Combined molecules

Abstract

The present disclosure provides improved binding molecules comprising an immunoglobulin-binding domain with affinity for a peptide tag moiety. The binding molecules are useful, for example, as part of a bispecific conjugate. They offer unexpected advantages as part of a biopharmaceutical product when compared with known binding molecules with similar binding affinities. Also provided are bispecific conjugates comprising the binding molecules, and complexes comprising the bispecific conjugates non-covalently linked to tag constructs comprising a peptide tag moiety and a cargo moiety comprising an antigen, for antigen delivery to immune cells. Medical uses of the binding molecules, conjugates, and complexes of the present disclosure are also provided.

Description

[Technical Field]

[0001] The present disclosure relates to improved binding molecules comprising an immunoglobulin-binding domain with affinity for a peptide tag moiety. The binding molecules are useful, for example, as part of a bispecific conjugate. They offer unexpected advantages as part of a biopharmaceutical product when compared with known binding molecules with similar binding affinities. Also provided are bispecific conjugates comprising the binding molecules, and complexes comprising the bispecific conjugates non-covalently linked to tag constructs comprising a peptide tag moiety and an antigen for antigen delivery to immune cells. Medical uses of the binding molecules, conjugates, and complexes of the present disclosure are also provided. [Background technology]

[0002] Monoclonal antibodies (mAbs) that modulate immune responses have proven highly effective in cancer therapy, and there is growing evidence that such responses can be harnessed to provide durable tumor eradication. Various antibodies against different targets, such as those targeting the immune checkpoints CTLA-4 and PD-1, have been developed, supporting the notion that T cell immunity can provide effective cancer therapy. Promising clinical data have also been obtained with immunostimulatory mAbs that agonistically bind to the costimulatory receptor CD40 on antigen-presenting cells (APCs).

[0003] Effective stimulation (or priming) of T cells requires not only application of the stimulus to the APC but also presentation of the antigen by the APC (in the context of MHC) for recognition and binding by the T cell receptor (TCR). Therefore, it is advantageous for the APC to cross-present the antigen to the T cell for the purpose of T cell stimulation, i.e., to take up, process, and present the (extracellularly derived) antigen to the T cell. However, antigenic material may not always be present (e.g., when a tumor is resected or in the context of a vaccine against an infectious disease), and CD40 agonists may be poorly effective in driving effective T cell stimulation in such situations. CD40 stimulation may also be insufficient for T cell activation (e.g., when there is dose-limiting toxicity of the CD40 agonist as an infusion product).

[0004] For these reasons, it is advantageous to activate CD40 on the APC surface using an agonist while simultaneously delivering an antigen to the APC. Bispecific conjugates for this purpose are described in WO2020 / 104690 and WO2021 / 239968. The bispecific conjugates described herein comprise two covalently linked binding proteins. The first binding protein is specific for CD40, and the second binding protein is not directly specific for the antigen but is instead specific for a tag moiety. Tag constructs are provided in which the tag moiety is covalently linked to the antigen and forms a complex with the bispecific conjugate via the binding affinity of the second binding protein in the conjugate for the tag moiety. Thus, a bispecific conjugate comprising a first binding protein covalently linked to a second binding protein indirectly binds to the antigen by binding the second binding protein to the tag moiety contained in the tag construct, providing a flexible modular approach in which the antigen contained in the tag construct can be varied, and there is no chemical bond between the antigen and the bispecific conjugate.

[0005] WO2020 / 104690 provides a complex formed between a bispecific conjugate and a tag construct, the complex providing both a CD40 agonist (for activation of APCs) and an antigen (for presentation by APCs), whereby APC activation by the CD40 agonist leads to activation of T cells specific for the target antigen, advantageously allowing flexibility in preparing conjugates and complexes for use in personalized medicine, and use of this flexible platform in vaccine development for vaccination of individuals using the CD40 pathway to initiate an effective anti-pathogen immune response.

[0006] The conjugates of WO2020 / 104690 can also stimulate B cell responses to antigens. The conjugates can form two interactions with B cells: the anti-CD40 binding protein contained in the bispecific conjugate can bind to CD40 on the surface of B cells, and the antigen contained in the tag construct can bind to a specific B cell receptor. The combination of these two interactions activates B cells that recognize the antigen. Indeed, as explained in WO2020 / 104690, B cells activated in this way using the conjugates may not require costimulation by helper T cells for full activation.

[0007] Rather than preparing conjugates containing an antigen directly fused to a CD40 agonist or an antigen-specific binding agent fused to an agonist, which requires laborious synthesis and the production of a separate conjugate for each patient (or at least for each different tumor antigen), bispecific conjugates can be tailored for individualized use by attaching different antigens but different tag constructs containing the same tag moiety, depending on the needs of a particular individual patient. This strategy allows bispecific conjugates to be tailored to accommodate personalized or indication-based strategies for vaccinating against tumors or pathogens with high antigenic drift, ensuring a flexible vaccination strategy. In this way, only the tailored tag construct is required for preparation, providing benefits in the ease and cost of preparing patient / pathogen-specific therapeutics. Furthermore, it is believed that indirect and non-covalent binding of an antigen to a CD40 agonist may be advantageous for the efficacy of the conjugate compared to conjugates containing an antigen directly covalently fused to a CD40 agonist or compared to providing the CD40 agonist and antigen separately.

[0008] WO2021 / 239968 discloses further agonistic antibodies (or related binding proteins) to CD40 that are shown to bind to CD40 with high affinity and exhibit potent agonistic activity. This CD40 binding protein is particularly suitable for use in the context of therapeutic bispecific conjugates, which in turn may be used in cancer therapy, or alternatively, in the treatment or vaccination of infectious diseases.

[0009] Both WO2020 / 104690 and WO2021 / 239968 disclose promising platforms for the development of personalized biopharmaceuticals, enabling therapeutic efficacy from CD40-mediated immune activation. However, the development and manufacture of candidate products places high demands on the yield, homogeneity, and other expression characteristics of all components or moieties contained in the product. Furthermore, to enable repeated dosing, it is generally necessary to avoid undesirable immunogenicity in the product. While functional and useful, the candidate second binding proteins in the bispecific conjugates disclosed in WO2020 / 104690 and WO2021 / 239968 have been found to be suboptimal in one or more of these considerations. Therefore, there is a need in the art to provide improved such binding proteins with maintained affinity for the tag moiety used in the platform. DETAILED DESCRIPTION OF THE INVENTION

[0010] The object of the present disclosure is to meet this need by providing a binding molecule that does not exhibit the drawbacks associated with murine scFv IBIIICI, but which essentially retains its binding properties.

[0011] It is another object of the present disclosure to provide alternative binding molecules that exhibit high levels of expression when produced in recombinant expression systems.

[0012] Another object of the present disclosure is to provide alternative binding molecules that, when used in the context of bispecific conjugates described, for example, in WO2020 / 104690 and WO2021 / 239968, exhibit increased compatibility with, for example, antibodies to CD40. An example of increased compatibility is that the binding molecule, when combined with an antibody in the bispecific conjugate, increases the monomer content achieved upon expression of the bispecific conjugate.

[0013] These and other objects apparent to those skilled in the art from this disclosure are met by the different aspects of the present invention as claimed in the appended claims and as generally disclosed herein.

[0014] Thus, in a first aspect, there is provided a binding molecule comprising: An immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and amino acid sequences having at least 90% identity thereto, provided that the heavy chain variable region (VH) comprises three complementarity-determining domains (CDRs): - VHCDR1 has the sequence set forth in SEQ ID NO: 7, -VHCDR2 has the sequence IGRIDPEX a X b DAEYVP (SEQ ID NO: 8) (wherein X a X b is selected from the group consisting of SG, GG, QG, DG, NA, and NG; - a VH, wherein VH CDR3 has the sequence set forth in SEQ ID NO: 9; An immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and amino acid sequences having at least 90% identity thereto, provided that the light chain variable region (VL) comprises three complementarity-determining domains (CDRs): - VLCDR1 has the sequence set forth in SEQ ID NO: 11, - VLCDR2 has the sequence set forth in SEQ ID NO: 12, - VL, wherein VLCDR3 has the sequence set forth in SEQ ID NO: 13.

[0015] While originally conceived as a humanized version of the known murine antibody IBIIICI as described in Example 1, the binding molecule defined herein was found to not only exhibit the advantage of having an increased "humanity" score, but also, when expressed, to exhibit unexpected and dramatic advantages over its progenitor, e.g., with respect to one or more of its protein expression yield, monomer content, and ease of purification. These advantages were evident both when testing the binding molecule alone in scFv format (Example 2, Figure 2) and when placing the scFv in the context of bispecific conjugates similar to those described in WO2020 / 104690 and WO2021 / 239968 (Example 6, Table 5, Figures 8 and 9). These unexpected improvements in developability parameters are of great importance when developing a commercial biopharmaceutical product.

[0016] The increased "humanity" of the binding molecules of the present disclosure also provides the benefit of being less prone to induce anti-drug antibodies over time when administered to a subject, e.g., a human patient, thus allowing for repeated dosing over time, which is advantageous compared to non-human proteins and other vector-based platforms that induce anti-drug antibodies (which limit the usefulness of the binding molecule over time).

[0017] Thus, the present disclosure provides binding molecules comprising immunoglobulin light and heavy chain variable regions having defined amino acid sequences. The term "binding molecule" is used herein to denote a binding molecule that comprises an antibody binding domain (i.e., a binding domain obtained or derived from an antibody, or based on an antibody binding domain). Thus, a binding molecule is an antibody-based or antibody-like molecule that comprises an antibody binding site or a binding site derived from an antibody.

[0018] As mentioned above, the binding domain of an antibody consists of a light chain variable domain and a heavy chain variable domain (thus, a classical bivalent antibody has two binding domains). Thus, a binding molecule can be a natural antibody or a fragment thereof, or an artificial or synthetic antibody, or an antibody construct or derivative (e.g., a single-chain antibody). In summary, a binding molecule of the present disclosure comprises a binding domain of an antibody, which in turn comprises a light chain variable domain and a heavy chain variable domain.

[0019] As detailed above, the binding molecules of the present disclosure comprise six CDR sequences. The light and heavy chain variable domains each comprise three CDRs, i.e., the heavy chain variable domain comprises VHCDR1, VHCDR2, and VHCDR3, and the light chain variable domain comprises VLCDR1, VLCDR2, and VLCDR3. The six CDRs have the following amino acid sequences: VHCDR1 has the sequence set forth in SEQ ID NO:7, VHCDR2 has the sequence set forth in SEQ ID NO:8, VHCDR3 has the sequence set forth in SEQ ID NO:9, VLCDR1 has the sequence set forth in SEQ ID NO: 11; VLCDR2 has the sequence set forth in SEQ ID NO: 12, VLCDR3 has the sequence set forth in SEQ ID NO:13.

[0020] As explained above, SEQ ID NO:8 corresponds to the amino acid sequence IGRIDPEX a X b DAEYVP, wherein the amino acid pair X a X b is selected from the group consisting of SG, GG, QG, DG, NA, and NG. In other words, the sequence of VHCDR2 of the heavy chain variable domain of the binding molecule is selected from the group consisting of SEQ ID NOs: 62 to 67. As shown in the Examples section herein, X a X bcan be varied in this manner without substantially affecting binding of the binding molecule to the amino acid sequence of its tag moiety counterpart. In particular embodiments of the binding molecules of the present disclosure, the six different types of VHCDR2 sequences are combined into subgroups including all possible subcombinations of 2, 3, 4, and 5 different VHCDR2 sequences. In even more specific embodiments, each individual VHCDR2 sequence represents an individual embodiment of the binding protein. The resulting disclosure of all possible combinations of SEQ ID NOS: 1-6 (which are equivalent to all possible combinations of SEQ ID NOS: 62-67) is shown in the itemized listing of embodiments below, items 2-63. All such combinations, each individually, are part of the disclosure herein.

[0021] A binding molecule of the present disclosure comprises a heavy chain variable domain having a sequence selected from SEQ ID NOs: 1-6, and any sequence having at least 90% identity to any one of SEQ ID NOs: 1-6, so long as the three CDR sequences consist of SEQ ID NOs: 7-9. In a specific embodiment, the sequence has at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99% identity to any one of SEQ ID NOs: 1-6.

[0022] A binding molecule of the present disclosure comprises a light chain variable domain having SEQ ID NO: 10, and any sequence having at least 90% identity to SEQ ID NO: 10, so long as the three CDR sequences consist of SEQ ID NOs: 11-13. In a specific embodiment, the sequence has at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identity to SEQ ID NO: 10.

[0023] As described herein, binding molecules having amino acid sequences with 90% or greater identity to the listed sequences are also contemplated as falling within the scope of the present disclosure. Such variant sequences may be modified compared to the listed sequences by one or more amino acid substitutions, insertions, and / or deletions.

[0024] Amino acid substitutions to the recited sequences may be conservative amino acid substitutions. The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, and therefore, conservative substitutions of amino acids important for the structure or function of a polypeptide may be expected to have less of an effect on polypeptide structure / function than non-conservative amino acid substitutions at the same position. Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, conservative amino acid substitutions can be considered substitutions in which a particular amino acid residue is replaced with a different amino acid within the same family. However, amino acid substitutions can also be non-conservative, in which one amino acid is replaced with another amino acid with a side chain belonging to a different family.

