by a first linker sequence, and (b) a Flag tag comprising the amino acid sequence set forth in SEQ ID NO: 61, optionally followed by a second linker sequence. 25. A pharmaceutical composition comprising the multivalent anti-CD40 antibody construct according to any one of items 1 to 24.

[0013] 26. The pharmaceutical composition according to item 25 or the multivalent anti-CD40 antibody construct according to any one of items 1 to 12 for use in treating cancer. 27. The pharmaceutical composition or multivalent anti-CD40 antibody construct according to item 26, wherein the cancer is a CD40-expressing cancer. 28. A nucleic acid or set of nucleic acids encoding the multivalent anti-CD40 antibody construct according to any one of items 1 to 24. 29. A recombinant cell comprising the nucleic acid or set of nucleic acids according to item 28 and expressing the multivalent anti-CD40 antibody construct according to any one of items 1 to 24. 30. A method for producing a multivalent anti-CD40 antibody construct according to any one of items 1 to 24, comprising expressing the multivalent anti-CD40 antibody construct from the nucleic acid or set of nucleic acids according to item 28 in a recombinant cell according to item 29, and optionally further comprising purifying the multivalent anti-CD40 antibody construct and formulating it into the pharmaceutical composition according to item 25. Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Brief explanation of the drawings]

[0014] [Figure 1](A-E) Characterization of anti-CD40 antibodies. (A) Effect of anti-CD40 IgG antibodies on CD40L binding. CD40-expressing cells were incubated with 200 ng / ml GpL-CD40L, a fusion protein of soluble CD40L and Gaussia princeps luciferase (GpL), and 5 μg / ml anti-CD40 antibodies for 1 hour. After washing three times with PBS to remove unbound molecules, the amount of GpL-CD40L bound to the cells was quantified using a luciferase assay and normalized to the antibody-free control. Averages from three independent experiments are shown. Antibody-treated groups were compared with the control group by ANOVA. ***p<0.001; *p<0.05; ns indicates not significant. (B) U2OS cells were treated with 10 μg / ml of the indicated antibodies together with HEK293 transfectants expressing memCD40L (293-mCD40L) or empty vector (293-EV). The following day, CD40-induced IL8 production was assessed by ELISA. (C) Specific binding of anti-CD40-IgG1-GpL fusion protein to HT1080 cells stably expressing (HT1080-CD40). Binding of anti-CD40-IgG1-GpL fusion protein to conventional HT1080 cells is considered nonspecific binding. Binding of anti-CD40-IgG1-GpL fusion protein to HT1080-CD40 cells resulted in total binding, and specific CD40 binding (open circles) was calculated by subtracting the nonspecific binding value (filled circles) from the corresponding total binding value (filled squares). The corresponding analysis of GpL-TNC-CD40L binding to CD40 (lower panel, right panel) served as a positive control. (D) Domain structure of CD40 and CD40 deletion mutants used in "E" below. (E) Anti-CD40 antibodies were loaded onto a protein G-coated ELISA plate. The protein G / anti-CD40 antibody complexes were finally incubated with C-terminal deletion mutants of the CD40 ectodomain carrying a C-terminal Gaussia princeps luciferase (GpL) reporter domain (see "D") or TNFR2(ed)-GpL as a negative control. Specific binding of the CD40 deletion mutant molecules was finally determined by subtracting the nonspecific TNFR2(ed)-GpL binding value from the total binding value of the various CD40-GpL fusion proteins. [Figure 2]Figure 1 shows that FcγR binding boosts IL8 production induced by anti-CD40 antibodies. (A) Specific binding of IgG1, IgG2, IgG4, and the IgG1(N297A) mutant of G28.5 to mouse FcγRIIb. (B) CD40-responsive U2OS cells were challenged with IgG1, IgG2, IgG4, and the IgG1(N297A) mutant of the indicated anti-CD40 antibodies along with HEK293 cells transfected with an empty vector (EV) or an expression plasmid encoding mouse FcγRIIb. The following day, cell supernatants were analyzed for IL8 production as a readout of CD40 activation. (C) Cocultures of U2OS cells and mouse A20 cells, which endogenously express FcγR, were stimulated with the anti-CD40 antibody CD40(Apexi)-IgG1, and IL8 production was measured again by ELISA the following day. [Figure 3] Figure 1 shows that FcγR binding enhances anti-CD40 antibody-induced p100 processing. (A) U2OS cells were stimulated overnight with HEK293 cells transfected with empty vector (EV) or an expression plasmid encoding mouse FcγRIIb and 150 ng / ml of the indicated antibody. Whole cell lysates were analyzed for p100 processing. (B) U2OS cells were stimulated overnight with mouse L929 cells, which do not express FcγR, or mouse A20 cells, which endogenously express FcγRIIb, and again with 150 ng / ml of the indicated antibody. Whole cell lysates were analyzed for p100 processing. [Figure 4]Oligovalent anti-CD40-IgG1(N297A) variants and anti-CD40-IgG1(N297A)-HC:scFvCD40 fusion proteins exert FcγR-independent CD40 agonism. (A) Domain structure (upper panel) and Western blotting (lower panel) of recombinant oligomerized anti-CD40-IgG1(N297A)-TNC and anti-CD40-IgG1(N297A-RGY) variants and tetravalent anti-CD40-IgG1(N297A)-HC:scFvCD40 fusion proteins. (B) HT1080-CD40 cells, which strongly produce the NFκB-regulated cytokine IL-8 after CD40 stimulation, were stimulated overnight with different anti-CD40 variants, and IL-8 production was finally recorded by ELISA. HT1080 cells were also challenged with membrane-transfected HEK293 cells transfected with CD40L. The resulting IL8 production was defined as maximal and was used to define half of the maximum possible IL8 response. Mean values ​​from four independent experiments are shown. [Figure 5] Agonism of oligovalent anti-CD40-IgG1(N297A) mutants and anti-CD40-IgG1(N297A)-HC:scFvCD40 fusion proteins is aggregation-independent. (A) The indicated constructs were purified by affinity chromatography on anti-Flag agarose, and purity was assessed by SDAS-PAGE and silver staining. (B) Gel filtration analysis of purified antibody fusion proteins. The dotted arrow indicates the remaining Flag peptide from affinity purification. (C) IL-8 induction in U2OS by purified IgG1(N297A)-scFv fusion proteins. [Figure 6] Oligomeric sdAb:CD40 variants display potent CD40 agonists. (A) Domain structure of oligomeric sdAb:CD40 variants. (B) Western blot analysis of sdAb:CD40 variants. (C) U2OS cells were stimulated overnight with different VHH:CD40 variants, and IL-8 production was determined by ELISA. (D, E) The selected constructs were purified by affinity chromatography and analyzed by SDS-PAGE (D) and gel filtration (E). [Figure 7](Figure 1) shows that endogenous agonism of anti-CD40(Seli)-N297A-HC:scFvSeli induces iDC maturation. Monocytes were cultured with GM-CSF / IL4 for 7 days to generate immature monocyte-derived dendritic cells (iDCs). iDCs were then treated with 200 ng / ml of anti-CD40(Seli)-N297A-HC:scFvSeli, and surface expression of CD14 and CD83 was analyzed by flow cytometry 2 days later. [Figure 8] Figure 1 shows induction of iDC maturation by VHH(V12t)-based multivalent CD40-specific constructs. Monocytes were cultured with GM-CSF / IL4 for 7 days to regenerate immature monocyte-derived dendritic cells (iDCs). (A) Two days after treatment of iDCs with 200 ng / ml of the indicated construct, cell surface expression of CD14 and CD83 was analyzed by flow cytometry. (B) iDCs were treated with 200 ng / ml of the indicated construct and analyzed the next day by Western blotting for the presence of the indicated proteins. [Figure 9] This figure shows that a multivalent variant of the CD40-specific antibody anti-CD40 (Seli)-N297A, which has intrinsic agonism, induces IL-8 production in iDCs. Monocytes were cultured with GM-CSF / IL-4 for 7 days to generate immature monocyte-derived dendritic cells (iDCs). The iDCs were then treated with 500 ng / ml of the indicated variant of anti-CD40 (Seli). The following day, cell culture supernatants were analyzed by IL-8 ELISA. [Figure 10] Figure 1 shows stimulation of IL8 production by VHH(V12t)-based multivalent CD40-specific constructs. Monocytes were cultured with GM-CSF / IL4 for 7 days to regenerate immature monocyte-derived dendritic cells (iDCs). iDCs were treated overnight with 8, 40, or 200 ng / ml of the indicated constructs, and finally, cell culture supernatants were analyzed for their IL8 content by ELISA. *p<0.05; **p<0.01, ***p<***. [Figure 11]Figure 1 shows the intrinsic agonism of tetravalent, hexavalent, and octavalent sdAb:CD40(V12t) variants on an IgG1(N297A) scaffold. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with different VHH:CD40-IgG1(N297A) variants, and IL-8 production was determined by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. [Figure 12] Figure 1 shows the intrinsic agonism of hexavalent, octavalent, and decavalent sdAb:CD40(V12t) variants on an IgG1(N297A) scaffold. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with different VHH:CD40-IgG1(N297A) variants, and IL8 production was determined by ELISA at the end. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. [Figure 13] Figure 1 shows the intrinsic agonism of octavalent and decavalent sdAb:CD40(V12t) variants on an IgG1(N297A) scaffold. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with different VHH:CD40-IgG1(N297A) variants and IL8 production was determined by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. [Figure 14] Figure 1 shows the intrinsic agonism of bivalent, trivalent, and tetravalent sdAb:CD40(V12t) variants on a Fab scaffold system. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with different VHH:CD40-Fab variants, and IL-8 production was determined by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. [Figure 15]Figure 1 shows the intrinsic agonism of bivalent sdAb:CD40(V12t) variants on an Fc(DANA) scaffold with different VHH domain configurations. Top panel: Domain structure of the constructs. Bottom panel: HT1080-CD40 cells were stimulated overnight with different VHH:CD40-Fc(DANA) variants, each containing a total of six VHH domains, but only two of which recognize CD40, and IL-8 production was determined in the cell culture supernatant by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. [Figure 16] Figure 1 shows the intrinsic agonism of tetravalent sdAb:CD40(V12t) variants on the Fc(DANA) scaffold system with different VHH domain configurations. Top panel: Domain structure of the constructs. Bottom panel: HT1080-CD40 cells were stimulated overnight with different VHH:CD40-Fc(DANA) variants, and finally, IL-8 production was determined in the cell culture supernatant by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 was used as a positive control. [Figure 17] Figure 1 shows the intrinsic agonism of trivalent, hexavalent, and nonavalent sdAb:CD40(V12t) variants on a tenascin trimerization (TNC) domain scaffold system. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with different VHH:CD40-TNC variants, and IL-8 production was determined by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. [Figure 18] Figure 1 shows the intrinsic agonism of bivalent, tetravalent, and hexavalent sdAb:CD40(V12t) variants on the Fc(DANA) scaffold system. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with different VHH:CD40-Fc(DANA) variants, and finally IL8 production was determined in the cell culture supernatant by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 was used as a positive control. [Figure 19]Figure 1 shows the intrinsic agonism of trivalent, tetravalent, and pentavalent sdAb:CD40(V12t) variants on a Fab scaffold system, with one sdAb domain on the opposite side of the scaffold from the other domains. Top panel: Domain structure of the constructs. Bottom panel: U2OS cells were stimulated overnight with the different VHH:CD40-Fab variants, and IL-8 production was determined by ELISA. Stimulation with soluble Flag-CD40L oligomerized with anti-Flag mAb M2 served as a positive control. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. Definitions and General Techniques Unless otherwise defined below, terms used herein have their ordinary meanings as known to those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications mentioned herein may be cited by identifying the full reference in the text, or by identifying the author and year of publication (e.g., "Kuramitsu et al. 2014") and identifying the corresponding full reference in the "References" section. All proteins according to the present invention, including the antibody constructs of the present invention, can be obtained by methods known in the art. Such methods include methods for producing recombinant proteins. The antibody constructs of the present invention can be expressed in recombinant host cells according to the present invention. The recombinant host cells of the present invention are preferably mammalian cells, such as CHO and HEK cells. It is understood that the antibody constructs of the present invention are meant to optionally include a secretory signal peptide sequence. Similarly, it is understood that the antibody constructs of the present invention are meant to optionally include an affinity tag, for example, to facilitate purification, and any protease cleavage site between the tag and the antibody construct, for example, to facilitate removal of the tag by protease cleavage. It is also understood that the antibody constructs of the present invention are meant to optionally include the respective propeptides. Additionally, any reference to amino acid sequences referred to herein includes not only the unmodified amino acid sequences but also common post-translational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, clipping of specific amino acids, or other post-translational modifications) that occur in cellular expression systems known in the art, including mammalian cells such as CHO cells or HEK cells.

