Binding molecules

Abstract

The present disclosure provides an improved binding molecule comprising an immunoglobulin binding domain having an affinity for a peptide tag moiety. The binding molecules can be used, for example, as part of bispecific conjugates. The binding molecules exhibit unexpected advantages as part of biopharmaceutical products as compared to known binding molecules with similar binding affinity. The disclosure also provides bispecific conjugates comprising a binding molecule and complexes comprising a bispecific conjugate non-covalently bound to a tag construct comprising a peptide tag moiety and a cargo domain comprising an antigen for delivery of the antigen to an immune cell. The disclosure also provides medical uses of the binding molecules, conjugates and complexes of the disclosure.

Description

Technical Field

[0001] The present disclosure relates to an improved binding molecule comprising an immunoglobulin binding domain with affinity for a peptide tag portion. The binding molecule can, for example, be used as a part of a bispecific conjugate. Compared with known binding molecules with similar binding affinity, the binding molecule shows unexpected advantages as a part of a biopharmaceutical product. The present disclosure also provides a bispecific conjugate comprising a binding molecule and a complex comprising a bispecific conjugate non-covalently bound to a tag construct, the tag construct comprising a peptide tag portion and a cargo domain (cargo moiety), and the payload portion comprising an antigen for delivering the antigen to an immune cell. The present disclosure also provides medical uses of the binding molecules, conjugates and complexes of the present disclosure. Background Art

[0002] Monoclonal antibodies (mAbs) that modulate immune responses have proven to be very effective in cancer treatment, and there is growing evidence that such responses can be used to provide durable clearance of tumors. A variety of antibodies have been developed against different targets, such as those targeting the immune checkpoints CTLA-4 and PD-1, supporting the view that T cell immunity can provide effective treatment for cancer. Promising clinical data have also been obtained with immunostimulatory mAbs that agonistically bind to the co-stimulatory receptor CD40 on antigen-presenting cells (APCs).

[0003] Effective stimulation (or sensitization) of T cells requires not only stimulation of APCs, but also presentation of antigens by APCs (in the context of MHC) for recognition and binding by T cell receptors (TCRs). Therefore, for the purpose of stimulating T cells, it is advantageous that APCs cross-present antigens to T cells, in other words, that APCs take up, process (extracellularly derived) antigens and present them to T cells. However, (for example, if a tumor has been resected or in the case of a vaccine against infection) antigenic material may not always be present, and in such cases, CD40 agonists may have poor efficacy in driving effective T cell stimulation. CD40 stimulation may also be insufficient to activate T cells (for example, if the CD40 agonist has dose-limiting toxicity as an infusion product).

[0004] For these reasons, it is advantageous to deliver the antigen to the APC while activating CD40 on the surface of the APC using an agonist. WO2020 / 104690 and WO2021 / 239968 describe bispecific conjugates for this purpose. The bispecific conjugates described therein comprise two binding proteins covalently coupled together. The first binding protein is specific for CD40; the second binding protein is not directly specific for the antigen, but is specific for the tag portion. A tag construct is provided, wherein the tag portion is covalently coupled to the antigen and forms a complex with the bispecific conjugate through the binding affinity of the second binding protein in the conjugate to the tag portion. Therefore, by binding the second binding protein to the tag portion contained in the tag construct, the bispecific conjugate comprising the first binding protein covalently coupled to the second binding protein indirectly binds to the antigen, thereby providing a flexible modular approach by which the antigen contained in the tag construct can be changed, and there is no chemical connection between the antigen and the bispecific conjugate.

[0005] WO2020 / 104690 provides a complex formed between a bispecific conjugate and a tag construct that provides a CD40 agonist (for activation of APCs) and an antigen (for presentation by APCs). This ensures that APC activation by the CD40 agonist results in activation of T cells specific for the target antigen, and this advantageously allows for the flexible preparation of conjugates and complexes for personalized medicine, as well as the use of this flexible vaccine development platform for vaccine development, for vaccination of individuals using the CD40 pathway to generate an effective anti-pathogen immune response.