[0025] As detailed above, according to the present disclosure, variants of the listed sequences share at least 90% sequence identity with each listed sequence. Sequence identity can be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform pairwise or multiple alignment of sequences are useful; for example, EMBOSS Needle or EMBOSS Stretcher (both Rice et al. (2000), Trends Genet., 16(6):276-277)) can be used for pairwise sequence alignment, while Clustal Omega (Sievers et al. (2011), Mol. Syst. Biol. 7:539) or MUSCLE (Edgar (2004), Nucleic Acids Res. 32(5):1792-1797) can be used for multiple sequence alignment, although any other suitable program can also be used. Whether the alignment is pairwise or multiple, it must be done globally (i.e., across the entire reference sequence) rather than locally.

[0026] Sequence alignments and percent identity calculations may be determined, for example, using standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1. Alternatively, standard EMBOSS Needle parameters may be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters may alternatively be used.

[0027] The binding molecules of the present disclosure can be synthesized by any method known in the art. Preferably, the binding molecules are synthesized using a protein expression system, such as a cellular expression system using prokaryotic (e.g., bacterial) cells or eukaryotic (e.g., yeast, fungal, insect, or mammalian) cells. An alternative protein expression system is a cell-free in vitro expression system, in which a nucleotide sequence encoding the binding molecule is transcribed into mRNA, and the mRNA is translated into protein in vitro. Cell-free expression system kits are widely available and can be purchased, for example, from ThermoFisher. Alternatively, the binding protein can be chemically synthesized in a non-biological system. Liquid-phase synthesis or solid-phase synthesis can be used to produce polypeptides that can form or be included within the binding molecules of the present disclosure.

[0028] Those skilled in the art can easily produce binding proteins using appropriate methodologies common in the art. In particular, binding molecules can be recombinantly expressed in mammalian cells, such as CHO cells. Binding molecules synthesized in protein expression systems can be purified using standard techniques in the art; for example, they can be synthesized with an affinity tag and purified by affinity chromatography. When the binding molecule is an antibody, it can be purified using affinity chromatography using one or more antibody-binding proteins, such as Protein G, Protein A, Protein A / G, or Protein L.

[0029] As mentioned above, the binding molecule is an antibody-based or antibody-like molecule. Thus, the binding molecule can be a natural antibody or a fragment thereof, or an artificial or synthetic antibody, or an antibody construct or derivative (e.g., a single chain antibody).

[0030] In one embodiment, the binding molecule is an antibody, particularly a monoclonal antibody. "Monoclonal antibody" refers to an antibody preparation consisting of a single antibody species, i.e., all antibodies in the preparation have the same amino acid sequence, including the same CDR, and therefore bind to the same epitope with the same effect on their target antigen ("target antigen" refers to the antigen that contains the epitope bound by a specific antibody). In other words, the antibody of the present disclosure is preferably not part of a polyclonal mixture of antibodies.

[0031] When the binding molecule is an antibody, the antibody can be of any isotype and subtype. Thus, it can be an IgA, IgD, IgE, IgG, or IgM antibody. The heavy chain constant domains corresponding to different immunoglobulin isotypes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different immunoglobulin isotypes are well known. Preferably, the antibody is an IgG antibody. As mentioned above, there are four subtypes of IgG antibodies: IgG1, IgG2, IgG3, and IgG4. The IgG anti-CD40 antibody of the present disclosure can be of any IgG subtype, i.e., it can be an IgG1, IgG2, IgG3, or IgG4 antibody.

[0032] In one embodiment, the binding molecule is a binding fragment of an antibody (i.e., an antibody fragment), i.e., a fragment that retains the ability of an antibody to specifically bind to its target antigen. Such fragments are well known and include, for example, Fab', Fab, F(ab')2, Fv, Fd, or dAb fragments, and can be prepared according to techniques well known in the art.

[0033] The Fab fragment consists of the antigen-binding domain of an antibody; i.e., an individual antibody can be recognized to contain two Fab fragments, each consisting of a light chain and the N-terminal portion of its associated heavy chain. Thus, the Fab fragment contains the entire light chain and the VH and CH1 domains of the heavy chain to which it is associated. Fab fragments can be obtained by digesting an antibody with papain.

[0034] An F(ab')2 fragment consists of two Fab fragments of an antibody plus the hinge region of the heavy chain, including the disulfide bond linking the two heavy chains together. In other words, an F(ab')2 fragment can be recognized as two covalently linked Fab fragments. An F(ab')2 fragment can be obtained by digesting an antibody with papain. Reduction of the F(ab')2 fragment results in two Fab' fragments, which can be recognized as Fab fragments containing additional sulfhydryl groups that may be useful for conjugating the fragment with other molecules.

[0035] In alternative and preferred embodiments, the binding molecule is a synthetic or artificial construct, i.e., an antibody-like molecule that contains a binding domain but is genetically engineered or artificially constructed. Such constructs include chimeric or CDR-grafted antibodies, as well as single-chain antibodies and other constructs, such as scFvs, dsFvs, ds-scFvs, dimers, minibodies, diabodies, single-domain antibodies (DABs), TandAb dimers, and heavy-chain antibodies such as VHHs. In certain embodiments, the binding molecule is a single-chain variable fragment (scFv). scFvs are fusion proteins in which a single polypeptide contains both the VH and VL domains of an antibody. scFv fragments generally contain a peptide linker covalently linking the VH and VL domains, contributing to the stability of the molecule. The linker can contain 1 to 20 amino acids, e.g., 1, 2, 3, or 4 amino acids, 5, 10, or 15 amino acids, or any other intermediate number conveniently ranging from 1 to 20. The peptide linker can be formed from any convenient amino acid residue, such as glycine and / or serine. One example of a suitable linker is G4S (SEQ ID NO: 70). Multimers of such linkers, for example, dimers, trimers, tetramers, or pentamers, can be used, i.e., (G4S)2 (SEQ ID NO:71), (G4S)3 (SEQ ID NO:72), ((G4S)4 (SEQ ID NO:14), or (G4S)5 (SEQ ID NO:73). In certain embodiments where the binding molecule of the disclosure is an scFv, the linker is a (G4S)4 linker (SEQ ID NO:14). However, the presence of a linker is not required, and the VL domain may instead be directly linked to the VH domain by a peptide bond. An scFv typically comprises a VH region connected, from N-terminus to C-terminus, to the VL region by a linker sequence. In other words, in such embodiments of the binding molecule of the disclosure, the scFv comprises a peptide linker connecting the C-terminus of the heavy chain variable region (VH) to the N-terminus of the light chain variable region (VL).

[0036] The preparation of scFv molecules is well known in the art, and one of skill in the art can readily prepare scFv binding molecules according to the present disclosure comprising the VH and VL sequences defined herein. Thus, as a non-limiting example, a binding molecule of the present disclosure can be an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 49-54, and sequences having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. The present disclosure also contemplates any individual scFv sequence or subgroup of sequences by analogy to the listing of individual pairs of VH and VL sequences in the "Itemized Listing of Embodiments" (see items 2-63). In a specific embodiment, a binding molecule of the present disclosure is an scFv comprising the amino acid sequence of SEQ ID NO: 49, designated "SG" or "IBIIICI CDR-grafted SG."

[0037] In one embodiment, the binding molecule is a humanized protein, particularly a humanized scFv. Humanized binding molecules may comprise VH and VL regions whose frameworks are derived from human germline immunoglobulin sequences. However, such proteins may contain amino acids not encoded by human germline Ig sequences, e.g., mutations introduced by random or site-directed mutagenesis, or amino acids derived from ancestral binding molecules generated in other species, e.g., mice.

[0038] In one embodiment of the binding molecule of the present disclosure, it can selectively bind to a peptide tag moiety included in a tag construct. "Selectively binding" means that the binding molecule binds to its target in a manner that can be distinguished from binding to non-target molecules; more specifically, the binding molecule binds to its target with a higher binding affinity than it binds to other molecules. That is, the binding molecule does not bind to other non-target molecules, or does not do so to a significant or significant extent, or binds to such other molecules with a lower affinity than it binds to the tag moiety. A binding molecule that "selectively binds" to a tag moiety can alternatively be referred to as being "directed against" or "recognizing" the tag moiety. In other words, the tag moiety is the antigen of the binding molecule of the present disclosure, and therefore the binding molecule is an "antigen-binding protein" in the sense that it binds to the tag moiety as its antigen. Those skilled in the art will understand that the terms "antigen" and "antigen-binding protein" in the preceding sentence are applied in a metaphorical context in an attempt to illustrate the binding relationship between the tag moiety and the binding molecule of the present disclosure. Those skilled in the art will also recognize that the foregoing does not indicate that the tag moiety is an antigen itself against which a non-specific or specific immune response is elicited, but rather that the tag moiety is a tailored binding partner for a binding molecule of the present disclosure.

[0039] In one embodiment of the present disclosure, the tag moiety to which a binding molecule can specifically bind comprises the amino acid sequence FIGITELK (SEQ ID NO: 43). Examples of such tag moieties comprising SEQ ID NO: 43 are tested in the experimental section below and form the group consisting of SEQ ID NOs: 26-35, 39-43, 45, and 47. A particular tag moiety to which a binding molecule herein can specifically bind consists of SEQ ID NO: 42, i.e., FIGITELKK.

[0040] In another embodiment, the tag portion comprises the amino acid sequence FIGITELL (SEQ ID NO: 74), e.g., selected from the group consisting of SEQ ID NOs: 44 and 74. In another embodiment, the tag portion comprises the amino acid sequence FIGITELH (SEQ ID NO: 75), e.g., selected from the group consisting of SEQ ID NOs: 36, 38, 46, and 75. In another embodiment, the tag portion comprises the amino acid sequence FIGISELK (SEQ ID NO: 76), e.g., selected from the group consisting of SEQ ID NOs: 37 and 76. In one embodiment, the tag portion comprises the amino acid sequence FIGITELLK (SEQ ID NO: 79). The tag portion comprises the amino acid sequence FIGITELHK (SEQ ID NO: 80).

[0041] As established in WO2020 / 104690 and WO2021 / 239968, the tag portion to which the second binding protein binds can advantageously form part of a tag construct. In one embodiment, a tag construct comprising a peptide tag portion further comprises at least one cargo portion. The at least one cargo portion can be, for example, an antigen portion and can be intended to elicit an immune response. The at least one cargo portion can be, for example, a peptide portion, such as an antigen peptide portion. For clarity, the "antigen portion" (an example of a type of "cargo portion") present in one embodiment of a tag construct is different from the "tag portion" contained in the construct, despite the fact that the tag portion can be considered an antigen to which a binding molecule of the present disclosure can selectively bind. In embodiments in which the tag construct comprises a peptide tag portion and a peptide cargo portion (e.g., a peptide antigen or "antigen portion"), the C-terminus of the tag portion can be covalently linked to the N-terminus of the cargo portion, or vice versa. In another embodiment, the cargo portion is a nucleic acid portion.

[0042] In embodiments in which at least one cargo moiety is a nucleic acid moiety, the tag moiety and cargo moiety can be arranged in any order in the polypeptide-nucleic acid combination. In one such embodiment, the cargo moiety is an siRNA molecule.

[0043] In one embodiment of the binding molecule of the present disclosure, it has a K D The value is up to 5 x 10 -9 M, e.g., up to 1 x 10 -10 M, e.g., up to 1 x 10 -11 M. One of skill in the art will understand that the binding interaction is between a binding protein of the present disclosure and a tag moiety.

[0044] In a second aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: at least one first moiety which is a binding molecule of the present disclosure; - at least one second moiety which is an antibody or an antigen-binding fragment thereof.

[0045] Design considerations for creating bispecific conjugates of this aspect of the disclosure are similar to those described for similar conjugates in WO2020 / 104690 and WO2021 / 239968. However, herein, the first moiety of the bispecific conjugate is a binding molecule according to the first aspect, which in one embodiment can advantageously be attached to a tag moiety. In one embodiment herein, the second moiety is an antibody or antigen-binding fragment thereof having binding affinity for an immunologically relevant target.

[0046] In one embodiment of a bispecific conjugate of the present disclosure, the second moiety is an antigen-binding fragment of an antibody, the fragment being selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fc fragment, an Fv fragment, a single-chain (scFv) fragment, an (scFv)2, and a domain antibody.

[0047] In one embodiment of the bispecific conjugate, the second moiety is optionally an antibody of the IgG2 subtype.

[0048] In one embodiment of the bispecific conjugate, the first moiety is covalently linked to the second moiety, optionally via a linker peptide, e.g., a linker selected from the linkers discussed above in connection with the first aspect. Thus, if present, the linker may comprise 1 to 20 amino acids and may be formed from any generally convenient amino acid residues, such as, for example, glycine and / or serine. One example of a suitable linker is G4S (SEQ ID NO:70) and its multimers, e.g., (G4S)2 (SEQ ID NO:71), (G4S)3 (SEQ ID NO:72), (G4S)4 (SEQ ID NO:14), or (G4S)5 (SEQ ID NO:73).

[0049] In one embodiment of the bispecific conjugate, the first moiety is covalently linked to the second moiety via a linker peptide.

[0050] In an alternative embodiment of the bispecific conjugate, the first moiety is directly linked to the second moiety.

[0051] In one embodiment of the bispecific conjugate, the first moiety is covalently linked (directly or via a linker) to the C-terminus or N-terminus of the light or heavy chain of the second moiety.

[0052] In one embodiment of the bispecific conjugate, it comprises two first moieties and one antibody as the second moiety, one first moiety conjugated to the CH3 domain of each heavy chain of the second partial antibody.

[0053] In another embodiment of the bispecific conjugate, it comprises two first moieties and one antibody as the second moiety, one first moiety conjugated to the CL domain of each light chain of the second partial antibody.