[0016] The term "antibody" as used herein is described in Chapter 7 of Paul, WE (Ed.): Fundamental Immunology 2nd Ed. Raven Press, Ltd., New York 1989. Without limitation, the term "antibody" includes antibodies from any suitable source species, including mammals such as chicken, mouse, goat, non-human primates, and humans. Preferably, the antibody is a humanized or human antibody. The antibody is preferably a monoclonal antibody, which can be prepared by methods well known in the art. The term "antibody" includes IgG-1, -2, -3, or -4, IgE, IgA, IgM, or IgD isotype antibodies. The term "antibody" includes monomeric antibodies (e.g., IgD, IgE, IgG) or oligomeric antibodies (e.g., IgA or IgM). The term "antibody" also includes, but is not limited to, isolated antibodies and altered (modified) antibodies, for example, recombinant antibodies, such as chimeric, humanized, or human antibodies. For example, the terms "IgG1," "IgG2," "IgG3," and "IgG4" used herein include chimeric, humanized, and human IgG1, IgG2, IgG3, and IgG4, respectively. As defined herein, the antibody constructs of the present invention are not native IgA or IgM molecules. As used herein, the term "native IgA or IgM molecule" includes any naturally occurring IgA or IgM molecule, as well as chimeric, humanized, or human forms thereof. That is, the antibody constructs of the present invention are neither naturally occurring IgA or IgM molecules nor chimeric, humanized, or human forms thereof. In contrast, it is understood that the term "native IgA or IgM molecule" does not include an IgA or IgM molecule fused to a single domain antibody (sdAb) comprising a binding site for CD40 or an scFv comprising a binding site for CD40, nor an IgA or IgM molecule in which a portion thereof has been replaced by a single domain antibody (sdAb) comprising a binding site for CD40 or an scFv comprising a binding site for CD40. Thus, in one embodiment of the present invention, the antibody construct of the invention may be an IgA or IgM molecule fused to, or a portion of, a single domain antibody (sdAb) comprising a binding site for CD40 or an scFv comprising a binding site for CD40, replaced by, or a single domain antibody (sdAb) comprising a binding site for CD40 or an scFv comprising a binding site for CD40.

[0017] The term "single domain antibody (sdAb)" should be understood according to its known meaning in the art. Single domain antibodies are often also referred to as "nanobodies". Single domain antibodies are engineered from heavy chain antibodies, such as those found in camelids. Single domain antibodies engineered from heavy chain antibodies are often referred to as "VHH". Thus, in a preferred embodiment, one, several or all occurrences of "single domain antibody (sdAb)" in connection with the present invention may optionally be replaced by "VHH".

[0018] The term "IgG" as used herein may refer to naturally occurring IgG or mutant IgG, as known in the art.

[0019] For IgG1, such as chimeric, humanized or human IgG1, known mutations include, but are not limited to, N297A, N297D, N297Q, N297G, E233P, L234A, L234F, L235A, L235E, P331S, P329A, P329G, P331S, P238S, LALA (LALA = leucine 234 to alanine and leucine 235 to alanine), A330S, G237A, M252Y, S254T, S228P, T256E, M252F, M252S, M252W, M252T, T256S, T256R, T256Q, T256D, H268A and combinations thereof. Known IgG1 mutations also include E345K or E430G, which promote IgG1 hexamerization. For IgG2, such as chimeric, humanized, or human IgG2, known mutations include, but are not limited to, V234A, G237A, P238S, H268A, H268Q, A330S, P331S, P233S, V309L, and combinations thereof. For IgG3, such as chimeric, humanized, or human IgG3, known mutations include, but are not limited to, substitution of the CH2 domain of IgG3 with the CH2 domain of IgG2, removal of Fc glycosylation, mutations at L322, L276, N297, and combinations thereof. For IgG4, such as chimeric, humanized, or human IgG4, known mutations include, but are not limited to, V234A, L235E, LALA (leucine 234 to alanine and leucine 235 to alanine), F234A, P329G, S228P, G237A, P238S, G236 deletion, and combinations thereof.

[0020] The nomenclature of antibodies, antibody fragments, and variants thereof follows terms known in the art. In the present invention, it will be understood by those skilled in the art that this known nomenclature also applies to the antibody constructs of the present invention. For example, the known nomenclature also applies to antibodies forming part of the antibody constructs of the present invention. For example, it will be understood that terms relating to antibody mutations such as "N297A" are used herein in relation to IgG1 forming part of the antibody constructs of the present invention, and that this term is based on known antibody nomenclature.