[0006] The complex of WO2020 / 104690 can also stimulate B cell responses to antigens. The complex 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, while 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. In fact, B cells activated in this way using the complex described in WO2020 / 104690 may not require co-stimulation with helper T cells to be fully activated.

[0007] Unlike the preparation of conjugates comprising an antigen fused directly to a CD40 agonist or an antigen-specific binding agent fused to an agonist, which requires the laborious synthesis and production of separate conjugates for each patient (or at least each different tumor antigen) or each pathogenic serotype, bispecific conjugates can be customized for individual use by combining different tag constructs containing different antigens but the same tag portion, according to the needs of a specific individual patient. Through this strategy, bispecific conjugates are customized to also adapt to personalized or indication-based strategies to vaccinate against tumors or pathogens with high antigenic drift, thereby ensuring flexible vaccination strategies. In this way, only a customized tag construct needs to be prepared, providing advantages in terms of convenience and cost of preparing patient / pathogen-specific therapeutics. It is further believed that the indirect and non-covalent binding of the antigen to the CD40 agonist may be beneficial for the efficacy of the complex compared to conjugates comprising an antigen directly and covalently fused to a CD40 agonist or compared to providing the CD40 agonist and antigen separately.

[0008] WO2021 / 239968 discloses another agonist antibody (or related binding protein) against CD40 that binds to CD40 with high affinity and exhibits strong agonistic activity. This CD40 binding protein is particularly suitable for use in the context of therapeutic bispecific conjugates, which can be similarly used for cancer treatment, or alternatively for the treatment of infections or vaccination against infections.

[0009] WO2020 / 104690 and WO2021 / 239968 together disclose a promising platform for the development of personalized biopharmaceuticals, enabling therapeutic efficacy to be achieved from CD40-mediated immune activation. However, the development and production of candidate products place high demands on the yield, homogeneity and other expression characteristics of all components or parts contained in the product. In addition, in order to enable repeated administration, it is generally necessary to avoid unnecessary immunogenicity in the product. Although the candidate second binding protein in the bispecific conjugates disclosed in WO2020 / 104690 and WO2021 / 239968 is functional and useful, it was found that it was not optimal in one or more of these considerations, and therefore there is a need in the art to provide improved such binding proteins having maintained affinity for the tag portion used in the platform. Summary of the Invention

[0010] It is an object of the present disclosure to meet this need by providing a binding molecule that does not exhibit the disadvantages associated with the mouse scFv IBIIICI while substantially retaining its binding properties.

[0011] It is another object of the present disclosure to provide an alternative binding molecule that exhibits high levels of expression when produced in a recombinant expression system.

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

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

[0014] Thus, in a first aspect, there is provided a binding molecule comprising:

[0015] an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 to SEQ ID NO: 6,

[0016] Provided that the heavy chain variable region (VH) comprises three complementarity determining regions (CDRs), wherein:

[0017] - VHCDR1 has the sequence shown in SEQ ID NO: 7;

[0018] -VHCDR2 has the sequence IGRIDPEX a X b DAEYVP (SEQ ID NO: 8), wherein X a X b Selected from the group consisting of SG, GG, QG, DG, NA and NG; and

[0019] - VHCDR3 has the sequence shown in SEQ ID NO: 9; and

[0020] an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 10,

[0021] Provided that the light chain variable region (VL) comprises three complementarity determining regions (CDRs), wherein:

[0022] - VLCDR1 has the sequence shown in SEQ ID NO: 11;

[0023] - VLCDR2 has the sequence shown in SEQ ID NO: 12; and

[0024] - VLCDR3 has the sequence shown in SEQ ID NO: 13.