[0054] In a particularly interesting embodiment of the bispecific conjugate, the second moiety is an anti-CD40 antibody or an antigen-binding fragment thereof.

[0055] In such embodiments, the second portion may be selected from the group consisting of, for example, CP-870,893, APX005M, ADC-1013, ChiLob7 / 4, SEA-CD40, and ABS-1150 / 1151, and antibodies comprising antigen-binding fragments derived from any one or more of said antibodies.

[0056] Alternatively, the second portion can be an anti-CD40 binding antibody or antigen-binding fragment thereof described in WO2021 / 239968, such as the antibody designated "A9" therein. In one such embodiment, the second portion is an anti-CD40 antibody or antigen-binding fragment thereof comprising the following six CDRs: VLCDR1: SEQ ID NO: 15, VLCDR2: SEQ ID NO: 16, VLCDR3: SEQ ID NO: 17, VHCDR1: SEQ ID NO: 18, VHCDR2: SEQ ID NO: 19, and VHCDR3: SEQ ID NO: 20. In a more specific such embodiment, the light chain variable domain (VL) of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 21 and amino acid sequences having at least 90% sequence identity thereto, and the heavy chain variable domain (VH) of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 22 and amino acid sequences having at least 90% sequence identity thereto. For example, such an anti-CD40 antibody may comprise a light chain (LC) comprising an amino acid sequence selected from SEQ ID NO: 23 and amino acid sequences having at least 90% sequence identity thereto, and a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO: 24 and amino acid sequences having at least 90% sequence identity thereto. By analogy to the sequences disclosed for the binding molecules according to the first aspect of the present disclosure, in specific embodiments, the VH, VL, HC, or LC sequence of the anti-CD40 antibody may have at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to any one of the respective recited SEQ ID NOs: 21-24.

[0057] Examples of individual bispecific conjugates of this aspect that have been tested in the experimental section of the present specification and found to exhibit desirable properties are those designated herein as SP007, SP019, and SP027. Each of these bispecific conjugates consists of a first moiety that is an scFv embodiment of the binding molecule of the first aspect, and two copies of a polypeptide chain comprising the antibody heavy chain of a particular anti-CD40 antibody, and two copies of a polypeptide chain that is the antibody light chain of the same anti-CD40 antibody (see Table 4 in Example 6).

[0058] In a third aspect of the present disclosure, there are provided polynucleotides encoding the binding molecules or bispecific conjugates described herein, expression vectors comprising the polynucleotides, and host cells comprising the expression vectors. Also encompassed by the present disclosure is a method of producing a binding molecule or bispecific conjugate of the present disclosure, the method comprising culturing the host cell under conditions that allow expression of the binding molecule or bispecific conjugate from the expression vector, and isolating the binding molecule or bispecific conjugate.

[0059] In a fourth aspect, the present disclosure provides a complex, such as a non-covalent complex, comprising the bispecific conjugate of the present disclosure and a tag construct of the present disclosure, which comprises a tag moiety to which the binding molecule of the first aspect can bind. In the complex embodiment, the tag construct is as defined above when considered in relation to the first aspect of the present disclosure. The complex of this aspect can be formed by contacting the bispecific conjugate with the tag construct, such that the tag construct is bound by the binding molecule of the conjugate via the affinity of the binding molecule for the tag moiety contained in the tag construct.

[0060] In a further aspect, the present disclosure provides a pharmaceutical composition comprising (i) a binding molecule of the present disclosure, as described above, (ii) a bispecific conjugate of the present disclosure, as described above, or (iii) a complex of the present disclosure, as described above. In addition to the binding protein, bispecific conjugate, or complex, the pharmaceutical composition also comprises at least one pharmaceutically acceptable carrier or excipient.

[0061] Also provided by the present disclosure are kits and articles of manufacture comprising, separately, the bispecific conjugate and tag construct as described above, in which the conjugate and tag construct may be provided separately in a composition containing a pharmaceutically acceptable carrier or excipient.

[0062] As used herein, "pharmaceutically acceptable carrier or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0063] Preferably, the carrier or excipient is suitable for parenteral, e.g., intradermal, intravenous, intramuscular, or subcutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the binding protein, bispecific conjugate, complex, or components thereof may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate or denature it.

[0064] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in pharmaceutical compositions, kits, and products include water, buffered water, and physiological saline. Other examples of carriers include ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyols such as mannitol and sorbitol, sodium chloride, etc.

[0065] The pharmaceutical compositions, products, or kits may also contain pharmaceutically acceptable antioxidants. They may also contain auxiliary substances such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that prolong absorption, such as aluminum monostearate and gelatin.

[0066] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0067] Sterile injectable solutions can be prepared by incorporating the active agent (e.g., complex) in the required amount in a suitable solvent containing one or a combination of the ingredients listed above, optionally followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which obtains a powder of the active agent plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0068] The pharmaceutical compositions, products, and kits may include additional active ingredients, as well as binding proteins, bispecific conjugates, complexes, or components thereof, e.g., they may include additional therapeutic or prophylactic agents. Thus, the complexes may be used as monotherapy or as part of a combination therapy, e.g., in the treatment of cancer. The kits or combination products described herein may additionally include instructions for use.

[0069] The binding proteins, bispecific conjugates, complexes, pharmaceutical compositions, kits, and combination products of the present disclosure can be used in therapy. Accordingly, the present disclosure provides binding proteins, bispecific conjugates, complexes, pharmaceutical compositions, or kits of the present disclosure for use in therapy. The term "therapy" refers to the treatment of a subject. As used herein, "therapy" refers to the treatment of any medical condition. Such treatment can be preventative (i.e., preventative), curative (or treatment intended to be curative), or palliative (i.e., treatment designed to merely limit, alleviate, or improve the symptoms of a condition). In curative and palliative applications, the conjugate or composition is administered to a subject already suffering from a disease or condition in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to accomplish this is defined as a "therapeutically effective amount." An effective amount for a given purpose will depend on the disease or condition being treated, its severity, and the size / weight and general condition of the subject.

[0070] Prophylactic treatment can include preventing a condition or delaying the onset or development of a condition. For example, the conjugates can be used to prevent infection or reduce the extent to which an infection may occur, or to prevent, delay, or reduce the extent of cancer onset or recurrence, or to prevent or reduce the extent of metastasis, for example.

[0071] As defined herein, a subject refers to any mammal, for example, a livestock animal such as a cow, horse, sheep, pig, or goat, a pet animal such as a rabbit, cat, or dog, or a primate such as a monkey, chimpanzee, gorilla, or human. Most preferably, the subject is a human.

[0072] The combination product of the present disclosure comprises a bispecific conjugate as defined herein and a tag construct as defined herein as a combined preparation for simultaneous or sequential use in therapy. That is, when the combination product disclosed herein is used according to the present disclosure, i.e., in therapy, the conjugate and tag construct are administered to a subject simultaneously or sequentially. Similarly, when the kit of the present disclosure is used in therapy, the bispecific conjugate and tag construct are administered to a subject simultaneously or sequentially. "Concurrent" administration, as used herein, means that the two components are administered to a subject simultaneously, or at least substantially simultaneously, by the same route of administration and at substantially the same site. "Sequential" administration, as used herein, means that the two components are administered to a subject at different times. In particular, administration of the first component is completed before administration of the second component begins.

[0073] Due to the nature of the present disclosure, sequential administration of the bispecific conjugate and tagged construct requires that both be administered by the same route and at substantially the same site. Furthermore, administration of the conjugate and tagged construct can be separated in time, but the interval between administrations should be such that a complex can be formed when both components are administered. Thus, for example, both components can be administered within one hour of each other, or more specifically, within 40, 30, 20, 15, 10, 8, 7, 6, 5, 4, 3, 2, or 1 minute of each other, or less than one minute.

[0074] Reference to a conjugate of the present disclosure (or a pharmaceutical composition comprising such a conjugate) administered to a subject for therapeutic purposes should be understood to refer to a pre-mixed composition (e.g., a solution) comprising both components of the conjugate (i.e., a bispecific conjugate of the present disclosure and a tag construct as described above), which may exist in a dynamic equilibrium comprising the conjugate and its two individual components.

[0075] In particular, the binding proteins, bispecific conjugates, complexes, compositions, kits, or combined products of the present disclosure can be used to treat or prevent cancer. Cancer refers to any malignant or pre-malignant neoplastic condition. Thus, cancer can be any cancer of any organ, tissue, or cell type. Included are cancers that present as solid tumors and cancers that do not present as solid tumors. Thus, hematopoietic cancers are included.

[0076] The cancer may be prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, rhabdomyosarcoma, neuroblastoma, multiple myeloma, leukemia, acute lymphoblastic leukemia, melanoma, bladder cancer, head and neck cancer, lymphoma, glioblastoma, or skin cancer. It may also be adrenal gland cancer, bone cancer, brain cancer, esophageal cancer, eye cancer, stomach cancer, oral cancer, penile cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer. Mast cell tumors and angiosarcomas may also be treated according to the present disclosure. The cancer may be newly diagnosed and untreated, or it may be relapsed or refractory, or relapsed and refractory, primary, or metastatic.

[0077] When present in the bispecific conjugate of the present disclosure, the CD40-specific second moiety activates the immune system by agonizing or stimulating CD40 on APCs, particularly dendritic cells. In particular, this can lead to T cell activation. The subsequent immune response exerts an anti-cancer effect on adjacent or accessible tumor cells, regardless of CD40 expression by the tumor. Therefore, the bispecific conjugate of this embodiment, or a complex containing it, can be effective against both CD40-positive and CD40-negative cancers. Such bispecific conjugates can also be effective as adjuvants in vaccination regimens against pathogens. When a vaccine platform (e.g., an attenuated virus or DNA / RNA-based vaccine) does not stimulate a sufficient immune response by itself, CD40 activation may be required to stimulate an effective anti-pathogen immune response that results in neutralizing antibody or T cell responses.

[0078] In addition to the agonistic immunostimulatory effect provided by the CD40-specific second moiety, in embodiments where it is present, the conjugates of the present disclosure also provide an antigen that can be presented to treated and activated T cells, thus priming the T cells to target cancer cells or virus-infected cells that express the antigen. This can be particularly beneficial in situations where the presence of cancer antigens is low or reduced, e.g., where tumors have been surgically removed, where anti-CD40 therapeutic agents cannot be delivered intratumorally, or where antigen presentation is not ideal at the tumor site due to suppressive factors secreted by the tumor. The conjugates according to this embodiment provide a means for presenting cancer antigens in proximity to agonistic activation signals, e.g., at non-tumor sites, ensuring the priming and activation of T cells that can then migrate to the tumor and exert their function.

[0079] In some embodiments, the cancer antigen delivered by the conjugate can be selected based on the subject and the particular cancer, thus enabling personalized medicine. For example, the subject's cancer can be subjected to genetic profiling, allowing suitable antigens to be selected. Banks or libraries of antigens, or tag constructs comprising the antigen in the form of a cargo moiety, can be provided from which suitable tag constructs can be prepared or selected depending on the type of cancer of the subject.

[0080] The bispecific conjugates and complexes (as well as kits and combination products) of the present disclosure may also be useful for treating or preventing infectious diseases. The present disclosure may be particularly useful in therapy for (e.g., vaccination against) viral infections, particularly infections caused by RNA viruses. Infections caused by RNA viruses that can be prevented (or treated by vaccination) according to the present disclosure include infections caused by coronaviruses (such as SARS-CoV1 (the causative agent of SARS), SARS-CoV2 (the causative agent of COVID-19), and MERS-CoV), influenza virus, Ebola virus, hepatitis C virus (HCV), hepatitis E virus (HEV), rabies virus, poliovirus, Ross River virus, and measles virus.

[0081] The present disclosure may also be used to treat or vaccinate against infections caused by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (e.g., HHV6), parvovirus B19, and human papillomavirus (HPV), although in principle any viral infection can be treated or prevented in accordance with the present disclosure.

[0082] Intracellular bacterial infections, such as brucellosis (caused by bacterial species of the genus Brucella), Q fever (caused by Coxiella burnetii), diseases caused by Chlamydiae species such as chlamydia (caused by Chlamydia trachomatis) and pneumonia (caused by Chlamydia pneumoniae), leprosy (caused by Mycobacterium leprae and Mycobacterium lepromatosis), and tuberculosis, including disseminated tuberculosis (caused by Mycobacterium tuberculosis), can be treated according to the present disclosure. Intracellular fungal or protozoal infections, including leishmaniasis (caused by trypanosomes of the genus Leishmania) and toxoplasmosis (caused by the apicomplexan Toxoplasma gondii), can also be treated according to the present disclosure. Thus, antigens can be derived from any of the aforementioned pathogens.

[0083] In the case of bispecific conjugates containing anti-CD40 antibodies or fragments thereof, CD40 agonism can activate the immune system to fight infectious diseases (based on principles similar to their use in cancer therapy). In the case of the conjugates of the present disclosure, as explained above, they can be provided with antigens derived from the target pathogen, thus inducing a specific immune response against the pathogen. Vaccine platforms can be beneficially adaptable, for example, for pandemic situations. This adaptability can be provided, in particular, by using tag constructs containing at least one cargo moiety that is an antigen that can be modified to address viral diversity and antigenic drift. Viral antigens can be selected based on HLA prevalence in specific regions, along with viral serotype determinants.