[0021] As is generally known in the art, each antibody monomer comprises two heavy chains and two light chains. Each heavy and light chain contains a variable domain (called VH for the heavy chain and VL for the light chain) that is important for antigen binding. These heavy and light chain variable domains comprise (from N- to C-terminus) the regions FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 (FR, framework region; CDR, complementarity-determining region, also known as hypervariable region). Identification and assignment of such antibody regions within an antibody sequence can generally be performed using the IMGT / V-QUEST software described by Kabat et al. (Sequences of proteins of immunological interest, US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. 1983) or Chothia et al. (Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21-28;342(6252):877-83), or see Giudicelli et al. (IMGT / V-QUEST, an integrated software program for immunoglobulin and T cell receptor VJ and VDJ rearrangement analysis. Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W435-40). Preferably, the antibody regions are identified and assigned using the IMGT / V-QUEST software.

[0022] A "monoclonal antibody" is an antibody derived from an essentially homogeneous antibody population, in which the antibodies are substantially identical in sequence (i.e., identical except for a small fraction of antibodies containing naturally occurring sequence modifications (e.g., amino acid modifications at their cDNA ends)). Unlike polyclonal antibodies, which contain a mixture of different antibodies directed against either a single epitope or many different epitopes, monoclonal antibodies are highly specific, because they are directed against the same epitope. The term "monoclonal antibody" includes, but is not limited to, antibodies obtained from a population of monoclonal cells derived from a single cell clone, such as, for example, antibodies produced by the hybridoma method described by Koehler and Milstein (Nature, 1975 Aug. 7;256(5517):495-7) or Harlow and Lane ("Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988). Monoclonal antibodies also include antibodies produced by the hybridoma method described by Clackson et al. (Nature. 1991 Aug. 15;352(6336):624-8) or Marks et al. (J. Mol. Biol. 1991 Dec. 2001). 5;222(3):581-97). Monoclonal antibodies may be antibodies optimized for antigen-binding properties, such as reduced Kd values, optimized association and dissociation kinetics, by methods known in the art. For example, Kd values ​​may be optimized by display methods, including phage display, resulting in affinity-matured monoclonal antibodies. The term "monoclonal antibody" is not limited to antibody sequences derived from a particular species of origin or from one single species of origin. Thus, the term "monoclonal antibody" includes chimeric monoclonal antibodies, such as humanized monoclonal antibodies and human antibodies.

[0023] A "humanized antibody" is an antibody that contains human sequences and a small portion of non-human sequences that confer binding specificity to an antigen of interest. Typically, humanized antibodies are generated by replacing hypervariable region sequences from a human acceptor antibody with hypervariable region sequences from a non-human donor antibody (e.g., a mouse, rabbit, or rat donor antibody) that binds to the antigen of interest. In some cases, framework region sequences of the acceptor antibody may also be replaced with the corresponding sequences from the donor antibody. In addition to the sequences derived from the donor and acceptor antibodies, a "humanized antibody" may or may not contain other (additional or replacement) residues or sequences. Such other residues or sequences serve to further improve antibody properties, such as binding characteristics (e.g., to reduce Kd value) and / or immunogenicity properties (e.g., to reduce antigenicity in humans). Non-limiting examples of methods for making humanized antibodies are known in the art, for example, from Riechmann et al. (Nature. 1988 Mar 24; 332(6162):323-7) or Jones et al. (Nature. 1986 May 29-Jun 4; 321(6069):522-5). The term "human antibody" relates to an antibody comprising human variable domain sequences and human constant domain sequences. This definition encompasses antibodies in which human sequences have single amino acid substitutions or modifications that may serve to further improve antibody properties, such as binding characteristics (e.g., to reduce Kd values) and / or immunogenic properties (e.g., to reduce antigenicity in humans). The term "human antibody" excludes humanized antibodies in which a portion of non-human sequence confers binding specificity for an antigen of interest.

[0024] As used herein, an "antigen-binding portion" of an antibody refers to a portion of an antibody that retains the antibody's function of specifically binding to an antigen. This function can be determined, for example, by determining the ability of the antigen-binding portion to compete with the antibody for specific binding to an antigen, using methods known in the art. An antigen-binding portion can comprise one or more fragments of an antibody. Antigen-binding portions can be produced by any suitable method known in the art, including, but not limited to, recombinant DNA methods and preparation by chemical or enzymatic fragmentation of antibodies. An antigen-binding portion can be a Fab fragment, a F(ab') fragment, a Fab2 fragment, a single-chain variable fragment (scFv), a single-domain antibody, a diabody, or any other portion(s) of an antibody that retains the function of an antibody specifically binding to an antigen. In the sense of the term "FAB2" as known in the art, it will be understood that the term "FAB2" is synonymous with the terms "FAB2," "FAB2," and "FAB2." Therefore, for purposes of this application, the term "FAB2" is used interchangeably with the terms "FAB2," "FAB2," and "FAB2."

[0025] An "antibody construct" according to the present invention is a protein comprising a binding site for CD40 as defined herein. The antibody construct is capable of binding to its specific target antigen, i.e., CD40. It is understood that the antibody construct according to the present invention may be a protein consisting of a single polypeptide chain, or a protein in which two or more polypeptide chains are linked together. They may be covalently linked together, for example, by a covalent bond. Such covalent bonds may be one or more disulfide bonds. Alternatively, the covalent bonds may be obtained by chemical conjugation (preferably chemical conjugation using click chemistry) and / or they may be any other covalent bond known in the art as a suitable linkage for proteins. Alternatively, the antibody construct according to the present invention may be a protein in which two or more polypeptide chains are non-covalently linked together (e.g., by a non-covalent interaction). For example, the IgG1 mutations E345K and / or E430G promote IgG1 hexamerization. According to the present invention, such IgG1 mutations may be included in the antibody construct of the present invention to obtain a hexameric antibody construct.

[0026] Generally, with respect to all antibody constructs of the present invention, it is understood that portions of the antibody construct may be fused using a linker sequence. In that case, the antibody construct of the present invention comprises such a linker sequence. Suitable linker sequences are known in the art and include, but are not limited to, peptide linkers. An "antibody construct" according to the present invention may be an antibody construct that has been derivatized or linked to a different molecule. For example, molecules that may be linked to an antibody construct are molecular labels (e.g., fluorescent, luminescent, colored or radioactive molecules) and / or pharmaceutical agents.

[0027] The terms "antigen-binding site" and "antigen-binding sites" as used herein are known in the art. Typically, an antigen-binding site contains six complementarity-determining regions (CDRs). The six complementarity-determining regions (CDRs) are usually located in the VH and VL domains, i.e., CDR1, CDR2, and CDR3 are located in the VH (i.e., heavy chain variable domain), and CDR1, CDR2, and CDR3 are located in the VL (i.e., light chain variable domain). Various forms of antigen-binding sites are known in the art, including, but not limited to, antigen-binding sites contained in the VH and VH of an antibody or antibody fragments thereof, such as scFv.

[0028] As used herein, the term "FcγR" refers to any member of the family of proteins that bind to the IgG antibody Fc domain and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys, and are preferably derived from humans. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0029] The term "mutation that reduces FcγR binding" as used herein in reference to the IgG Fc domain of an antibody construct of the invention includes any mutation that reduces binding of the antibody construct to at least one FcγR receptor compared to a corresponding reference antibody construct in which the mutation is not present in the Fc domain. Whether a mutation reduces FcγR binding to an Fcγ receptor can be determined by methods known in the art, such as comparative surface plasmon resonance measurements of the binding of the antibody construct and the reference antibody construct to immobilized recombinant Fcγ receptors, respectively. Mutations that reduce FcγR binding are well known in the art.

[0030] For IgG1, such as chimeric, humanized, or human IgG1, known mutations that reduce FcγR binding include N297A, N297D, N297Q, N297G, E233P, L234A, L234F, L235A, L235E, P331S, P329A, P329G, P331S, P238S, LALA (LALA = leucine 234 to alanine and leucine 234), and LALA (LALA = leucine 234 to alanine and leucine 234). Examples of the nucleotide sequences include, but are not limited to, a nucleotide sequence mutation (a nucleotide sequence mutation of 235 to alanine), A330S, G237A, M252Y, S254T, S228P, T256E, M252F, M252S, M252W, M252T, T256S, T256R, T256Q, T256D, H268A, DANA (=D265A and N297A) and combinations thereof. For IgG2, such as chimeric, humanized, or human IgG2, known mutations that reduce FcγR binding include, but are not limited to, V234A, G237A, P238S, H268A, H268Q, A330S, P331S, P233S, V309L, and combinations thereof. For IgG3, such as chimeric, humanized, or human IgG3, known mutations that reduce FcγR binding include, but are not limited to, substitution of the CH2 domain of IgG3 with the CH2 domain of IgG2, removal of Fc glycosylation, mutations at L322, L276, N297, and combinations thereof. For IgG4, such as chimeric, humanized, or human IgG4, known mutations that reduce FcγR binding include, but are not limited to, V234A, L235E, LALA (leucine 234 to alanine and leucine 235 to alanine), F234A, P329G, S228P, G237A, P238S, G236 deletion, and combinations thereof.