[0025] Although the binding molecules defined herein were initially considered to be humanized versions of the known mouse antibody IBIIICI described in Example 1, they were found to exhibit not only the advantage of having an increased "human origin" score, but also unexpected significant advantages over their ancestors when expressed, such as in one or more aspects of protein expression yield, monomer content, and ease of purification. Whether in the study of the binding molecules in scFv form alone (Examples 2, Figure 2 ), also when placing this scFv in the context of bispecific conjugates similar to those described in WO2020 / 104690 and WO2021 / 239968 (Example 6, Table 5, Figure 8 and Figure 9 These advantages are significant. Unexpected improvements in these developability parameters have important implications when developing commercial biopharmaceutical products.

[0026] The increased "humanity" of the binding molecules of the present disclosure also makes them less prone to inducing anti-drug antibodies over time when administered to a subject, such as a human patient, thereby providing the benefit of allowing repeated dosing over time. This is advantageous compared to non-human proteins and other vector-based platforms that induce anti-drug antibodies, which can limit the usefulness of the binding molecules over time.

[0027] Therefore, the present disclosure provides a binding molecule comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region having a defined amino acid sequence. The term "binding molecule" is used herein to refer to a binding molecule comprising the binding domain of an antibody (that is, a binding domain obtained or derived from an antibody, or a binding domain based on an antibody). Therefore, a binding molecule is an antibody-based or antibody-like molecule comprising the binding site of an antibody or a binding site derived from an antibody.

[0028] As described above, the binding domain of an antibody is composed of a light chain variable region and a heavy chain variable region (thus, a classic bivalent antibody has two binding domains). Therefore, the binding molecule can be a natural antibody or a fragment thereof, or an artificial or synthetic antibody, or an antibody construct, or a derivative (e.g., a single-chain antibody). In summary, the binding molecules of the present disclosure comprise the binding domain of an antibody, wherein the binding domain of the antibody comprises a light chain variable region and a heavy chain variable region.

[0029] As described in detail above, the binding molecules of the present disclosure comprise six CDR sequences. The light chain variable region and the heavy chain variable region each comprise three CDRs: the heavy chain variable region comprises VHCDR1, VHCDR2, and VHCDR3, and the light chain variable region comprises VLCDR1, VLCDR2, and VLCDR3. The six CDRs have the following amino acid sequences:

[0030] VHCDR1 has the sequence shown in SEQ ID NO:7;

[0031] VHCDR2 has the sequence shown in SEQ ID NO:8;

[0032] VHCDR3 has the sequence shown in SEQ ID NO:9;

[0033] VLCDR1 has the sequence shown in SEQ ID NO: 11;

[0034] VLCDR2 has the sequence shown in SEQ ID NO: 12; and

[0035] VLCDR3 has the sequence shown in SEQ ID NO:13.

[0036] As mentioned above, SEQ ID NO: 8 is the amino acid sequence IGRIDPEX a X b DAEYVP, where 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 VHCDR2 sequence of the heavy chain variable region of the binding molecule is selected from the group consisting of SEQ ID NO: 62 to SEQ ID NO: 67. As shown in the Examples section herein, X a X bCan be changed in this way without significantly affecting the binding of the binding molecule to the amino acid sequence of its tag portion counterpart. In various embodiments of the binding molecules disclosed herein, six different versions of the VHCDR2 sequence are combined into subgroups, each of which contains 2, 3, 4, and 5 possible subcombinations of different VHCDR2 sequences. In even more specific embodiments, each individual VHCDR2 sequence represents a separate embodiment of the binding protein. The final disclosure of all possible combinations of SEQ ID NO: 1 to SEQ ID NO: 6 (equivalent to all possible combinations of SEQ ID NO: 62 to SEQ ID NO: 67) is given in the detailed list of embodiments below, items 2 to 63. All of these combinations are individually part of the disclosure herein.