[0084] In one embodiment, a bispecific conjugate of the present disclosure may comprise an antagonistic anti-CD40 antibody or antigen-binding fragment thereof. In such an embodiment, the bispecific conjugate is useful for tolerizing the immune system to targets that drive autoimmune disease. In one embodiment of the conjugate of the present disclosure, it comprises a bispecific conjugate whose second moiety is a CD40 antagonist and a tag construct comprising at least one cargo moiety that is an antigen known to be a driver of autoimmune disease, such as T cell-mediated autoimmune disease. In this case, the purpose of the bispecific conjugate in complex with the tag construct comprising at least one cargo moiety that is an antigen is to reeducate the immune system to tolerate the disease-causing antigen, thereby preventing excessive destruction of healthy tissue. Examples of antigens useful for tag constructs according to such embodiments are selected from the group consisting of antigens associated with SLE, type 1 diabetes, rheumatoid arthritis, vasculitis, myositis, multiple sclerosis, psoriasis, and allergies.

[0085] At least one cargo moiety that is an antigen (e.g., a cancer antigen, a pathogen-derived antigen, or an antigen associated with an immune-mediated disease) can be selected to be recognized by a particular subset of T cells in the subject to be treated, the T cells expressing a TCR known to recognize the selected antigen. In particular, the antigen can be selected based on its recognition by T cells to be used in adoptive cell therapy in the subject to be treated.

[0086] For example, in adoptive cell therapy, T cells can be obtained from a subject, and T cells that recognize an antigen of interest can be isolated. The isolated T cells can then be expanded and / or otherwise treated to stimulate their effector functionality, and then reinfused into the subject to be treated. In this context, the antigen recognized by the reinfused T cells can be used in a tag construct by being included in the tag construct as at least one cargo moiety. The complex of the present disclosure can then be administered to the subject so that the antigen activates the reinfused T cells.

[0087] Alternatively, T cells can be obtained from the subject or donor to be treated and genetically modified to express a TCR that recognizes a target antigen. The genetically modified T cells can then be expanded and / or otherwise treated to stimulate their effector functionality, and then infused (or re-infused) into the subject to be treated. In this context, the antigen recognized by the genetically modified T cells can be used in a tag construct by being included in the tag construct as the at least one cargo moiety. A conjugate of the present disclosure can then be administered to the subject so that the antigen activates the infused T cells. Methods in which administration of a conjugate of the present disclosure is combined with adoptive cell therapy are particularly useful for treating cancer, in which case the antigen used in the tag construct is a cancer antigen by being included in the tag construct as the at least one cargo moiety.

[0088] Accordingly, the present disclosure provides a method of treating or preventing cancer, the method comprising administering to a subject a binding molecule of this disclosure, a bispecific conjugate of this disclosure, a complex of this disclosure, or a pharmaceutical composition of this disclosure.

[0089] In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising: (i) obtaining T cells from a subject; (ii) isolating T cells that recognize the target cancer antigen and, optionally, expanding the isolated T cells; (iii) re-infusing the subject with the isolated T cells; and (iv) administering to the subject a conjugate of the present disclosure, wherein the tagged construct comprises the target cancer antigen. Equivalently, in step (iv), the subject may alternatively be administered a bispecific conjugate of the present disclosure and a tagged construct comprising the target cancer antigen separately.

[0090] In another embodiment, the present disclosure provides a method of treating cancer in a subject, the method comprising: (i) obtaining T cells from a subject or donor; (ii) genetically modifying T cells to express a TCR that recognizes a target cancer antigen, and optionally expanding the T cells before or after genetic modification; (iii) infusing the genetically modified T cells into the subject; (iv) administering to the subject a conjugate of the present disclosure, wherein the tagged construct comprises a target cancer antigen. Equivalently, in step (iv), the subject may alternatively be administered a conjugate of the present disclosure and a tagged construct comprising the target cancer antigen separately.

[0091] The present disclosure also provides the use of a binding molecule of the present disclosure, a bispecific conjugate of the present disclosure, or a complex of the present disclosure in the manufacture of a medicament for the treatment of cancer prevention.

[0092] Similarly, the present disclosure provides a method of treating or preventing an infectious disease, the method comprising administering to a subject a binding molecule of this disclosure, a bispecific conjugate of this disclosure, a complex of this disclosure, or a pharmaceutical composition of this disclosure.

[0093] The present disclosure also provides the use of a binding molecule of the present disclosure, a bispecific conjugate of the present disclosure, or a complex of the present disclosure in the manufacture of a medicament for the treatment of prophylaxis against an infectious disease.

[0094] The present disclosure also provides a method of treating or preventing an autoimmune disease, the method comprising administering to a subject a binding molecule of this disclosure, a bispecific conjugate of this disclosure, a complex of this disclosure, or a pharmaceutical composition of this disclosure.

[0095] The present disclosure also provides the use of a binding molecule of the present disclosure, a bispecific conjugate of the present disclosure, or a complex of the present disclosure in the manufacture of a medicament for the treatment of prophylaxis of an autoimmune disease.

[0096] Throughout the above embodiments, references to the use of a complex of the present disclosure include the combined use of a bispecific conjugate and tag construct of the present disclosure, which are administered separately or sequentially.

[0097] In an alternative embodiment, the conjugates of the present disclosure may be used in gene therapy. In this embodiment, a gene therapy vector or delivery system encoding both the bispecific conjugate of the present disclosure and the corresponding tag construct may be administered to a subject. Upon uptake by the subject's cells, the conjugate and tag construct are expressed and secreted, forming a complex in vivo.

[0098] As described above, the binding molecules, bispecific conjugates, or complexes of the present disclosure can be used as monotherapy or in combination with other therapeutic agents. Thus, in the treatment of cancer, the other therapeutic agent can be an anti-cancer agent, e.g., a chemotherapeutic agent, many classes of which are known in the art, or an immunological agent, including, for example, interferon, immune checkpoint inhibitors (e.g., anti-PD-1, -PD-L1, or -CTLA4 antibodies), and other immune-enhancing agents (e.g., anti-OX40 agonist antibodies). Other therapeutic agents, such as antiproliferative or anti-inflammatory cytokines, as well as antiproliferative, immunomodulatory, or blood coagulation-influencing factors, or angiogenesis inhibitors, can be beneficial in the treatment of cancer or infectious diseases. For the treatment of infectious diseases, the other (or second) therapeutic agent can be an antimicrobial agent, e.g., an antibiotic, antifungal, or antiviral agent.

[0099] A binding molecule, bispecific conjugate, complex, or pharmaceutical composition comprising a binding molecule, bispecific conjugate, or complex (or a conjugate and its tagged construct component) can be administered via one or more routes of administration using one or more of a variety of methods known in the art. Similarly, the conjugate and tagged construct can be administered separately by these same methods. As will be understood by those of skill in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, e.g., directly at the site of a tumor, e.g., by injection or infusion.

[0100] As used herein, the phrase "parenteral administration" refers to forms of administration other than enteral and topical administration, usually by injection. Alternatively, parenteral routes, such as topical, epidermal, or mucosal administration routes, can be used. Local administration is preferred, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracavity injection, intravesical administration, and inhalation. However, the binding molecule, bispecific conjugate, complex, or composition can also be administered systemically.

[0101] In embodiments in which the bispecific conjugate and tag construct are administered separately, i.e., they form a complex without first being premixed, the conjugate and tag construct must be administered via the same route. Preferably, they are both administered topically, e.g., intradermally, to the same (or substantially the same) site so that the two components mix and thus combine to form a complex rapidly after administration. In these embodiments, the two components must be administered to a subject simultaneously or quickly one after the other to avoid a delay between the administration of the first component and the administration of the second component. This ensures that the second component is administered and allows complex formation before the first component has time to degrade or diffuse excessively away from the administration site.

[0102] Suitable dosages of the specific binding molecules, bispecific conjugates, or complexes of the present disclosure can be determined by one of ordinary skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions and products of the present disclosure can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular subject, i.e., patient, without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular complex / conjugate used, the route of administration, the time of administration, the rate of excretion of the complex, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0103] Suitable doses of binding molecules, bispecific conjugates, or complexes of the disclosure can range, for example, from about 0.1 μg / kg to about 100 mg / kg of body weight of the patient being treated. For example, suitable dosages can be from about 0.1 μg / kg to about 10 mg / kg of body weight per day, or from about 10 μg / kg to about 5 mg / kg of body weight per day.

[0104] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions into dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the necessary pharmaceutical carrier.

[0105] The binding molecule, bispecific conjugate, or complex (or combination of conjugate and tag construct) may be administered in a single dose or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different sites. Alternatively, the complex may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency may vary depending on the half-life of the administered species in the patient and the desired duration of treatment. The dosage and frequency of administration may also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage may be administered at relatively infrequent intervals over an extended period of time. In therapeutic applications, a relatively high dosage may be administered, for example, until the patient shows partial or complete improvement in disease symptoms. In an exemplary dosage regimen, the complex (or combination of conjugate and tag construct) is administered to a subject once a week, once every two weeks, or once every three weeks, in a cycle repeated 2 to 10 times.

[0106] The combined administration of two or more agents can be achieved in several different ways. In one embodiment, the conjugate and the other agent can be administered together in a single composition. In another embodiment, the conjugate and the other agent can be administered in separate compositions as part of a combination therapy. For example, the conjugate can be administered before, after, or simultaneously with the other agent. The conjugates of the present disclosure can be administered in combination with or sequentially with tumor-targeting antibodies, targeted therapies, pathway inhibitors, or other immunomodulatory antibodies targeting, for example, PD-1, PD-L1, CD137, GITR, OX40, CTLA-4, CD27, HVEM, LTβR, and LAG3. Furthermore, the conjugates can also be combined with local radiation. Similarly, such additional therapies can be co-administered when the conjugate and tagged construct are administered separately to the subject.

[0107] The present disclosure also provides an in vitro or ex vivo method for activating T cells that express a TCR that recognizes an antigen, the method comprising: i) a bispecific conjugate of the present disclosure and a tag construct as defined above, wherein the tag construct comprises an antigen recognized by the TCR; or ii) contacting with a complex of the present disclosure, wherein the tag construct of the complex comprises an antigen recognized by the TCR.

[0108] Thus, the conjugates or bispecific conjugates of the present disclosure can be used to activate APCs in vitro or ex vivo, as well as in vivo. Thus, the conjugates and bispecific conjugates (in combination with the tag peptide contained in the tag construct) have both medical and non-medical uses, and all such uses are encompassed herein. For example, isolated or cultured APCs can be contacted with the conjugate, for example, in a laboratory setting, for example, for research, development, or testing purposes. This can be achieved by premixing the conjugate and tag construct to form a complex, and then applying the complex to the APC. Alternatively, the conjugate and tag construct can be applied separately to the APC so that the complex forms within the APC culture.

[0109] The complex can be used to activate T cells expressing a TCR that recognizes the antigen contained in the tag construct. Specifically, the TCR recognizes the antigen when presented by an APC (i.e., in the context of an MHC). Thus, an APC activated by the complex, or activated for activation by the complex, can be contacted with a T cell. Thus, the APC can be cultured or incubated in the presence of the complex (or a component thereof), and then the APC can be contacted with a T cell, e.g., co-cultured, or further incubated in the presence of a T cell. Alternatively, the complex (or a component thereof), the APC, and the T cell can be incubated or co-cultured together. Thus, the antigen is delivered to the APC and presented to the T cell, resulting in their activation.

[0110] The invention is further illustrated by the following non-limiting figures and examples. [Brief explanation of the drawings]