[0031] The term "CD40 agonist" refers to a substance capable of inducing CD40 signal transduction or some aspect of CD40 signal transduction (e.g., one pathway of a set of pathways involved in memCD40L-induced CD40 signal transduction). CD40 signal transduction can be induced by the CD40 ligand (CD40L / CD154), a trimeric transmembrane protein of the TNF superfamily (TNFSF), which occurs in two forms: transmembrane CD40L (memCD40L) or soluble CD40L (sCD40L). MemCD40L is primarily expressed on activated CD4+ T cells, and sCD40L is released from memCD40L by proteolytic processing, including but not limited to, by metalloproteases. CD40L also binds to the integrins αMβ2, α5β1, α4β1, αIIbβ3, and αvβ3 (Aloui et al., 2014; Takada et al., 2019). CD40L binding to integrins is not competitive with CD40 but rather results in a ternary CD40L-CD40-integrin complex. Binding of sCD40L to CD40 and integrin (α5β1) stimulated classical NFκB signaling and B cell activation (Takada et al., 2019). Therefore, whether an antibody construct is a CD40 agonist can be determined by methods known in the art, including, but not limited to, methods for measuring IL8 secretion in CD40-expressing cells (e.g., by ELISA) and methods for measuring p100, the precursor protein of the NFκB transcription factor subunit p52. CD40-expressing cells are known and available in the art and include, for example, U2OS or HT-1080-CD40 cells, but also B cells, dendritic cells, and macrophages.

[0032] The cancers treated by the present invention are preferably solid cancers. A "solid cancer" is a cancer that forms one or more solid tumors. Such solid cancers that form solid tumors are generally known in the art. The term "solid cancer" includes the primary tumor formed by the cancer and possible secondary tumors, also known as metastases. Known solid cancers that can be treated by the present invention include, but are not limited to, melanoma, colorectal cancer, prostate cancer, head and neck cancer, urothelial cancer, gastric cancer, pancreatic cancer, liver cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, brain tumor, breast cancer, stomach cancer, renal cell carcinoma, Ewing's sarcoma, non-small cell lung cancer, and small cell lung cancer.

[0033] According to the present invention, the terms "cancer treatment" or "treating cancer," etc., refer to therapeutic treatment. Evaluating whether a therapeutic treatment works can be done, for example, by evaluating whether cancer growth is inhibited in the treated patient(s). Preferably, the inhibition is statistically significant, as assessed by a suitable statistical test known in the art. Inhibition of cancer growth can be assessed by comparing cancer growth in a group of patients treated according to the present invention with a control group of untreated patients, or by comparing a group of patients treated according to the present invention in addition to a standard cancer treatment in the art with a control group of patients receiving only the standard cancer treatment in the art. Such studies for evaluating inhibition of cancer growth are designed according to accepted standards for clinical studies, e.g., double-blind, randomized studies with sufficient statistical power. The term "treating cancer" includes inhibition of cancer growth in which cancer growth is partially inhibited (i.e., cancer growth in the patient is slowed compared to a control group of patients), completely inhibited (i.e., cancer growth in the patient is halted), and reversed (i.e., the cancer shrinks). Preferably, the assessment of whether a therapeutic treatment is effective is based on the classification of responders and non-responders using the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1) (Eisenhauer et al.: New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). In: Eur. J. Cancer. 45, No. 2, January 2009, pp. 228-47). Alternatively, or in addition, the assessment of whether a therapeutic treatment is effective can be based on known clinical indicators of cancer progression. Cancer treatment according to the present invention may be first line, second line, or third line, or more than third line therapy, as the meaning of such terms is known in the art and in accordance with terminology commonly used by the National Cancer Institute. Whether a cancer is a CD40-expressing cancer can be determined by methods known in the art, including, but not limited to, immunohistochemistry using anti-CD40 antibodies to analyze a cancer biopsy sample, or flow cytometry or binding studies using CD40-specific reagents (CD40L, antibodies). According to the present invention, the term "comprising" may optionally be replaced with the term "consisting of" respectively.

[0034] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or antibody-related methods) are carried out according to procedures known in the art, such as those described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual," 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology," Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0035] Protein-protein binding, e.g., antibody binding to each target protein, can be assessed by methods known in the art, preferably by surface plasmon resonance, spectroscopy, flow cytometry, and Gaussia princeps luciferase (GpL) fusion proteins of the protein of interest (e.g., CD40, antibody constructs).

[0036] Sequence alignment of the sequences according to the present invention can be performed using the BLAST algorithm (Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215, pp. 403-410; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402). Suitable parameters for sequence alignment of short peptides using the BLAST algorithm, which are suitable for the peptide antigens according to the present invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust parameters for sequence alignment of short input sequences. In one embodiment, the following parameters are used: maximum target sequence 10; word size 3; BLOSUM 62 matrix; gap cost: presence 11, extension 1; conditional composition score matrix adjustment. Therefore, when used in connection with sequences, terms such as "identity" or "identical" preferably refer to the identity value obtained by using the BLAST algorithm.

[0037] Preparation of the Pharmaceutical Composition of the Present Invention The pharmaceutical compositions of the present invention are prepared according to known standards for preparing pharmaceutical compositions. For example, the pharmaceutical compositions are prepared so that they can be appropriately stored and administered. Therefore, the pharmaceutical compositions of the present invention may contain pharmaceutically acceptable ingredients, such as carriers, excipients, and / or stabilizers. The pharmaceutically acceptable ingredients are not toxic in the amounts used when administering the pharmaceutical composition to human patients. The pharmaceutically acceptable ingredients added to the pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the specific intended use of the pharmaceutical composition, and the route of administration. Generally, pharmaceutically acceptable ingredients used in connection with the present invention are used according to knowledge available in the art, for example, from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.

[0038] array Preferred amino acid sequences referred to in this application may be independently selected from the following sequences: The sequences are presented in N-terminal to C-terminal order; represented in the single letter amino acid code. The following non-limiting exemplary sequences were used in the experimental examples of this application:

[0039] Table 1: Amino acid sequences represented in heavy and light chain variants of CD40-specific antibody constructs

[0040] [Table 1] TIFF2025531618000002.tif190170 TIFF2025531618000003.tif248170 TIFF2025531618000004.tif165170 TIFF2025531618000005.tif159170 TIFF2025531618000006.tif195170 TIFF2025531618000007.tif202170 TIFF2025531618000008.tif186170TIFF2025531618000009.tif84170TIFF20255316180 00010.tif255170TIFF2025531618000011.tif254170TIFF2025531618000012.tif254170 TIFF2025531618000013.tif255170TIFF2025531618000014.tif254170TIFF20255316180 00015.tif254170TIFF2025531618000016.tif254170TIFF2025531618000017.tif254170 TIFF2025531618000018.tif17170 For each of plasmid numbers 1-5, 9, 13, 17, 21, 24, 36, 43, 47, 51, 55, and 56, the encoded amino acid sequences are as follows: The indicated amino acids of SEQ ID NOs: 1-6, 9, 13, 17, 21, 24, 36, 43, 47, 51, 55, and 56 are each preceded by an N-terminal amino acid consisting of (in order from N-terminus to C-terminus): a leader sequence (SEQ ID NO: 30: MNFGFSLIFLVLVLKGVQCEVKLVPR), a first restriction site encoding a 2AA linker sequence consisting of amino acids QL, a Flag tag (SEQ ID NO: 59: DYKDDDDK), and a second restriction site encoding a 2AA linker sequence consisting of amino acids EL.

[0041] The encoded amino acid sequences for each of plasmid numbers 6, 7, 8, 10, 11, 12, 14, 15, 16, 18, 19, 20, 44, 45, 46, 48, 49, 50, and 52-54 are as follows: The indicated amino acids in SEQ ID NOs: 7, 8, 10, 11, 12, 14, 15, 16, 18, 19, 20, 44, 45, 46, 48, 49, 50, and 52-54 each contain (in order from N-terminus to C-terminus): a leader sequence (SEQ ID NO: 30: MNFGFSLIFLVLVLKGVQCEVKLVPR), a first restriction site encoding a 2AA linker sequence consisting of amino acids QL, a Flag tag (SEQ ID NO: 59: DYKDDDDK), and a second restriction site encoding a 2AA linker sequence consisting of amino acids EF, followed by the N-terminal amino acid.

[0042] The encoded amino acid sequences for each of plasmid numbers 22, 23, 25, 26, and 37 to 41 are as follows: Each of the designated amino acids in SEQ ID NOs: 22, 23, 25, 26, and 37 to 41 is preceded by an N-terminal amino acid consisting of (in order from N-terminus to C-terminus) a leader sequence (SEQ ID NO: 30: MNFGFSLIFLVLVLKGVQCEVKLVPR), a first restriction site encoding a 2AA linker sequence consisting of the amino acid QL, a flag tag (SEQ ID NO: 61: DYKDDDDK), and a second restriction site encoding a 2AA linker sequence consisting of the amino acid GT.