[0037] The binding molecules of the present disclosure comprise a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and any sequence with at least 90% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 6, provided that the three CDR sequences consist of SEQ ID NO: 7 to SEQ ID NO: 9. In a specific embodiment, the sequence is 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% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0038] The binding molecules of the present disclosure comprise a light chain variable region having a sequence selected from 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 NO: 11 to SEQ ID NO: 13. In a specific embodiment, the sequence is 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% identical to SEQ ID NO: 10.

[0039] As described herein, binding molecules having amino acid sequences with 90% or greater identity to the listed sequences are also considered to fall within the scope of the present disclosure. Such variant sequences can be modified by substitution, insertion, and / or deletion of one or more amino acids relative to the listed sequences.

[0040] Amino acid substitutions relative to the listed sequences can be conservative amino acid substitutions. As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which an amino acid residue is substituted by another amino acid residue with a similar side chain. Amino acids with similar side chains generally have similar properties, and therefore, it can be expected that conservative substitutions of amino acids important to the structure or function of a polypeptide will have less impact on the 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, including 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), non-polar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, conservative amino acid substitutions can be considered to be substitutions in which a particular amino acid residue is substituted by a different amino acid in the same family. However, amino acid substitutions may also be non-conservative substitutions, in which one amino acid is replaced by another amino acid having a side chain belonging to a different family.

[0041] As mentioned above, according to the present disclosure, the variant of the listed sequence has at least 90% sequence identity with the corresponding listed sequence. Sequence identity can be evaluated by any convenient method. However, in order to determine the degree of sequence identity between sequences, a computer program for carrying out paired or multiple comparisons of sequences can be used, such as EMBOSS Needle or EMBOSS stretcher (all referring to Rice et al. (2000), Trends Genet., 16 (6): 276-277) for paired sequence comparisons, and Clustal Omega (Sievers et al. (2011), Mol.Syst.Biol.7: 539) or MUSCLE (Edgar (2004), Nucleic Acids Res.32 (5): 1792-1797) for multiple sequence comparisons, but any other suitable program can also be used. Regardless of whether the comparison is paired or multiple, it must be performed globally (i.e., across the entire reference sequence), rather than locally.

[0042] Sequence alignments and % identity calculations can be determined using, for example, standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1. Alternatively, standard EMBOSS Needle parameters can be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters can be used alternatively.

[0043] Binding molecules of the present disclosure can be synthesized by any method known in the art. Preferably, protein expression system is used to synthesize binding molecules, for example, using a cell expression system of prokaryotic (for example bacteria) cells or eukaryotic (for example yeast, fungi, insects or mammals) cells. Alternative protein expression system is a cell-free in vitro expression system, wherein, in vitro, the nucleotide sequence encoding the binding molecule is transcribed into mRNA, and the mRNA is translated into protein. Cell-free expression system test kits are widely available and can be purchased from companies such as ThermoFisher. Alternatively, the binding protein can be chemically synthesized in abiotic systems. Liquid phase synthesis or solid phase synthesis can be used to generate the polypeptide that can be formed or included in the binding molecules of the present disclosure.

[0044] Those skilled in the art can easily prepare binding proteins using appropriate methods common in the art. In particular, binding molecules can be recombinantly expressed in mammalian cells such as Chinese hamster ovary celIs. Binding molecules synthesized in protein expression systems can be purified using standard techniques in the art, for example, they can be synthesized together with affinity tags and purified by affinity chromatography. If the binding molecule is an antibody, one or more antibody-binding proteins, such as protein G, protein A, protein A / G or protein L, can be used to purify it using affinity chromatography.

[0045] As mentioned above, the binding molecule is an antibody-based or antibody-like molecule. Thus, the binding molecule can be a natural antibody or fragment thereof, or an artificial or synthetic antibody, or an antibody construct or derivative (e.g., a single-chain antibody).