[0111] [Figure 1A]Figure 1 shows a sequence alignment illustrating the design and construction of a humanized version of murine scFv IBIIICI as described in Example 1. From top to bottom: VH and VL sequences from wild-type murine scFv IBIIICI ("VH_murine": SEQ ID NO: 55, "VL_murine": SEQ ID NO: 56), selected human germline framework sequences, sequences resulting from grafting murine scFv CDRs onto the human germline framework (VH: SEQ ID NO: 6) and VL (SEQ ID NO: 10), respectively. [Figure 1B] Figure 1 shows a sequence alignment illustrating the design and construction of a humanized version of murine scFv IBIIICI as described in Example 1. From top to bottom: VH and VL sequences from wild-type murine scFv IBIIICI ("VH_murine": SEQ ID NO: 55, "VL_murine": SEQ ID NO: 56), selected human germline framework sequences, sequences resulting from grafting murine scFv CDRs onto the human germline framework (VH: SEQ ID NO: 6) and VL (SEQ ID NO: 10), respectively. [Figure 2] Figure 1 shows an SDS-PAGE gel of expressed and purified scFvs as described in Example 2. M: molecular weight ladder. Lane 1: IBIIICI mouse scFv (SEQ ID NO: 25). Lane 2: IBIIICI CDR-grafted scFv (SEQ ID NO: 54). [Figure 3A-3B] 1 shows sensorgrams obtained from SPR affinity measurements of the interaction of immobilized IBIIICI mouse scFv (left side; A, C, E, SEQ ID NO: 25) or IBIIICI CDR-grafted scFv (right side; B, D, F, SEQ ID NO: 54) with a panel of 14 peptides (A-B: UU0070-UU0074, SEQ ID NOs: 27-31; C-D: UU0075-UU0078, SEQ ID NOs: 32-35, and E-F: UU0024, UU0080-UU0083, SEQ ID NOs: 26, 36-39), as described in Example 3. [Figure 3C-3D]1 shows sensorgrams obtained from SPR affinity measurements of the interaction of immobilized IBIIICI mouse scFv (left side; A, C, E, SEQ ID NO: 25) or IBIIICI CDR-grafted scFv (right side; B, D, F, SEQ ID NO: 54) with a panel of 14 peptides (A-B: UU0070-UU0074, SEQ ID NOs: 27-31; C-D: UU0075-UU0078, SEQ ID NOs: 32-35, and E-F: UU0024, UU0080-UU0083, SEQ ID NOs: 26, 36-39), as described in Example 3. [Figures 3E-3F] 1 shows sensorgrams obtained from SPR affinity measurements of the interaction of immobilized IBIIICI mouse scFv (left side; A, C, E, SEQ ID NO: 25) or IBIIICI CDR-grafted scFv (right side; B, D, F, SEQ ID NO: 54) with a panel of 14 peptides (A-B: UU0070-UU0074, SEQ ID NOs: 27-31; C-D: UU0075-UU0078, SEQ ID NOs: 32-35, and E-F: UU0024, UU0080-UU0083, SEQ ID NOs: 26, 36-39), as described in Example 3. [Figure 4] Figure 1 shows a sequence alignment of the VH sequences of murine IBIIICI (SEQ ID NO: 55), the original IBIIICI CDR-grafted VH (SEQ ID NO: 6), and five CDR-grafted variant VHs with mutations at predicted deamidation sites, designated IBIIICI CDR-grafted GG (SEQ ID NO: 2), IBIIICI CDR-grafted DG (SEQ ID NO: 4), IBIIICI CDR-grafted SG (SEQ ID NO: 1), IBIIICI CDR-grafted QG (SEQ ID NO: 3), and IBIIICI CDR-grafted NA (SEQ ID NO: 5). CDR boundaries and residue numbering are as defined by the IMGT nomenclature (Lefranc et al., 2003, Dev Comp Immunol 27(1):55-77). [Figure 5] 1 shows an SDS-PAGE gel of the indicated scFv variants expressed and purified as described in Example 4. [Figure 6A]Figure 1 shows sensograms obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interactions with the five peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0024 (SEQ ID NO: 26), B) UU0084 (SEQ ID NO: 40), C) UU0085 (SEQ ID NO: 41), D) UU0086 (SEQ ID NO: 42), and E) UU0087 (SEQ ID NO: 43). [Figure 6B] Figure 1 shows sensograms obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interactions with the five peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0024 (SEQ ID NO: 26), B) UU0084 (SEQ ID NO: 40), C) UU0085 (SEQ ID NO: 41), D) UU0086 (SEQ ID NO: 42), and E) UU0087 (SEQ ID NO: 43). [Figure 6C] Figure 1 shows sensograms obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interactions with the five peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0024 (SEQ ID NO: 26), B) UU0084 (SEQ ID NO: 40), C) UU0085 (SEQ ID NO: 41), D) UU0086 (SEQ ID NO: 42), and E) UU0087 (SEQ ID NO: 43). [Figure 6D]Figure 1 shows sensograms obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interactions with the five peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0024 (SEQ ID NO: 26), B) UU0084 (SEQ ID NO: 40), C) UU0085 (SEQ ID NO: 41), D) UU0086 (SEQ ID NO: 42), and E) UU0087 (SEQ ID NO: 43). [Figure 6E] Figure 1 shows sensograms obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interactions with the five peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0024 (SEQ ID NO: 26), B) UU0084 (SEQ ID NO: 40), C) UU0085 (SEQ ID NO: 41), D) UU0086 (SEQ ID NO: 42), and E) UU0087 (SEQ ID NO: 43). [Figure 7A] Figure 1 shows SPR sensorgrams obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interaction with the four peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0090 (SEQ ID NO: 44), B) UU0091 (SEQ ID NO: 45), C) UU0092 (SEQ ID NO: 46), and D) negative control p003 (SEQ ID NO: 48). [Figure 7B]Figure 1 shows SPR sensorgrams obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interaction with the four peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0090 (SEQ ID NO: 44), B) UU0091 (SEQ ID NO: 45), C) UU0092 (SEQ ID NO: 46), and D) negative control p003 (SEQ ID NO: 48). [Figure 7C] Figure 1 shows SPR sensorgrams obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interaction with the four peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0090 (SEQ ID NO: 44), B) UU0091 (SEQ ID NO: 45), C) UU0092 (SEQ ID NO: 46), and D) negative control p003 (SEQ ID NO: 48). [Figure 7D] Figure 1 shows SPR sensorgrams obtained from the six IBIIICI CDR-grafted scFv variants indicated when analyzed for interaction with the four peptides described in Example 5. The scFv variants analyzed are designated WT (SEQ ID NO: 54), SG (SEQ ID NO: 49), GG (SEQ ID NO: 50), QG (SEQ ID NO: 51), DG (SEQ ID NO: 52), and NA (SEQ ID NO: 53). The peptides analyzed were A) UU0090 (SEQ ID NO: 44), B) UU0091 (SEQ ID NO: 45), C) UU0092 (SEQ ID NO: 46), and D) negative control p003 (SEQ ID NO: 48). [Figure 8] Figure 1 shows the results of Western blot experiments for the indicated bispecific conjugate constructs SP031 and SP007, as described in Example 6. Analysis was performed on supernatants from 2.5 ml expression cultures harvested 5 days after transfection. [Figure 9]1 shows the results of a biolayer interferometry experiment for the indicated bispecific conjugate constructs SP031 and SP007, as described in Example 6. [Figure 10] 1 is a survival graph showing the survival probability in the three groups of mice indicated in the experiment described in Example 7. HD: high dose. LD: low dose. Example 1

[0112] The following examples disclose the development of a novel binding molecule, IBIIICI CDR-grafted scFv, created by grafting the complementarity-determining regions (CDRs) from the known murine scFv IBIIICI onto human germline variable heavy (VH) and variable light (VL) frameworks and additional point mutations. The amino acid sequences of the engineered humanized VH and VL domains described herein are listed in the Sequence Listing as SEQ ID NOS: 1-6 (VH sequences) and 10 (VL sequence), and the complete scFv sequences are listed as SEQ ID NOS: 49-54. These examples further describe the expression and characterization of scFv polypeptides and variants thereof, as well as bispecific conjugates and complexes comprising them, and demonstrate their surprisingly superior properties with respect to expression and function in both bacterial and mammalian expression settings.

[0113] Example 1 Design and construction of a humanized version of the IBIIICI murine scFv Mangsbo et al. (Mol Immunol 93:115-124, 2018) describe the generation of murine scFv IBIIICI, which is further tested in the bispecific conjugate context in WO2020 / 104690 and WO2021 / 239968. The complete amino acid sequence of murine scFv IBIIICI is listed herein as SEQ ID NO: 25 (SEQ ID NO: 48 in WO2020 / 104690). In the present disclosure, humanization of this ancestral murine scFv was performed via CDR grafting into human germline variable heavy and variable light (VH / VL) frameworks, as described below.

[0114] Materials and Methods IBIIICI Design of CDR-grafted scFv The human germline VH sequence IGHV1-46, which has 59.4% sequence identity to the IBIIICI mouse VH (residues 1-104, IMGT / DomainGapAlign), was selected as the VH framework. Sequence identity was considered for VL selection, but VH / VL pairs with favorable biophysical properties were also considered (Tiller et al., 2013, mAbs 5(3):445-470). Collectively, this resulted in the selection of the human germline gene IGKV1-39 for VL grafting. Based on IMGT / DomainGapAlign domain searches, IGHJ4 and IKVJ2 were selected as the binding fragments for VH and VL, respectively.

[0115] Construction of IBIIICI CDR-grafted scFv The IBIIICI CDR graft was generated by grafting the six antigen-binding loops of the murine sequence onto a human IGHV1-46 / IGKV1-39 scaffold (Figure 1). A gene encoding the VH fused to the VL via a glycine-serine linker ((G4S)4, SEQ ID NO: 14) was designed. Nucleotides encoding two additional amino acids (R and T, both parts of the light chain constant domain) were added to the end of the VL gene to include a BsiWI restriction site. Synthesis and subcloning of the scFv gene were performed without codon optimization (GenScript, Piscataway, USA). For the framework regions, the nucleotide sequence found in the human germline gene was used. For the CDRs, the codons found in the original murine antibody were used. After synthesis, the scFv gene was cloned into an in-house vector using the restriction enzymes SfiI and BsiWII, providing an scFv with a triple-FLAG tag and a hexahistidine tag at the C-terminus when expressed.

[0116] result IBIIICI Design of CDR-grafted scFv The degree of humanness introduced during the humanization procedure of IBIIICI murine scFv was calculated using a T20 score analyzer (Gao et al., 2013, BMC Biotechnology 13:55). Briefly, the T20 score is scaled from 0 to 100, with higher scores representing more human-like antibodies. The T20 scores obtained for the original IBIIICI murine scFv and the IBIIICI CDR-grafted scFv are presented in Table 1.

[0117] [Table 1]

[0118] The T20 scores for the VH and VL sequences of the humanized scFv IBIIICI CDR-grafts all showed a clear increase in humanness compared to the T20 scores of their respective murine counterparts. The T20 scores for the VH and VL frameworks for the IBIIICI CDR-grafts were both above 90, exceeding the criterion of a T20 score of above 85 to be considered "human-like." Based on a comparison of the T20 scores between the original IBIIICI murine scFv and the resulting IBIIICI CDR-draft scFv, the humanization procedure was determined to be successful.

[0119] Construction of IBIIICI CDR-grafted scFv After gene synthesis and cloning, 4 μg of vector encoding the IBIIICI CDR-grafted scFv was obtained, and the correct sequence was verified by DNA sequencing.

[0120] Example 2 Small-scale expression and purification of IBIIICI murine scFv and IBIIICI CDR-grafted scFv This example describes the small-scale expression and purification of both the known IBIIICI murine scFv (SEQ ID NO: 25) and a newly designed and constructed humanized version, the IBIIICI CDR-grafted scFv (SEQ ID NO: 54). To determine whether the humanization process affected the developability of the scFv constructs, expression and purification were performed and the results compared.

[0121] Materials and Methods Vectors encoding the scFv variants described in Example 1 were transformed into Top10 E. coli and the intended sequences were confirmed by sequencing.

[0122] 50 ml cultures containing each scFv vector were grown overnight, centrifuged, and the pellet resuspended in B-Per Protein Extraction Buffer (Pierce). Following another round of centrifugation, Ni-NTA Sepharose resin slurry (GE Healthcare) was added to the clarified supernatant. After incubation, the mixture was transferred to an empty gravity-flow column. After washing, the expressed scFv protein was eluted with an imidazole-containing buffer, and the sample was buffer-exchanged and concentrated to approximately 0.5 ml in PBS. Protein concentration was determined using an Implen Nanophotometer, and purity and integrity were verified by SDS-PAGE.

[0123] result The IBIIICI CDR-grafted scFv showed satisfactory purity as assessed by SDS-PAGE, as indicated by the presence of one major band corresponding to the expected molecular weight of the scFv (approximately 30 kDa, Figure 2). In addition, the measured protein concentration of the IBIIICI CDR-grafted scFv sample was relatively high (1.2 mg / ml). On the other hand, the IBIIICI mouse scFv showed poor sample purity, with multiple protein bands derived from E. coli. Furthermore, the protein band corresponding to the scFv (approximately 30 kDa) of the IBIIICI mouse scFv was extremely weak compared to the other bands on the gel, indicating a much lower concentration of this product than the measured value (0.2 mg / ml).

[0124] conclusion Small-scale expression experiments demonstrated that the IBIIICI CDR-grafted scFv has a superior expression profile compared to its murine ancestor.

[0125] Example 3 Measurement of the binding properties of murine IBIIICI scFv and IBIIICI CDR-grafted scFv using surface plasmon resonance The binding kinetics of the new IBIIICI CDR-grafted scFv was compared to that of the IBIIICI murine scFv to a panel of peptides using surface plasmon resonance (SPR).

[0126] Materials and Methods Affinity measurements of the IBIIICI murine scFv and IBIIICI CDR-grafted scFv variants obtained as described in Examples 1 and 2 were performed by SPR using a Biacore T200 instrument (GE Healthcare) and single-cycle kinetics. Anti-FLAG M2 antibody (Sigma-Aldrich) was immobilized on a Series S CM5 chip by primary amine coupling using NHS-EDC chemistry according to the manufacturer's instructions, allowing capture of each scFv via the FLAG tag provided by the expression vector. A 5-fold dilution series containing five concentrations (0.16 nM to 100 nM) of different peptides (Table 2) was sequentially injected into the flow cell to allow binding to the captured scFv. After the dissociation step, surface regeneration was achieved under acidic conditions using 10 mM glycine-HCl, pH 2.1. Response unit sensorgrams for all peptides were obtained by subtracting the response curve of a surface with immobilized anti-FLAG antibody (reference surface). Data were analyzed using BIAeval v.3.1 (GE Healthcare).

[0127] [Table 2]

[0128] result The results are presented in Figure 3. Overall, the binding properties of the IBIIICI murine scFv (Figures 3A, 3C, and 3E) and the humanized IBIIICI CDR-grafted scFv (Figures 3B, 3D, and 3F) were very similar, with low nanomolar binding affinities for most target peptides in the panel tested, indicating successful humanization.

[0129] Example 4 Generation of five IBIIICI CDR-grafted scFv variants by point mutation A putative deamidation site is present in CDR-H2 at positions 62-63 of the IBIIICI VH sequence. Five point mutation variants were generated to mutate this site without negatively affecting the binding properties or other important biophysical parameters of the humanized IBIIICI scFv.