[0043] For plasmid numbers 27, 31, 34, and 42, the encoded amino acid sequences are as follows: Each of the given amino acids in SEQ ID NOs: 27 and 42 is preceded by an N-terminal amino acid consisting of (in order from N to C-terminus) a leader sequence (SEQ ID NO: 30: MNFGFSLIFLVLVLKGVQCEVKLVPR) and a restriction site encoding a 2AA linker sequence consisting of amino acids QL.

[0044] The encoded amino acid sequences for each of plasmid numbers 28-29, 32, 33, 35, 57, and 58 are as follows: Each of the predetermined amino acid sequences of SEQ ID NOs: 28-29 is preceded by an N-terminal amino acid sequence consisting of (in order from N-terminus to C-terminus) a leader sequence (SEQ ID NO: 30: MNFGFSLIFLVLVLKGVQCEVKLVPR) and a restriction site encoding a 2AA linker sequence consisting of the amino acids GT.

[0045] It should be noted that in the present invention, the above-mentioned restriction sites encoding 2AA linker sequences are cloning-related amino acid linker sequences, which may be independently removed or independently replaced with another linker sequence comprising 1 to 20 amino acids. Similarly, in the present invention, the above-mentioned internal linker sequence STKGPKLEEGEFSEA in the amino acid sequences of SEQ ID NOs: 8, 12, 16, 20, 45, 50, and 54, and the above-mentioned internal linker sequence GGGGSGGGGSGGGGSGGGGSGGGGGS in the amino acid sequences of SEQ ID NOs: 22, 23, 25, 26, 28, 28, 32, 33, 35, 37, 38, 40, 41, 57, and 58, are internal linker sequences, respectively, which may be independently removed or independently replaced with another linker sequence comprising 1 to 20 amino acids.

[0046] Table 2: CD40 agonist proteins

[0047] [Table 2] TIFF2025531618000020.tif140170 Note that "X" can be any single domain antibody (sdAb).

[0048] [Example] The present invention is further illustrated by the following non-limiting examples. Materials and Methods cell culture HEK293, U2OS, L929, A20J, and HT1080 cells (ATCC, Rockville, MD, USA) and HT1080-CD40 transfectants (Wyzgol et al., 2009) were cultured at 37°C and 5% CO2 with regular splitting twice weekly. HEK293, A20J, and HT1080 / HT1080-CD40 cells were cultured in RPMI 1640 medium (Gibco), while U2OS and L929 cells were cultured in DMEM containing high-glucose medium (Glutamax, Gibco). The culture medium was supplemented with 10% fetal calf serum (FCS; Life Technologies, Karlsruhe, Germany). Expression plasmids Standard cloning techniques, DNA amplicons, and synthetic DNA fragments were used to generate pCR3-based expression plasmids encoding the proteins and antibody chains listed in Table 1. The antibody variants used in this study are listed in Table 2. The amino acid sequences represented by the building blocks of the proteins encoded by the plasmids in Table 1 are listed in Table 3. Table 3. Amino acid sequences represented by building blocks of proteins encoded by the plasmids in Table 1

[0049] [Table 3] TIFF2025531618000022.tif38170 1 Therapeutic structural antibody database Recombinant Protein Production and Purification All recombinant proteins were produced by transfecting HEK293 cells with the corresponding expression plasmids using polyethyleneimine (PEI; Polyscience Inc., Warrington, WA, USA) as described in detail elsewhere (Kums et al., 2017). A 1:1 mixture of the corresponding expression plasmids was used to generate recombinant antibody variants containing light and heavy chains. Five to seven days after transfection, cell culture supernatants from transfected cells were collected and centrifuged at 5,000 g for 10 minutes to remove cell debris. The concentrations of the recombinant proteins were estimated by Western blot analysis of the supernatants with Flag-tagged protein standards of known concentrations using anti-Flag antibody M2 (Sigma-Aldrich, St. Louis, CA, USA) and goat anti-mouse IgG1 IRDye800CW antibody (Licor, Lincoln, CA, USA).

[0050] Purification of antibody variants by affinity chromatography To purify the Flag-tagged antibody variants, affinity chromatography using anti-Flag M2 agarose and 3xFlag peptide (both Sigma-Aldrich, Steinheim, Germany) was performed as described by the manufacturer. After elution, protein purity was analyzed by SDS-PAGE and silver staining using the Pierce Silver Stain Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. The concentration of the purified protein was estimated by comparison with a protein of known concentration using the Amersham LMW Calibration Kit for SDS electrophoresis (GE Healthcare UK Limited, Little Chalfont, UK).

[0051] Co-culture, ELISA and ECL Western blot To assess CD40 activation, CD40-responsive cells (HT1080-CD40 or U2OS) were grown overnight on flat-bottom cell culture plates. In parallel, HEK293 cells were transfected with expression plasmids encoding FcγRIIb or memCD40L, or with an empty vector as a negative control, using the PEI method (see above). The following day, CD40-responsive cells were mixed (1:1) with mouse FcγRIIb and EV transfectants, and the resulting cocultures were stimulated with the anti-CD40 antibody construct of interest. For analysis of CD40-induced IL8 production, coculture experiments were performed in 96-well plates (2 × 10 cells per well). 4 +2×10 4 Cells). After overnight challenge with the antibody constructs, the supernatants were analyzed for IL-8 content by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, San Diego, USA) according to the manufacturer's protocol. Co-culture with memCD40L-expressing transfectants helped define the maximal IL-8 response. To assess CD40-mediated activation, p100 processing and TRAF1 induction were analyzed by Western blotting in U2OS cells. For Western blot experiments, co-culture experiments were performed in 6-well plates (1 × 10 6 +1×10 6) after overnight stimulation. After overnight stimulation, the cocultures were harvested by scraping with a rubber policeman in ice-cold PBS. Cells were then washed twice with fresh ice-cold PBS, pelleted, and resuspended in Laemmli buffer. Samples were sonicated for 25 seconds at 100% amplitude using an ultrasonication probe (UP100H Ultrasonic Processor, Hierscher, Germany), heated at 95°C for 5 minutes, and subjected to SDS-PAGE separation. Proteins were transferred to nitrocellulose membranes and then subjected to Western blot analysis using anti-p100 / p52 (Millipore), anti-TRAF1 (Cell Signaling Technology, Valerie, MA, USA), anti-β-actin (Sigma-Aldrich), and horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse antibody (Dako, Glostrup, Denmark) or HRP-conjugated anti-rabbit antibody (Cell Signaling Technology, Valerie, MA, USA). Finally, the membranes were developed by chemiluminescent Western blot detection.

[0052] Combined research For binding studies, 2 x 10 4 Cells / well were cultured overnight in clear, flat-bottom black cell culture plates (Greiner Bio-One). To analyze the inhibitory effect of anti-CD40 antibodies on CD40L binding, HT1080-CD40 cells were incubated with anti-CD40 antibodies for 30 min at 37°C in triplicate and then supplemented with GpL-TNC-CD40L (100 ng / ml). After an additional 1 h, the cells were washed five times with ice-cold PBS, and cell-associated luciferase activity was measured. To determine the specific binding of GpL-linked anti-CD40 antibodies, the latter were added in pairs at increasing concentrations to HT1080 (nonspecific binding) and HT1080-CD40 (total binding) cells. After 1 h at 37°C, cells were washed five times with fresh ice-cold PBS, and GpL activity was finally measured. Specific binding was calculated by subtracting the nonspecific binding value from the corresponding total binding value. To determine the binding of GpL-tagged deletion mutants of the CD40 ectodomain to various anti-CD40 antibodies, black high-binding 96-well plates (Greiner Bio-One) were coated overnight with 1 μg / ml protein G. After blocking remaining free binding sites with 10% FCS in PBS and washing steps with ice-cold PBS, anti-CD40 antibodies (1 μg / ml) were added to the culture medium for 30 min. After removing free antibodies by two washing steps with ice-cold PBS, GpL-linked CD40 deletion mutants were added for 1 h at 37°C. Cells were then washed five times with ice-cold PBS, and luminescence was measured. GpL activity was measured by adding 25 μl of 1.5 μM coelenterazine substrate in PBS to the cells. [Example]

[0053] FcγR-binding anti-CD40 antibodies consistently exhibit strong agonism regardless of isoform and idiotype Given the complex and sometimes even contradictory findings regarding the FcγR dependence of CD40 antibody agonism, we first defined a panel of anti-CD40 antibodies with widely differing properties. This panel included the well-established and long-known anti-CD40 antibody G28.5 (Clark et al., 1988) and several anti-CD40 antibodies currently being studied in clinical trials and / or disclosed in several patents (CP-870,893 / Selicrelumab, Vonderheide et al., 2007; APX005M / Sotigalimab, Filbert et al., 2021; ADC-1013 / Mitazalimab, Non-Patent Document 5; ChiLob7.4, Non-Patent Document 15). All antibodies in the panel recognize the human CD40 molecule but have different effects on CD40L binding. ADC-1013, APX005M, and the IgG1 variant of G28.5 significantly inhibited the binding of a soluble CD40L GpL fusion protein (GpL-CD40L) to CD40-expressing cells, whereas CP-870,893 and ChiLob7.4 had no effect on GpL-CD40L binding to CD40 (Figure 1A). Consistent with this, the latter two antibodies had no effect on memCD40L-induced production of IL-8, a readily measurable chemokine whose expression is predominantly regulated by the canonical NFκB signaling pathway and, therefore, is efficiently induced by CD40 activation (Figure 1B). In contrast, ADC-1013, APX005M, and G28.5 significantly inhibited this CD40 response (Figure 1B). The antibodies in the panel further differed with respect to CD40 affinity and the region in the extracellular domain of CD40 with which they interacted (Fig. 1C-E).

[0054] Anti-CD40 antibodies currently in clinical development are IgG1, IgG2, or IgG4 antibodies. The different in vitro agonism of these antibodies as well as their different in vivo tolerability are, at least in part, due to differences in isotype (Vonderheide and Glennie 2013; Non-Patent Document 1). Therefore, we investigated the anti-CD40 antibodies in our panel not only in their original isotypes but also in the other two of the three isotypes mentioned above. Furthermore, we also analyzed an IgG1 variant of the antibody with a point mutation (N297A) (IgG1(N297A)), which has been shown to strongly reduce binding to FcγRI and abrogate binding to the remaining FcγRs (Wang et al., 2018). First, we evaluated the ability of the conventional IgG variants in our anti-CD40 antibody panel to induce FcγR-dependent CD40 activation. For this purpose, we used a simple in vitro system in which CD40-expressing responder cells (U2OS or HT1080-CD40 transfectants), which secrete large amounts of IL-8 after CD40 stimulation, were cocultured with HEK293 transfectant cells. The latter express little IL-8 and were transiently transfected with mouse FcγRIIb (Figure 2A), which can bind IgG1, IgG2, and weakly IgG4, or with an empty vector. The use of FcγR transfectants offered three advantages over the use of cells with endogenous FcγR expression. First, we could analyze one type of FcγR without possible interference from other FcγR types. Second, there was a strict negative control, i.e., EV-transfected HEK293 cells. Third, the high expression levels achieved by transient transfection minimized the risk that CD40 binding by FcγR-binding anti-CD40 antibodies would be underestimated simply due to the fact that the number of CD40 molecules in the system exceeded the number of FcγRs available for anti-CD40 presentation. All wild-type anti-CD40 IgG variants, except for Lob7.4, efficiently stimulated IL8 production in cocultures with HEK293 transfectants expressing mouse FcγRIIb, reaching a maximal response similar or nearly identical to that obtained with memCD40L transfectants (Figure 2B, lower panel).The agonistic activity of IgG1 and IgG4 Lob7.4 mutants was still significantly increased in coculture with FcγRIIb-expressing HEK293 cells, but did not fully reach the level of memCD40L. In sharp contrast, coculture with HEK293 empty vector control transfectants showed no or significantly weaker IL8 induction at high concentrations. As expected, anti-CD40 IgG1(N297A) mutants of various antibodies that do not bind FcγRIIb showed little or no agonism in both cocultures with FcγRIIb-transfected HEK293 cells (Figure 2B, lower panel) and with control transfectants (Figure 2B, upper panel). Similar results were largely obtained using cocultures of CD40-responsive cells with murine A20 cells, which endogenously express FcγR (Figure 2C). A similar response pattern for anti-CD40-induced IL8 production was also observed when CD40-mediated activation of the alternative NFκB signaling pathway was investigated. The alternative NFκB pathway essentially activates a different set of NFκB transcription factors and uses a different signaling pathway to achieve this (Hayden and Gosh, 2012). Activation of the alternative NFκB pathway is characterized by processing of the precursor protein p100 to the NFκB transcription factor subunit p52. In cocultures with FcγRIIb-expressing HEK293 transfectants and U2OS cells, all anti-CD40 antibodies efficiently processed p100 to p52 and upregulated the expression of TRAF1 protein, which is induced by both NFκB signaling pathways (Figure 3). In contrast, there was little or no evidence of activation of the alternative NFκB signaling pathway in cocultures with HEK293 control transfectants. Thus, these results demonstrated that the agonism of plasma membrane-presented anti-CD40 antibody-FcγR complexes is regularly much higher than that of anti-CD40 IgG antibodies, regardless of the epitope recognized by the specific anti-CD40 antibody.Furthermore, because the maximum IL8 induction achievable by FcγR-bound anti-CD40 antibodies is comparable to the IL8 production induced by membrane CD40L, it appears that FcγR-bound anti-CD40 antibodies, but not "free" anti-CD40 antibodies, can stimulate the maximum possible CD40 activity. [Example]

[0055] Recombinant anti-CD40 antibody oligomers are potent agonists It is well established that secondary cross-linking of anti-TNFR antibodies can enhance agonistic activity (Non-Patent Document 4). In this study, we investigated whether the defined oligomerization of anti-CD40 antibodies by genetic engineering would result in agonistic molecules. To this end, we generated trimeric (hence hexavalent) and hexameric (hence dodecavalent) variants of all IgG1(N297A) antibodies in our anti-CD40 antibody panel. The hexavalent variants were obtained by genetically fusing the trimerization domain of the tenascin-C molecule, containing approximately 30 amino acids, to the C-terminus of the heavy chain of the IgG1(N297A) molecule (Figure 4A). Point mutations that promote the assembly of hexameric IgG1 molecules were introduced into the IgG1(N297A) heavy chain to obtain the dodecavalent variant (Diebolder et al., 2014) (Figure 4A). Various anti-CD40-IgG1(N297A)-TNC variants induced half-maximal IL-8 responses in benchmark memCD40L-expressing cells at concentrations ranging from 0.2 to 2 μg / ml (Figure 3B). The anti-CD40-IgG1(N297A-RGY) variant also acted as an efficient CD40 agonist, regularly being slightly more efficient in CD40-dependent IL-8 induction than the anti-CD40-IgG1(N297A)-TNC variant (Figure 4B). Indeed, all anti-CD40-IgG1(N297A-RGY) constructs, except for the Lob7.4-based variant, induced half-maximal CD40-dependent IL-8 production at concentrations below 100 ng / ml (Figure 4B). A tetravalent variant of the anti-CD40-IgG1(N297A) antibody, obtained by fusing an scFv domain generated from the antibody variable domain to the C-terminus of the antibody heavy chain (Figure 4B), was also examined. These IgG1(N297A)-HC:scFv mutants also exhibited potent agonistic activity against all CD40 antibodies, exhibiting activity comparable to that of the anti-CD40-IgG1(N297A-RGY) mutant. The data presented thus far were obtained using cell culture supernatants containing the anti-CD40 variants of interest. To verify that i) the observed agonistic activity was indeed associated with oligomerized antibody molecules and ii) the anti-CD40 fusion proteins maintained their integrity, the CP-870,893 mutants were illustratively purified by gravity-flow affinity chromatography on anti-Flag antibody M2 (Figure 5A).Indeed, gel filtration analysis revealed high-molecular-weight peaks corresponding to oligomerized antibody molecules, but in the case of CP-870,893-IgG1(N297A)-RGY and CP-870,893-IgG1(N297A)-TNC, peaks corresponding to the parental, non-aggregated antibody molecules were also present (Figure 5B). The most significant purified protein samples remained potent CD40 agonists (Figure 5C). [Example]

[0056] Recombinant oligovalent CD40-specific single-domain antibodies are potent agonists When protein scaffolds were oligomerized using human CD40-specific "single-domain antibodies (sdAbs)" (de Weerdt et al., 2021) as building blocks, similar results to those of oligomerized conventional antibodies were obtained (Figure 6A, B). Similar to conventional bivalent anti-CD40 IgG antibodies, bivalent sdAb-Fc and trivalent sdAb-TNC fusion proteins proved to be poorly active / inactive (Figure 6C). However, when three sdAb domains were sequentially cloned into the Fc domain of human IgG1 or the trimerization domain of TNC, hexavalent and nonavalent molecules exhibited strong CD40 stimulatory activity (Figure 6C). Tetravalent sdAb variants, obtained by replacing the IgG1 variable domain with CD40-specific sdAb domains, showed some activity, but were much less active than the hexavalent and nonavalent variants. Gel filtration analysis of the purified proteins showed no aggregates for the tetravalent and nonavalent variants, but some aggregates for the hexavalent molecules. [Example]