[0130] Materials and Methods Design and construction of five IBIIICI CDR-grafted scFv variants Five different types of CDR-grafted VH sequences with mutated deamidation sites were designed. The diversity found in the natural antibody repertoire at these positions was taken into consideration (Kirik et al. 2017, Front Immunol 8:1433; Persson et al. 2018, Front Immunol 9:1391). Conservative substitutions, i.e., substitution of amino acids with different amino acids with similar biochemical properties, were then performed. Alignments of the VH sequences of the original mouse scFv, the humanized scFv described in Examples 1-3, and the five point mutation variants are shown in Figure 3. The amino acid sequences of the VH domains of the five point mutation variants and the original humanized variant are represented by SEQ ID NOs: 1-6 in the Sequence Listing. The complete scFv sequences of the five point mutation variants were designated IB CDR-grafted scFv SG (SEQ ID NO: 49), IB CDR-grafted scFv GG (SEQ ID NO: 50), IB CDR-grafted scFv QG (SEQ ID NO: 51), IB CDR-grafted scFv DG (SEQ ID NO: 52), and IB CDR-grafted scFv NA (SEQ ID NO: 53). The "original" humanized IBIIICI CDR-grafted scFv (SEQ ID NO: 54), designated "IB CDR-grafted scFv NG" or "IB CDR-grafted scFv WT," contains the original amino acids NG in CDR-H2 at positions 62-63 of the VH sequence (Figure 3).

[0131] Genes encoding scFv variants were constructed by introducing point mutations into the IBIIICI CDR-grafted scFv vector designed as described in Example 2. The vector provided scFv clones with a triple FLAG tag and a hexahistidine tag at the C-terminus.

[0132] Small-scale expression and purification Vectors encoding the designed scFv constructs were transformed into Top10 E. coli and the intended sequences were confirmed by sequencing.

[0133] 50 ml cultures of the six different humanized scFv variants and the original IBIIICI murine scFv were grown overnight, the cultures were centrifuged, and the pellets were resuspended in B-Per Protein Extraction Buffer (Pierce). After another round of centrifugation, Ni-NTA Sepharose resin slurry (GE Healthcare) was added to the clarified supernatant. After incubation, the mixture was transferred to an empty gravity-flow column. After washing, the polypeptides were eluted with an imidazole-containing buffer, and the sample was buffer-exchanged and concentrated to approximately 0.4 ml in PBS. Protein concentration was determined using an Implen Nanophotometer, and purity and integrity were verified by SDS-PAGE.

[0134] result The bacterial expression levels between the five different point-mutated IBIIICI CDR-grafted scFv variants and the IBIIICI CDR-grafted WT scFv ("Variant NG") were relatively high and very similar, in contrast to the IBIIICI murine scFv, which was relatively poorly produced (as previously shown in Example 2).

[0135] The SDS-PAGE gel is shown in Figure 5. The IBIIICI mouse scFv showed low sample purity, with multiple E. coli-derived protein bands. Furthermore, the protein band corresponding to the size of the scFv (approximately 30 kDa) in the IBIIICI mouse scFv lane was extremely weak compared to the other bands on the gel, indicating a much lower concentration of this clone. All point mutation variants exhibited satisfactory purity, as evidenced by a single major band corresponding to the expected molecular weight of the scFv (approximately 30 kDa). In addition, the measured protein concentrations of these samples were relatively high (2.3–3.3 mg / ml). Expression and purification of the variants were unaffected compared to the IBIIICI CDR-grafted WT scFv ("Variant NG"). Therefore, the generation of the point mutation variants was considered successful in terms of expression and purification.

[0136] Example 5 Biophysical characterization of IBIIICI CDR-grafted scFv variants The five IBIIICI CDR-grafted scFv point mutation variants generated as described in Example 4 and the IBIIICI CDR-grafted WT scFv described in Examples 1-3 were characterized using three different biophysical characterization methods: SPR, nanoDSF, and size exclusion chromatography (SEC).

[0137] Materials and Methods Surface plasmon resonance Affinity measurements of IBIIICI CDR-grafted WT scFv and IBIIICI CDR-grafted scFv point mutation variants were performed by SPR using a Biacore T200 instrument (GE Healthcare) and single-cycle kinetics. Anti-FLAG antibody M2 (Sigma-Aldrich) was immobilized on a CM5 S chip by primary amine coupling using NHS-EDC chemistry, allowing capture of each scFv via its FLAG tag. A 5-fold dilution series (Table 2) consisting of a panel of tested peptides at five concentrations (0.16 nM to 100 nM) was sequentially injected over the flow cell to allow binding to each captured scFv. After the dissociation step, surface regeneration was performed under acidic conditions using 10 mM glycine-HCl, pH 2.1. Response unit sensorgrams for all peptides were obtained by subtracting the response curve of a reference surface with immobilized anti-FLAG antibody. Data were analyzed using BIAeval v.3.1 (GE Healthcare).

[0138] Determination of melting temperature (Tm) by nanoDSF To assess the stability of purified scFv variants, melting temperatures were determined by nanoDSF using a Prometheus NT.48 instrument (NanoTemper Inc.) The nanoDSF technique measures the intrinsic fluorescence of a protein while it is subjected to thermal denaturation, thereby characterizing protein unfolding under native conditions.

[0139] Six purified IBIIICI CDR-grafted scFv variants were diluted to 1.0 mg / ml in PBS and applied to a high-sensitivity capillary tube (NanoTemper, #PR-C006) by capillary force. A melting gradient was set from 20°C to 95°C and heated at 1°C / min. Tryptophan release was measured at 330 nm and 350 nm, and the calculated ratio was plotted against temperature to obtain a melting curve for each variant. Tm values ​​were estimated using the software PR ThermoControl (NanoTemper Inc.).

[0140] Stability testing and SEC To further assess any differences in stability between the variants, a stability study was performed. Six purified IBIIICI CDR-grafted scFv variants were analyzed by SEC HPLC using a Bio SEC-3 3 μm 300A column (Agilent). Samples were analyzed at two time points: t = 0 h and t = 24 h after incubation at 37°C.

[0141] result Surface plasmon resonance The results are shown in Figures 6-7. Overall, the binding properties of the point-mutated IBIIICI CDR-grafted scFv variants to a panel of peptides were very similar to each other and to the IBIIICI CDR-grafted WT (variant "NG"), indicating that the point mutations introduced at positions 62 and 63 of the VH sequence did not negatively affect binding. Figures 6A-E show binding sensograms of different IBIIICI CDR-grafted scFv variants to peptides UU0024, UU0084, UU0085, UU0086, and UU0087, and Figures 7A-C show the corresponding binding to peptides UU0090, UU0091, and UU0092 (see Table 2 for peptide sequences). None of the variants showed any binding to the negative control peptide p003 (Figure 7).

[0142] NanoDSF measurements The measured Tm values ​​(inflection point #1) are shown in Table 3 and were between 60 and 65 °C. The determined Tm values ​​are useful as a measure of protein stability. The data showed somewhat higher stability for variant IBIIICI CDR-grafted scFv SG (64 °C) compared to the other analyzed scFv variants. In contrast, variant IBIIICI CDR-grafted scFv NA had the lowest Tm value (61 °C) within the set.

[0143] [Table 3]

[0144] SEC HPLC analysis To examine the integrity and stability of the CDR-grafted variants, six IBIIICI CDR-grafted scFv variants were analyzed by SEC HPLC before and after 24 h of incubation at 37° C. Compared to the starting sample, there was no detectable change in monomer content in any of the IBIIICI CDR-grafted scFvs after 24 h.

[0145] conclusion The point mutations introduced into CDR-H2 had minimal effect on the binding of the scFv variants to the tested peptides or on the stability of the scFv variants. Each of the five CDR-grafted variants, designed to remove putative deamidation sites, exhibits very similar binding patterns compared to both the initial IBIIICI CDR-grafted scFv (variant "NG") and the original IBIIICI murine scFv.

[0146] Example 6 Construction, expression, and characterization of bispecific conjugates of murine and humanized IBIIICI scFv with full-length antibodies In this example, a humanized scFv according to the present disclosure is placed in the bispecific conjugate context described in WO2020 / 104690 and WO2021 / 239968. That is, the humanized scFv is designed to form part of a bispecific conjugate construct in which the peptide-binding scFv is conjugated to another binding molecule, e.g., a full-length antibody against CD40. See Figure 6A of WO2020 / 104690 for a schematic diagram of the construct design. The anti-CD40 antibody used in this example is the antibody designated "A9" disclosed in WO2021 / 239968. Both the IBIIICI murine scFv and two humanized versions, IBIIICI CDR-grafted scFv NG and IBIIICI CDR-grafted scFv SG, were expressed and purified in the conjugate context, and the results were compared.

[0147] Materials and Methods Expression of murine and humanized scFv-A9 conjugates The four bispecific conjugate constructs (Table 4) were expressed in ExpiCHO-S™ cells (Gibco, #A29127) according to the manufacturer's instructions, using a standard expression protocol for 7 days. Bispecific conjugates were expressed in a transfection volume of 2.5 ml and incubated at 37°C, 5% CO2, humidified air, and 250 rpm. Supernatants were collected 10 days post-transfection for purification.

[0148] The conjugate was then transfected and expressed in a scaled-up 25 ml culture, incubated at 37°C, 5% CO2, humidified air, and 120 rpm, and harvested for affinity purification 7 days post-transfection.

[0149] Western blot A total of 20 μl of supernatant collected 5 days posttransfection was mixed with 3x loading buffer (0.1 M Tris-HCl, 45% glycerol, 0.03% bromophenol blue, 0.3% SDS) for non-reducing conditions. Samples were run on 4-20% Criterion™ TGX Stain-Free™ protein gels (Bio-Rad Laboratories, #5678095) according to the manufacturer's protocol. The gels were transferred to PVDF membranes (BioRad Transfer-Blot Turbo transfer system) for Western blot analysis. The membranes were incubated for 1 hour in 5% TBST milk buffer, followed by 1 hour of incubation with goat anti-human HRP antibody (ThermoFisher). The membranes were washed 3x with TBST for 5 minutes, then incubated with chemiluminescent substrate (Merck), and the blots were analyzed using a Chemidoc XRS+ system (BioRad).

[0150] Biolayer Interferometry Titers in supernatants from day 5 posttransfection were determined by biolayer interferometry using an Octet® RED96e system equipped with a Dip and Read™ Protein A biosensor (Fortebio Biologics by Molecular Devices, #10819702) according to the manufacturer's instructions. Supernatants were diluted 1:1 with equilibration buffer to a final volume of 200 μl in a 96-well black plate, with a final concentration of 20 mM citric acid, pH 4.0, 0.1% BSA (w / v), 0.1% Tween-20, and 0.5 M NaCl. A standard curve was prepared with IgG (IgG1 Protein A Standard, Sartorius #18-1118) over a concentration span of 1 to 700 μg / ml.

[0151] [Table 4]

[0152] Purification of murine and humanized scFv-A9 conjugates The expressed conjugate was purified by Protein A affinity chromatography on an AktaSTART system using a mAbSelect SuRe column (GE Healthcare, #11003493). 20 mM sodium phosphate, 0.15 M sodium chloride (pH 7.3) buffer was used as the binding and washing buffer, 0.1 M glycine (pH 2.5) as the elution buffer, and 1 M Tris-HCl (pH 8.5) as the neutralization buffer.

[0153] Conjugate purity determination using size exclusion chromatography A total of 25 μg of each conjugate in 100 μl was injected onto a Superdex Increase 200 10 / 30 GL gel filtration column (GE Healthcare, #28989336) connected to an Agilent 1200 Series HPLC system. SEC runs were performed at a flow rate of 0.5 ml / min using PBS as the running buffer. Proteins were detected by online absorbance measurement at 280 nm. Data analysis and peak integration were performed using GraphPad Prism 8.0.

[0154] result Western blot and biolayer interferometry of expressed conjugates Western blot results of 2.5 ml cultures of SP007 and SP031 are shown in Figure 8. SP007 gave stronger intensity than SP031. Overall, the Western blots showed that the construct containing the humanized scFv, SP007, had higher titers than the construct containing the murine scFv, SP031.

[0155] Biolayer interferometry titration of supernatants 5 days post-transfection is shown in Figure 9. The titer of SP007 was approximately 3-fold higher than that of SP031, mirroring the results observed from the Western blot.

[0156] Conjugate purity determination using size exclusion chromatography Expression and purification of the SP031 conjugate resulted in a monomer population clearly below the quality control cutoff for monomer content, set at 95%, as determined by SEC. Analysis of the chromatogram for SP031 resulted in a monomer content of 85.3% after affinity purification, with a distinct peak representing a smaller, non-native population of low molecular weight species comprising 14.7% of the total sample. All other tested conjugates containing humanized scFvs (SP007, SP019, and SP027) showed superior SEC profiles after purification compared to the ancestral murine forms (Table 5).

[0157] [Table 5]

[0158] conclusion Constructs containing the murine scFv IBIIICI (variant SP031) showed significantly lower expression levels compared to constructs containing humanized scFvs. Furthermore, the SEC profile of SP031 exhibited significant amounts of undesired species and had a total monomer population of 85.3%. From a developability perspective, improved profiles were observed for all conjugates containing humanized scFvs: SP007, SP019, and SP027, with monomer populations greater than 95% as observed in the SEC results. Therefore, conjugates containing humanized scFvs with anti-CD40 antibodies were considered superior from a developability perspective compared to conjugates containing non-humanized scFvs.

[0159] Example 7 Tumor treatment effectiveness We investigated the antitumor response induced by repeated vaccination with a peptide in combination with the bispecific conjugate SP027 in the TC-1 tumor model. By local vaccination at a non-tumor site, a tagged construct containing a tumor-associated antigen (derived from a virus or neoantigen) was delivered to dendritic cells via binding to CD40 of the bispecific conjugate, which then internalized and released the antigenic peptide. The antigenic peptide was processed and presented to T cells. Upon repeated injections, the antigen-specific T cells proliferated, migrated, and targeted tumor cells. The antitumor effect of the complex of the bispecific conjugate and tagged construct was compared with that of the peptide alone and / or the bispecific conjugate alone, similarly injected at a non-tumor site.

[0160] Materials and Methods Mice of strain B-hCD40 (Biocytogen) (9-12 weeks old) were divided into three treatment groups (6-7 mice per group): Group 1: a high dose of 30 μg of antigenic peptide UU0171 (SEQ ID NO: 77, a synthetic long peptide derived from immunogenic E7, HPV16 E7 [HPV16 E7 44-62]); Group 2: bispecific binding conjugate SP027 alone, 50 μg for the first injection on day 5 and 30 μg for injections on days 10 and 15; Group 3: bispecific binding conjugate SP027 complexed with tag construct UU0169 (SEQ ID NO: 78), comprising a 9 aa tag moiety linked to an HPV peptide and administered at a low dose of 3 μg in the conjugate mixture per dose, 50 μg for the first injection on day 5 and 30 μg for injections on days 10 and 15. Approximately 5×10 5 Tumor cells were injected sc into the right flank on day 0. Mice received treatment injections sc into the left ankle on days 5, 10, and 15; no therapy was administered at the site of tumor growth. Tumor growth and survival were monitored three times weekly, with tumor size reaching a maximum of 1000 mm as the experimental endpoint. 3 Mice were sacrificed when they reached a humane endpoint (e.g., health status in terms of wounds, weight, and appearance) or when they reached a humane endpoint. Mice that reached a humane endpoint while not reaching the experimental endpoint were excluded.

[0161] result The results are shown in Figure 10. The TC-1 peptide derived from the oncogenic HPV protein E7, coupled to a 9-mura tag moiety (UU0169) and delivered in a complex with the bispecific conjugate construct SP027, induced a strong immune response and significantly reduced tumor volume compared to control groups receiving either the bispecific conjugate SP027 alone or a high dose of the antigenic peptide without the tag moiety (UU0171), even though the therapy was injected at a low dose at a non-tumor site.

[0162] Unofficial sequence listing SEQ ID NO: 1 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 2 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEGGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 3 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO:4 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEDGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO:5 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO:6 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO:7 FNIKDFNI SEQ ID NO: 8) IGRIDPEX a X b DAEYVP SEQ ID NO:9 TTGSYDLDVE SEQ ID NO: 10 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 11 HASQNINVWLS SEQ ID NO: 12 KASTLHT SEQ ID NO: 13 QQGQSYPLT SEQ ID NO: 14 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 15 QSISSY SEQ ID NO: 16 AAS SEQ ID NO: 17 QQGYPYPFT SEQ ID NO: 18 GFTFSSYA SEQ ID NO: 19 ISGYSGST SEQ ID NO: 20 ARYYSYYGYYYFDY SEQ ID NO: 21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK SEQ ID NO: 22 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSS SEQ ID NO: 23 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 25 EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSSGGGG SGGGGSGGGGSGGGGSELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK SEQ ID NO: 26 FIGITELKKLESKINKVFK-Biotin SEQ ID NO: 27 FIGITELKKLESKINK SEQ ID NO: 28 FIGITELKKLESKIN SEQ ID NO: 29 FIGITELKKLESKI SEQ ID NO: 30 FIGITELKKLESK SEQ ID NO: 31 FIGITELKKLES SEQ ID NO: 32 LEQLESIINFEKLLAAAAKFIGITELKKLES SEQ ID NO: 33 FIGITELKKLESLEQLESIINFEKLLAAAAAK SEQ ID NO: 34 Biotin-(PEG3)FIGITELKKLES SEQ ID NO: 35 FIGITELKKLES(PEG3)K-Biotin SEQ ID NO: 36 FIGITELHHLESK-Biotin SEQ ID NO: 37 FIGISELKKLESK-Biotin SEQ ID NO: 38 FIGITELHKLESK-Biotin SEQ ID NO: 39 FIGITELKHLESK-Biotin SEQ ID NO: 40 FIGITELKKLE SEQ ID NO: 41 FIGITELKKL SEQ ID NO: 42 FIGITELKK SEQ ID NO: 43 FIGITELK SEQ ID NO: 44 FIGITELLEQLESIINFEKLAAAAAK SEQ ID NO: 45 FIGITELKLEQLESIINFEKLAAAAAK SEQ ID NO: 46 FIGITELHLEQLESIINFEKLAAAAAK SEQ ID NO: 47 ANSKFIGITELK SEQ ID NO: 48 Biotin-IDIKNDLYEKTLNDYKAIANKLSQV SEQ ID NO: 49 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO:50 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEGGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO:51 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO:52 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEDGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO:53 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO:54 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 55 EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSS SEQ ID NO:56 ELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK SEQ ID NO:57 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSEVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRAT MTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK SEQ ID NO:58 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:59 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 60 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 61 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 62 IGRIDPESGDAEYVP SEQ ID NO: 63 IGRIDPEGGDAEYVP SEQ ID NO: 64 IGRIDPEQGDAEYVP SEQ ID NO: 65 IGRIDPEDGDAEYVP SEQ ID NO: 66 IGRIDPENADAEYVP SEQ ID NO: 67 IGRIDPENGDAEYVP SEQ ID NO: 68 QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPSDNFSNLNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLTVSS SEQ ID NO: 69 SDIVMTQATPSVLVTPGEAVSISCRASRSLLHSNGITYLYWFLQRPGQSPQVLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEFPYTFGGGTKLEIK SEQ ID NO: 70 GGGGS SEQ ID NO: 71 GGGGSGGGGS SEQ ID NO:72 GGGGSGGGGSGGGGS SEQ ID NO: 73 GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:74 FIGITELL SEQ ID NO: 75 FIGITELH SEQ ID NO:76 FIGISELK SEQ ID NO:77 QAEPDRAHYNIVTFCCKCD SEQ ID NO:78 FIGITELKKQAEPDRAHYNIVTFCCKCD SEQ ID NO:79 FIGITELLK SEQ ID NO: 80 FIGITELHK