[0057] Recombinant oligomeric CD40-specific variants stimulate maturation of immature human dendritic cells Monocytes were cultured with GM-CSF / IL4 for 7 days to generate immature monocyte-derived dendritic cells (iDCs). When iDCs were treated with the described oligovalent CD40-specific variants, their maturation was clearly demonstrated by upregulation of CD83 as measured by flow cytometry and enhanced production of IL-8 and A20 as measured by ELISA and Western blotting (Figures 7-10). [Example]

[0058] Not only the number but also the location of CD40-binding domains determines agonist activity Various sdAb constructs with 2, 3, 4, 6, 8, 9, 10 or 12 parallel-organized CD40-binding domains were obtained by fusing one, two or three sdAb domains to the N-terminus of a human IgG1 Fc domain (N297A) based or substituting the variable domains of an IgG1 antibody (again N297 based) or Fab1. With the exception of the moderately active hexavalent 2xsdAb-TNC fusion protein (construct 26, Figure 17), all constructs with six or more sdAb domains (constructs 17 and 18, Figure 11; constructs 19-21, Figure 12; constructs 22 and 23, Figure 13; and construct 30, Figure 18) were highly active at low concentrations, reaching the activity of the benchmark oligomerized recombinant soluble CD40L. All bivalent and trivalent sdAb variants were largely inactive (constructs 33 and 34, Figure 14; constructs 8 / 992, 8 / 995, and 9 / 50, Figure 15; construct 25, Figure 17; and construct 28, Figure 18). It is worth noting that the agonism of the tetravalent constructs varied considerably, from poor activity (construct 35, Figure 14) to moderate activity (construct 16, Figure 11) to activity almost identical to that of the hexavalent constructs (constructs 8 / 983, 8 / 994, and 8 / 998, Figure 16). The large variability in the activity of the tetravalent constructs may be due to the different simultaneous binding capabilities of the four sdAb domains within a particular construct or to different binding capabilities to CD40 molecules expressed by two adjacent cells (which may also be the cause of the hexavalent system 2xVHH-TNC). Indeed, there is evidence that cell-cell contact conditions significantly enhance the ability of soluble ligand molecules and antibodies to bind to TNFRSF receptors, including CD40 (NPL 4). To demonstrate the latter, we included one additional sdAb domain at the C-terminus (constructs 36-38, Figure 19) in the bivalent, trivalent and tetravalent sdAb-Fab1 systems (constructs 33-35, Figure 14). Indeed, this significantly enhanced the activity of the inactive constructs 34 and 35 in Figure 14.

[0059] Conclusions A) Six parallel-oriented CD40 binding sites robustly enhance CD40 agonism. Adding more binding sites has a comparable, lesser effect. The construction of four and possibly five binding sites defines a transition region where small structural differences can have a strong effect on agonism. Conclusion B) Bidirectionally oriented CD40 binding sites can compensate for the number of CD40 binding sites to some extent. [Industrial Applicability]

[0060] The pharmaceutical compositions, polypeptides, nucleic acids, cells, and products for use in the present invention may be industrially applicable, for example, they may be used in the manufacture of pharmaceuticals or as pharmaceuticals. References

[0061] [Table 4] TIFF2025531618000024.tif223170TIFF2025531618000025.tif235170TIFF2025531618000026.tif234170

Claims

1. A multivalent anti-CD40 antibody construct comprising at least four antigen binding sites for CD40, wherein the multivalent anti-CD40 antibody construct is not a natural IgA or IgM molecule.

2. 2. The multivalent anti-CD40 antibody construct of claim 1, comprising at least one IgG molecule comprising two of the antigen binding sites for CD40.

3. 2. The multivalent anti-CD40 antibody construct of claim 1, comprising a modified IgG molecule in which each of the two variable domains of the heavy chain has been replaced with at least one single domain antibody (sdAb) comprising one of the antigen binding sites for CD40, and in which each of the two variable domains of the light chain has been replaced with at least one single domain antibody (sdAb) comprising one of the antigen binding sites for CD40.

4. 4. The multivalent anti-CD40 antibody construct of claim 2 or 3, further comprising an scFv or single domain antibody (sdAb) covalently attached to the C-terminus of one of the two heavy chains of the IgG molecule or modified IgG molecule, wherein the scFv or single domain antibody (sdAb) comprises one of the antigen binding sites for CD40.

5. The multivalent anti-CD40 antibody construct of claim 4, further comprising the scFv or single domain antibody (sdAb) covalently linked to the C-terminus of the other of the two heavy chains of the IgG molecule or modified IgG molecule, wherein the scFv or single domain antibody (sdAb) comprises one of the antigen binding sites for CD40.

6. 2. The multivalent anti-CD40 antibody construct of claim 1, comprising an Fc fragment of an IgG molecule, wherein each of the two N-termini and / or each of the two C-termini of said Fc fragment is covalently linked to a polypeptide chain comprising one, two or three, preferably three, single domain antibodies (sdAbs), each of said one, two or three, preferably three, single domain antibodies (sdAbs) comprising one of said antigen binding sites for CD40.

7. The multivalent anti-CD40 antibody construct of any one of claims 1 to 6, comprising six antigen-binding sites for CD40.

8. The multivalent anti-CD40 antibody construct of any one of claims 1 to 7, comprising 12 antigen-binding sites for CD40.

9. The multivalent anti-CD40 antibody construct according to any one of claims 1 to 8, wherein any two of the antigen-binding sites for CD40 are oriented in parallel.

10. 10. The multivalent anti-CD40 antibody construct of any one of claims 2 to 9, comprising trimers of three of the IgG molecules or modified IgG molecules formed by a trimerization domain fused to the C-terminus of each heavy chain of each of the IgG molecules or modified IgG molecules.

11. The multivalent anti-CD40 antibody construct of any one of claims 4 to 10, wherein the trimerization domain is the trimerization domain of tenascin-C.

12. 11. The multivalent anti-CD40 antibody construct of claim 10, wherein the trimerization domain comprises an amino acid sequence set forth in SEQ ID NO: 60, or a sequence at least 70% identical thereto, at least 80% identical thereto, at least 85% identical thereto, at least 90% identical thereto, at least 93% identical thereto, or at least 96% identical thereto.

13. The multivalent anti-CD40 antibody construct of any one of claims 2 to 12, wherein the IgG molecule or modified IgG molecule comprises an oligomerization mutation.

14. 14. The multivalent anti-CD40 antibody construct of claim 13, wherein the oligomerization mutations are E345R / E430G / S440Y mutations.

15. 15. The multivalent anti-CD40 antibody construct of any one of claims 1 to 14, comprising one or more Fab1 domains, scFv domains, and / or sdAb domains, each comprising one of said antigen binding sites for CD40.

16. The multivalent anti-CD40 antibody construct of any one of claims 2 to 15, wherein the IgG molecule or modified IgG molecule is IgG1, IgG2, IgG3 or IgG4.

17. The multivalent anti-CD40 antibody construct of any one of claims 2 to 16, wherein the IgG molecule or modified IgG molecule is IgG2 or IgG4.

18. 18. The multivalent anti-CD40 antibody construct of any one of claims 2 to 17, wherein the Fc domain of the IgG molecule or modified IgG molecule or Fc fragment of the IgG molecule comprises a mutation that reduces FcγR binding.

19. 19. The multivalent anti-CD40 antibody construct of claim 18, wherein the mutation is an N297A mutation.

20. A multivalent anti-CD40 antibody construct comprising at least two antigen binding sites for CD40, which are not natural IgA or IgM molecules, and wherein two of the antigen binding sites for CD40 are oriented antiparallel.

21. 21. The multivalent anti-CD40 antibody construct of claim 20, wherein the domains comprising the binding sites are linked by a linker sequence or a peptide linker sequence.

22. The multivalent anti-CD40 antibody construct of any one of claims 1 to 21, which is an Fc-independent CD40 agonist.

23. below: a) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 1 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 1 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 1 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence represented by SEQ ID NO: 1 and an amino acid sequence represented by SEQ ID NO: 2; b) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 1 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 1 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 1 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence that is represented by SEQ ID NO: 1 and an amino acid sequence that is represented by SEQ ID NO: 3; c) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:1 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO:4, an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4, an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence set forth in SEQ ID NO:1 and an amino acid sequence set forth in SEQ ID NO:4: d) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:5 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:6, an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6, an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:5 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence set forth in SEQ ID NO:5 and an amino acid sequence set forth in SEQ ID NO:6; e) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:5 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:7, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:5 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:7, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:5 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:6, or an amino acid sequence represented by SEQ ID NO:5 and an amino acid sequence represented by SEQ ID NO:7; f) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:5 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:8, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:5 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:8, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:5 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:8, or an amino acid sequence represented by SEQ ID NO:5 and an amino acid sequence represented by SEQ ID NO:8; g) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:10, an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:10, an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence set forth in SEQ ID NO:10; h) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:11, an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:11, an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:11, or an amino acid sequence set forth in SEQ ID NO:9 and an amino acid sequence set forth in SEQ ID NO:11; i) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:9 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:12, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:9 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:12, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:9 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:12, or an amino acid sequence represented by SEQ ID NO:9 and an amino acid sequence represented by SEQ ID NO:12; j) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 14, an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14, an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 13 and at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence set forth in SEQ ID NO: 13 and an amino acid sequence set forth in SEQ ID NO: 14; k) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 13 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 13 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 13 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 15, or an amino acid sequence represented by SEQ ID NO: 13 and an amino acid sequence represented by SEQ ID NO: 15; l) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 13 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 13 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 13 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 16, or an amino acid sequence represented by SEQ ID NO: 13 and an amino acid sequence represented by SEQ ID NO: 16; m) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 17 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 17 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 17 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence represented by SEQ ID NO: 17 and an amino acid sequence represented by SEQ ID NO: 18; n) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 17 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 19, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 17 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 19, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 17 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 19, or an amino acid sequence represented by SEQ ID NO: 17 and an amino acid sequence represented by SEQ ID NO: 19; o) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 17 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 20, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 17 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 20, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 17 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence represented by SEQ ID NO: 17 and an amino acid sequence represented by SEQ ID NO: 20; p) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:24, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:24, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:24, or an amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence represented by SEQ ID NO:24; q) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:25, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:25, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:25, or an amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence represented by SEQ ID NO:25; r) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:26, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:26, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:26, or an amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence represented by SEQ ID NO:26; s) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:24, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:24, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:24, or an amino acid sequence represented by SEQ ID NO:22 and an amino acid sequence represented by SEQ ID NO:24; t) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:25, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:25, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:25, or an amino acid sequence represented by SEQ ID NO:22 and an amino acid sequence represented by SEQ ID NO:25; u) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:26, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:26, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:26, or an amino acid sequence represented by SEQ ID NO:22 and an amino acid sequence represented by SEQ ID NO:26; v) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:24, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:24, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:24, or an amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence represented by SEQ ID NO:24; w) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:25, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:25, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:25, or an amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence represented by SEQ ID NO:25; x) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:26, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:26, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:26, or an amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence represented by SEQ ID NO:26; y) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:27, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:27, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO:27, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO:27, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:27, or the amino acid sequence represented by SEQ ID NO:27; z) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:28, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:28, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO:28, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO:28, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:28, or the amino acid sequence represented by SEQ ID NO:28; aa) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:29, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:29, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO:29, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO:29, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:29, or the amino acid sequence represented by SEQ ID NO:29; bb) an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 31, an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 31, an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 31, an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 31, an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 31, or the amino acid sequence set forth in SEQ ID NO: 31; cc) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 32, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 32, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 32, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 32, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 32, or the amino acid sequence represented by SEQ ID NO: 32; dd) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 33, or the amino acid sequence represented by SEQ ID NO: 33; ee) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 34, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 34, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 34, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 34, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence represented by SEQ ID NO: 34; ff) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 35, or an amino acid sequence represented by SEQ ID NO: 35; gg) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:36, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:36, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:36, or an amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence represented by SEQ ID NO:36; hh) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:36, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:36, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:36, or an amino acid sequence represented by SEQ ID NO:22 and an amino acid sequence represented by SEQ ID NO:36; ii) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:36, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:36, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:36, or an amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence represented by SEQ ID NO:36; jj) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:39, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:39, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:39, or an amino acid sequence that is represented by SEQ ID NO:21 and an amino acid sequence that is represented by SEQ ID NO:39; kk) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:39, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:39, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:39, or an amino acid sequence represented by SEQ ID NO:22 and an amino acid sequence represented by SEQ ID NO:39; ll) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:39, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:39, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:39, or an amino acid sequence that is represented by SEQ ID NO:23 and an amino acid sequence that is represented by SEQ ID NO:39; mm) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:40, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:40, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:40, or an amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence represented by SEQ ID NO:40; nn) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 40, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 40, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence represented by SEQ ID NO: 22 and an amino acid sequence represented by SEQ ID NO: 40; oo) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:40, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:40, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:40, or an amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence represented by SEQ ID NO:40; pp) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:41, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:41, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:41, or an amino acid sequence represented by SEQ ID NO:21 and an amino acid sequence represented by SEQ ID NO:41; qq) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:41, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:41, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:22 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:41, or an amino acid sequence represented by SEQ ID NO:22 and an amino acid sequence represented by SEQ ID NO:41; rr) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:41, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:41, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:23 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:41, or an amino acid sequence represented by SEQ ID NO:23 and an amino acid sequence represented by SEQ ID NO:41; ss) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 42, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 42, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 42, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 42, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence represented by SEQ ID NO: 42; tt) an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence represented by SEQ ID NO: 39; uu) an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 41, an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 41, an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence of a single domain antibody (sdAb) and an amino acid sequence represented by SEQ ID NO: 41; vv) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 43 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 44, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 43 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 44, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 43 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 44, or an amino acid sequence represented by SEQ ID NO: 43 and an amino acid sequence represented by SEQ ID NO: 44; ww) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 43 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 45, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 43 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 45, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 43 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence represented by SEQ ID NO: 43 and an amino acid sequence represented by SEQ ID NO: 45; xx) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 43 and at least 85% identical to the amino acid sequence represented by SEQ ID NO: 46, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 43 and at least 90% identical to the amino acid sequence represented by SEQ ID NO: 46, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 43 and at least 99% identical to the amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence represented by SEQ ID NO: 43 and an amino acid sequence represented by SEQ ID NO: 46; yy) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:48, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:48, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:48, or an amino acid sequence that is represented by SEQ ID NO:47 and an amino acid sequence that is represented by SEQ ID NO:48; zz) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:49, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:49, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:49, or an amino acid sequence that is represented by SEQ ID NO:47 and an amino acid sequence that is represented by SEQ ID NO:49; aaa) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:50, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:50, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:47 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:50, or an amino acid sequence that is represented by SEQ ID NO:47 and an amino acid sequence that is represented by SEQ ID NO:50; bbb) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:51 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:52, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:51 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:52, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:51 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:52, or an amino acid sequence represented by SEQ ID NO:51 and an amino acid sequence represented by SEQ ID NO:52; ccc) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:51 and at least 85% identical to the amino acid sequence represented by SEQ ID NO:53, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:51 and at least 90% identical to the amino acid sequence represented by SEQ ID NO:53, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:51 and at least 99% identical to the amino acid sequence represented by SEQ ID NO:53, or an amino acid sequence represented by SEQ ID NO:51 and an amino acid sequence represented by SEQ ID NO:53; ddd) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:51 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:54, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:51 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:54, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:51 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:54, or an amino acid sequence that is represented by SEQ ID NO:51 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:54; eee) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:55 and an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:56, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:55 and an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:56, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:55 and an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:56, or an amino acid sequence represented by SEQ ID NO:55 and an amino acid sequence represented by SEQ ID NO:56; fff) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO: 57, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 57, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 57, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 57, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 57, or an amino acid sequence represented by SEQ ID NO: 57; or ggg) an amino acid sequence that is at least 85% identical to the amino acid sequence represented by SEQ ID NO:58, an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:58, an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO:58, an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO:58, an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO:58, or the amino acid sequence represented by SEQ ID NO:58; 23. The multivalent anti-CD40 antibody construct of any one of claims 1 to 22, comprising:

24. Furthermore, the following (a) and (b): (a) a leader sequence comprising the amino acid sequence set forth in SEQ ID NO: 30, optionally followed by a first linker sequence; and (b) a flag tag comprising the amino acid sequence set forth in SEQ ID NO: 61, optionally followed by a second linker sequence; 24. The multivalent anti-CD40 antibody construct of any one of claims 1 to 23, comprising an N-terminal amino acid sequence comprising one or two of:

25. A pharmaceutical composition comprising the multivalent anti-CD40 antibody construct of any one of claims 1 to 24.

26. A pharmaceutical composition according to claim 25 or a multivalent anti-CD40 antibody construct according to any one of claims 1 to 12 for use in the treatment of cancer.

27. 27. The pharmaceutical composition or multivalent anti-CD40 antibody construct of claim 26, wherein the cancer is a CD40-expressing cancer.

28. A nucleic acid or set of nucleic acids encoding the multivalent anti-CD40 antibody construct of any one of claims 1 to 24.

29. A recombinant cell comprising a nucleic acid or set of nucleic acids according to claim 28 and expressing a multivalent anti-CD40 antibody construct according to any one of claims 1 to 24.

30. 27. A method for producing a multivalent anti-CD40 antibody construct according to any one of claims 1 to 24, comprising expressing said multivalent anti-CD40 antibody construct from a nucleic acid or set of nucleic acids according to claim 28 in a recombinant cell according to claim 29, and optionally further comprising purifying and formulating said multivalent anti-CD40 antibody construct into a pharmaceutical composition according to claim 25.

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