[0163] Itemized List of Embodiments 1. A binding molecule comprising: An immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and amino acid sequences having at least 90% identity thereto, provided that the heavy chain variable region (VH) comprises three complementarity-determining domains (CDRs): - VHCDR1 has the sequence set forth in SEQ ID NO: 7, -VHCDR2 has the sequence IGRIDPEX a X b DAEYVP (SEQ ID NO: 8) (wherein X a X b is selected from the group consisting of SG, GG, QG, DG, NA, and NG; - a VH, wherein VH CDR3 has the sequence set forth in SEQ ID NO: 9; An immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and amino acid sequences having at least 90% identity thereto, provided that the light chain variable region (VL) comprises three complementarity-determining domains (CDRs): - VLCDR1 has the sequence set forth in SEQ ID NO: 11, - VLCDR2 has the sequence set forth in SEQ ID NO: 12, - a VL, wherein VLCDR3 has the sequence set forth in SEQ ID NO: 13. 2. The binding molecule according to item 1, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6 and amino acid sequences having at least 90% identity thereto. 3. The binding molecule according to item 1, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, and 6, and amino acid sequences having at least 90% identity thereto. 4. The binding molecule according to item 1, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, and 6, and amino acid sequences having at least 90% identity thereto. 5. The binding molecule according to item 1, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, and 6, and amino acid sequences having at least 90% identity thereto. 6. The binding molecule according to item 1, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 6, and amino acid sequences having at least 90% identity thereto. 7. The binding molecule according to item 1, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and amino acid sequences having at least 90% identity thereto. 8. The binding molecule according to any one of items 2 to 3, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6 and amino acid sequences having at least 90% identity thereto. 9. The binding molecule according to any one of items 2 and 4, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, and 6, and amino acid sequences having at least 90% identity thereof. 10. The binding molecule according to any one of items 2 and 5, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, and 6, and amino acid sequences having at least 90% identity thereof. 11. The binding molecule according to any one of items 2 and 6, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, and 6, and amino acid sequences having at least 90% identity thereof. 12. The binding molecule according to any one of items 2 and 7, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and amino acid sequences having at least 90% identity thereto. 13. The binding molecule of any one of items 3 and 4, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, and 6, and amino acid sequences having at least 90% identity thereof. 14. The binding molecule according to any one of items 3 and 5, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 6, and amino acid sequences having at least 90% identity thereof. 15. The binding molecule according to any one of items 3 and 6, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6, and amino acid sequences having at least 90% identity thereto. 16. The binding molecule according to any one of items 3 and 7, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 5, and amino acid sequences having at least 90% identity thereof. 17. The binding molecule according to any one of items 4 and 5, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 5, and 6, and amino acid sequences having at least 90% identity thereof. 18. The binding molecule according to any one of items 4 and 6, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, and 6, and amino acid sequences having at least 90% identity thereof. 19. The binding molecule according to any one of items 4 and 7, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, and 5, and amino acid sequences having at least 90% identity thereof. 20. The binding molecule according to any one of items 5 and 6, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 6, and amino acid sequences having at least 90% identity thereof. 21. The binding molecule according to any one of items 5 and 7, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 5, and amino acid sequences having at least 90% identity thereof. 22. The binding molecule according to any one of items 6 and 7, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and amino acid sequences having at least 90% identity thereto. 23. The binding molecule according to any one of items 8, 9, and 13, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6 and amino acid sequences having at least 90% identity thereto. 24. The binding molecule of any one of items 8, 10, and 14, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 6, and amino acid sequences having at least 90% identity thereof. 25. The binding molecule of any one of items 8, 11, and 15, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 6, and amino acid sequences having at least 90% identity thereof. 26. The binding molecule of any one of items 8, 12, and 16, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 5, and amino acid sequences having at least 90% identity thereof. 27. The binding molecule of any one of items 9, 10, and 17, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 6, and amino acid sequences having at least 90% identity thereof. 28. The binding molecule of any one of items 9, 11, and 18, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, and amino acid sequences having at least 90% identity thereof. 29. The binding molecule of any one of items 9, 12, and 19, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 5, and amino acid sequences having at least 90% identity thereof. 30. The binding molecule according to any one of items 10, 11, and 20, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 6, and amino acid sequences having at least 90% identity thereof. 31. The binding molecule of any one of items 10, 12, and 21, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 5, and amino acid sequences having at least 90% identity thereof. 32. The binding molecule according to any one of items 11, 12, and 22, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4 and amino acid sequences having at least 90% identity thereto. 33. The binding molecule according to any one of items 13, 14, and 17, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, and 6, and amino acid sequences having at least 90% identity thereof. 34. The binding molecule of any one of items 13, 15, and 18, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 6, and amino acid sequences having at least 90% identity thereof. 35. The binding molecule according to any one of items 13, 16, and 19, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 5, and amino acid sequences having at least 90% identity thereof. 36. The binding molecule according to any one of items 14, 15, and 20, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 6, and amino acid sequences having at least 90% identity thereof. 37. The binding molecule according to any one of items 14, 16, and 21, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5, and amino acid sequences having at least 90% identity thereof. 38. The binding molecule according to any one of items 15, 16, and 22, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 4, and amino acid sequences having at least 90% identity thereof. 39. The binding molecule according to any one of items 17, 18, and 20, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 6, and amino acid sequences having at least 90% identity thereto. 40. The binding molecule according to any one of items 17, 19 and 21, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 5 and amino acid sequences having at least 90% identity thereof. 41. The binding molecule according to any one of items 18, 19, and 22, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 4, and amino acid sequences having at least 90% identity thereof. 42. The binding molecule according to any one of items 18, 19, and 22, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and amino acid sequences having at least 90% identity thereto. 43. The binding molecule according to any one of items 23, 24, 27, and 33, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and amino acid sequences having at least 90% identity thereto. 44. The binding molecule according to any one of items 23, 25, 28, and 34, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 6, and amino acid sequences having at least 90% identity thereof. 45. The binding molecule according to any one of items 23, 26, 29, and 35, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 5 and amino acid sequences having at least 90% identity thereto. 46. ​​The binding molecule of any one of items 24, 25, 30, and 36, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 6, and amino acid sequences having at least 90% identity thereof. 47. The binding molecule according to any one of items 24, 26, 31, and 37, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 5, and amino acid sequences having at least 90% identity thereto. 48. The binding molecule according to any one of items 25, 26, 32, and 38, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 4 and amino acid sequences having at least 90% identity thereto. 49. The binding molecule according to any one of items 27, 28, 30, and 39, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 6, and amino acid sequences having at least 90% identity thereof. 50. The binding molecule according to any one of items 27, 29, 31, and 40, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 5, and amino acid sequences having at least 90% identity thereto. 51. The binding molecule according to any one of items 28, 29, 32, and 41, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4, and amino acid sequences having at least 90% identity thereto. 52. The binding molecule according to any one of items 30, 31, 32, and 42, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-3 and amino acid sequences having at least 90% identity thereto. 53. The binding molecule according to any one of items 33, 34, 36, and 39, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6, and amino acid sequences having at least 90% identity thereof. 54. The binding molecule according to any one of items 33, 35, 37, and 40, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 5, and amino acid sequences having at least 90% identity thereto. 55. The binding molecule according to any one of items 34, 35, 38, and 41, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4, and amino acid sequences having at least 90% identity thereof. 56. The binding molecule according to any one of items 36, 37, 38, and 42, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3 and amino acid sequences having at least 90% identity thereof. 57. The binding molecule according to any one of items 39 to 42, wherein the heavy chain variable region (VH) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and amino acid sequences having at least 90% identity thereto. 58. The binding molecule according to any one of items 43, 44, 46, 49, and 53, wherein the heavy chain variable region (VH) consists of the amino acid sequence SEQ ID NO: 6 and an amino acid sequence having at least 90% identity thereto. 59. The binding molecule according to any one of items 43, 45, 47, 50, and 54, wherein the heavy chain variable region (VH) consists of the amino acid sequence SEQ ID NO: 5 and an amino acid sequence having at least 90% identity thereto. 60. The binding molecule according to any one of items 44, 45, 48, 51, and 55, wherein the heavy chain variable region (VH) consists of the amino acid sequence SEQ ID NO: 4 and an amino acid sequence having at least 90% identity thereto. 61. The binding molecule according to any one of items 46 to 48, 52, and 56, wherein the heavy chain variable region (VH) consists of the amino acid sequence SEQ ID NO: 3 and an amino acid sequence having at least 90% identity thereto. 62. The binding molecule according to any one of items 49 to 52 and 57, wherein the heavy chain variable region (VH) consists of the amino acid sequence SEQ ID NO: 2 and an amino acid sequence having at least 90% identity thereto. 63. The binding molecule according to any one of items 53 to 57, wherein the heavy chain variable region (VH) consists of the amino acid sequence SEQ ID NO: 1 and an amino acid sequence having at least 90% identity thereto. 64. A binding molecule according to any preceding item which is an antibody construct. 65. The binding molecule of any preceding item, selected from the group consisting of antibodies and antigen-binding fragments thereof, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, Fv fragments, single-chain (scFv) fragments, (scFv)2, and domain antibodies. 66. The binding molecule of any preceding item, which is an scFv. 67. The binding molecule of any preceding item, further comprising a peptide linker connecting the C-terminus of the heavy chain variable region (VH) to the N-terminus of the light chain variable region (VL). 68. The binding molecule according to item 67, wherein the peptide linker consists of the amino acid sequence SEQ ID NO: 14. 69. A binding molecule according to any preceding item, which is capable of selectively binding to a peptide tag moiety contained in a tag construct. 70. The binding molecule according to item 69, wherein the tag construct further comprises at least one cargo moiety. 71. The binding molecule according to item 70, wherein the at least one cargo moiety is an antigenic moiety. 72. The binding molecule according to any one of items 70 to 71, wherein the at least one cargo moiety is a peptide moiety or a nucleic acid moiety. 73. The binding molecule according to item 72, wherein the C-terminus of the tag moiety is covalently linked to the N-terminus of the at least one peptide cargo moiety. 74. The binding molecule according to item 72, wherein the N-terminus of the tag moiety is covalently linked to the C-terminus of the at least one peptide cargo moiety. 75. The binding molecule according to any one of items 71 to 74, wherein the at least one cargo moiety is a peptide antigen moiety comprising a target antigen amino acid sequence. 76. K for Interaction D The value is up to 5 x 10 -9 M, e.g., up to 1 x 10 -10 M, e.g., up to 1 x 10 -11 76. The binding molecule according to any one of items 69 to 75, which is capable of binding to the tag moiety such that M is 77. A bispecific conjugate comprising: - at least one first moiety which is a binding molecule according to any one of the preceding items; - at least one second moiety which is an antibody or an antigen-binding fragment thereof. 78. The bispecific conjugate according to item 77, wherein the second portion is an antigen-binding fragment of an antibody selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fc fragment, an Fv fragment, a single-chain (scFv) fragment, an (scFv)2, and a domain antibody. 79. The bispecific conjugate according to item 77, wherein the second moiety is an antibody, optionally of the IgG2 subtype. 80. The bispecific conjugate according to any one of items 77 to 79, wherein the first moiety is covalently linked to the second moiety. 81. The bispecific conjugate according to item 80, wherein the first moiety is covalently linked to the second moiety via a linker peptide. 82. The bispecific conjugate according to item 80, wherein the first moiety is directly linked to the second moiety. 83. The bispecific conjugate according to any one of items 80 to 82, wherein the first moiety is covalently linked to the C-terminus of the light chain of the second moiety. 84. The bispecific conjugate according to any one of items 80 to 82, wherein the first moiety is covalently linked to the N-terminus of the light chain of the second moiety. 85. The bispecific conjugate according to any one of items 80 to 82, wherein the first moiety is covalently linked to the C-terminus of the heavy chain of the second moiety. 86. The bispecific conjugate according to any one of items 80 to 82, wherein the first moiety is covalently linked to the N-terminus of the heavy chain of the second moiety. 87. The bispecific conjugate according to any one of items 77 to 86, wherein the conjugate comprises two first moieties and one antibody as second moiety, one first moiety being conjugated to the CH3 domain of each heavy chain of the second partial antibody. 88. The bispecific conjugate according to any one of items 77 to 86, wherein the conjugate comprises two first moieties and one antibody as second moiety, one first moiety being conjugated to the CL domain of each light chain of the second partial antibody. 89. The bispecific conjugate according to any one of items 77 to 88, wherein the second moiety is an anti-CD40 antibody or an antigen-binding fragment thereof. 90. The bispecific conjugate according to item 89, wherein the anti-CD40 antibody is selected from the group consisting of CP-870,893, APX005M, ADC-1013, ChiLob7 / 4, SEA-CD40, and ABS-1150 / 1151, and antigen-binding fragments derived from any one or more of said antibodies. 91. The anti-CD40 antibody or antigen-binding fragment thereof comprises six complementarity-determining domains (CDRs); VLCDR1 has the sequence set forth in SEQ ID NO: 15; VLCDR2 has the sequence set forth in SEQ ID NO: 16; VLCDR3 has the sequence set forth in SEQ ID NO: 17, VHCDR1 has the sequence set forth in SEQ ID NO: 18, VHCDR2 has the sequence set forth in SEQ ID NO: 19, 89. The bispecific conjugate according to item 89, wherein VHCDR3 has the sequence set forth in SEQ ID NO: 20. 92. the light chain variable domain of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 21 and an amino acid sequence having at least 90% sequence identity thereto; 92. The bispecific conjugate of item 91, wherein the heavy chain variable domain of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 22 and an amino acid sequence having at least 90% sequence identity thereto. 93. The anti-CD40 antibody is a light chain comprising an amino acid sequence selected from SEQ ID NO: 23 and an amino acid sequence having at least 90% sequence identity thereto; 93. The bispecific conjugate of item 92, comprising a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 and amino acid sequences having at least 90% sequence identity thereto. 94. A complex - a bispecific conjugate according to any one of items 77 to 93, - a tag construct comprising a peptide tag moiety. 95. The conjugate of item 94, wherein the tag construct further comprises a cargo moiety, the cargo moiety being a peptide antigen moiety comprising a target antigen amino acid sequence, and the C-terminus of the tag moiety is covalently linked to the N-terminus of the antigen moiety in the tag construct. 96. A polynucleotide encoding a binding molecule or conjugate according to any one of items 1 to 93. 97. An expression vector comprising the polynucleotide according to item 96. 98. A host cell comprising the expression vector according to item 97. 99. A method for producing a binding molecule or conjugate according to any one of items 1 to 93, the method comprising: - culturing a host cell according to item 98 under conditions allowing expression of the binding molecule or conjugate from the vector; - isolating said binding molecule or conjugate. 100. A composition comprising a binding molecule according to any one of items 1 to 76 and at least one pharmaceutically acceptable excipient or carrier. 101. A composition comprising the conjugate according to any one of items 77 to 93 and at least one pharmaceutically acceptable excipient or carrier. 102. A composition comprising the conjugate according to any one of items 94 to 95 and at least one pharmaceutically acceptable excipient or carrier. 103. A kit comprising: - a conjugate according to any one of items 77 to 93, a peptide tag construct comprising a peptide tag moiety; -Instructions for forming the complex according to item 94. 104. The kit of item 103, wherein the tag construct further comprises a cargo moiety, the cargo moiety being a peptide antigen moiety comprising a target antigen amino acid sequence, the C-terminus of the tag moiety being covalently linked to the N-terminus of the antigen moiety in the tag construct, and the instructions are instructions for forming the complex of item 95. 105. A binding molecule according to any one of items 1 to 76, a conjugate according to any one of items 77 to 93, a complex according to any one of items 94 to 95, a composition according to any one of items 100 to 102 or a kit according to any one of items 103 to 104 for use in therapy. 106. A binding molecule, conjugate, complex, composition or kit according to item 105 for use in the treatment or prevention of cancer, an infectious disease or an autoimmune disease. 107. A method for treating or preventing cancer, an infectious disease, or an autoimmune disease, comprising administering to a subject in need thereof an effective amount of a binding molecule according to any one of items 1 to 76, a conjugate according to any one of items 77 to 93, a complex according to any one of items 94 to 95, or a composition according to any one of items 100 to 102. 108. Use of a binding molecule according to any one of items 1 to 76, a conjugate according to any one of items 77 to 93, a complex according to any one of items 94 to 95, or a composition according to any one of items 100 to 102 in the manufacture of a medicament.

Claims

1. A binding molecule comprising: An immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and amino acid sequences having at least 90% identity thereto, provided that the heavy chain variable region (VH) comprises three complementarity determining domains (CDRs); - VHCDR1 has the sequence set out in SEQ ID NO: 7, - VHCDR2 has the sequence IGRIDPEX ａ X ｂ DAEYVP (SEQ ID NO: 8) ａ X ｂ is selected from the group consisting of SG, GG, QG, DG, NA, and NG; - a VH, wherein VHCDR3 has the sequence set out in SEQ ID NO: 9; An immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and amino acid sequences having at least 90% identity thereto, the light chain variable region (VL) comprises three complementarity-determining domains (CDRs); - VLCDR1 has the sequence set forth in SEQ ID NO: 11, - VLCDR2 has the sequence set forth in SEQ ID NO: 12, - a VL, wherein the VLCDR3 has the sequence set forth in SEQ ID NO:

13.

2. The binding molecule of claim 1 which is an scFv.

3. 10. A binding molecule according to any one of the preceding claims, capable of selectively binding to a peptide tag moiety comprised in a tag construct, said tag construct optionally further comprising at least one cargo moiety.

4. The binding molecule of claim 3 , wherein the cargo moiety is a peptide antigen moiety comprising a target antigen amino acid sequence.

5. K of Interaction Ｄ The value is up to 5 x 10 －９ M, for example, up to 1 × 10 －１０ M, for example, up to 1 × 10 －１１ 5. The binding molecule of claim 3 or 4, which is capable of binding to the tag moiety such that M is

6. 1. A bispecific conjugate comprising: - at least one first moiety which is a binding molecule according to any one of the preceding claims; - at least one second moiety which is an antibody or antigen-binding fragment thereof, for example an anti-CD40 antibody or antigen-binding fragment thereof.

7. the second portion is an anti-CD40 antibody or antigen-binding fragment thereof comprising six complementarity-determining domains (CDRs); VLCDR1 has the sequence set forth in SEQ ID NO: 15; VLCDR2 has the sequence set forth in SEQ ID NO: 16; VLCDR3 has the sequence set forth in SEQ ID NO: 17; VHCDR1 has the sequence set forth in SEQ ID NO: 18; VHCDR2 has the sequence set forth in SEQ ID NO: 19; 7. The bispecific conjugate of claim 6, wherein the VHCDR3 has the sequence set forth in SEQ ID NO:

20.

8. the light chain variable domain of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 21 and an amino acid sequence having at least 90% sequence identity thereto; 8. The bispecific conjugate of claim 7, wherein the heavy chain variable domain of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 22 and amino acid sequences having at least 90% sequence identity thereto.

9. the anti-CD40 antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 23 and an amino acid sequence having at least 90% sequence identity thereto; and a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 and amino acid sequences having at least 90% sequence identity thereto.

10. A complex, - a bispecific conjugate according to any one of claims 6 to 9, - a tag construct comprising a peptide tag portion.

11. 11. The conjugate of claim 10, wherein the tag construct further comprises a cargo moiety, the cargo moiety being a peptide antigen moiety comprising a target antigen amino acid sequence, the C-terminus of the tag moiety being covalently linked to the N-terminus of the antigen moiety in the tag construct.

12. A polynucleotide(s) encoding a binding molecule or conjugate according to any one of claims 1 to 9.

13. 12. A composition comprising a binding molecule according to any one of claims 1 to 5, a conjugate according to any one of claims 6 to 9 or a complex according to claim 10 or 11, and at least one pharmaceutically acceptable excipient or carrier.

14. A kit comprising: - a conjugate according to any one of claims 6 to 9, - a peptide tag construct comprising a peptide tag moiety; - instructions for forming the complex of claim 10 or 11.

15. 15. A binding molecule according to any one of claims 1 to 5, a conjugate according to any one of claims 6 to 9, a complex according to claim 10 or 11, a composition according to claim 13, or a kit according to claim 14 for use in therapy.

16. 16. A binding molecule, conjugate, complex, composition or kit for use as claimed in claim 15 in the treatment or prevention of cancer, infectious diseases or autoimmune diseases.

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