[0046] In one embodiment, the binding molecule is an antibody, particularly a monoclonal antibody. A "monoclonal antibody" refers to an antibody preparation composed of a single antibody species, i.e., all antibodies in the preparation have the same amino acid sequence, including the same CDRs, so that they bind to the same epitope on their target antigen with the same efficacy ("target antigen" refers to the antigen containing the epitope bound by a specific antibody). In other words, the antibodies of the present disclosure are preferably not part of a polyclonal antibody mixture.

[0047] If the binding molecule is an antibody, the antibody may be of any isotype and subtype. Thus, the antibody may be an IgA, IgD, IgE, IgG, or IgM antibody. The heavy chain constant regions corresponding to the different immunoglobulin isotypes are referred to as 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 described above, IgG antibodies have four subtypes: IgG1, IgG2, IgG3, and IgG4. The IgG anti-CD40 antibody of the present disclosure may be of any IgG subtype, i.e., it may be an IgG1, IgG2, IgG3, or IgG4 antibody.

[0048] 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 examples include Fab', Fab, F(ab')2, Fv, Fd, or dAb fragments, which can be prepared according to techniques well known in the art.

[0049] The Fab fragment consists of the antigen-binding domain of an antibody. That is, a single antibody can be considered to contain two Fab fragments, each consisting of a light chain and the N-terminal portion of the heavy chain to which it is attached. Thus, a Fab fragment contains the entire light chain as well as the VH and CH1 regions of the heavy chain to which it is bound. Fab fragments can be obtained by digesting antibodies with papain.

[0050] A F(ab')2 fragment consists of two Fab fragments of an antibody plus the hinge region of the heavy chain region, which includes a disulfide bond linking the two heavy chains. In other words, a F(ab')2 fragment can be considered as two covalently linked Fab fragments. F(ab')2 fragments can be obtained by digesting antibodies with pepsin. Reduction of the F(ab')2 fragments yields two Fab' fragments, which can be considered as Fab fragments containing an additional sulfhydryl group that can be used to conjugate the fragment to other molecules.

[0051] In alternative and preferred embodiments, the binding molecule is a synthetic construct or an artificial construct, that is, an antibody molecule comprising a binding domain but genetically engineered or artificially constructed. Such constructs include chimeric antibodies or CDR-grafted antibodies, as well as single-chain antibodies and other constructs, such as scFv, dsFv, ds-scFv, dimers, miniantibodies, diabodies, single domain antibodies (DABs), TandAbs dimers, and heavy chain antibodies such as VHH. In a specific embodiment, the binding molecule is a single-chain variable fragment (scFv). scFv is a fusion protein comprising the VH and VL regions of an antibody in a single polypeptide. The scFv fragment typically includes a peptide linker covalently linking the VH and VL regions, which contributes to the stability of the molecule. The linker may comprise 1 to 20 amino acids, such as 1, 2, 3, or 4, 5, 10, or 15 amino acids, or, if appropriate, other intermediate numbers within the range of 1 to 20. The peptide linker can be formed by any commonly convenient amino acid residue, such as glycine and / or serine. An example of a suitable linker is G4S (SEQ ID NO: 70). A polymer of such a linker can be used, such as a dimer, trimer, tetramer or pentamer, 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 a specific embodiment in which the binding molecule of the present disclosure is a scFv, the linker is a (G4S)4 linker (SEQ ID NO: 14). However, the presence of a linker is not essential, and the VL region can also be alternatively directly connected to the VH region via a peptide bond. scFv generally includes a VH region from N-terminal to C-terminal, and the VH region is connected to the VL region via a linker sequence. In other words, in this embodiment of the binding molecules of the present 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).

[0052] The preparation of scFv molecules is well known in the art, and those of ordinary skill in the art can easily prepare scFv binding molecules according to the present disclosure, which include VH sequences and VL sequences defined herein. Therefore, as non-limiting examples, the binding molecules of the present disclosure can be scFv, which include the amino acid sequence of the group consisting of sequences selected from SEQ ID NO:49 to SEQ ID NO:54 and SEQ ID NO:49 to SEQ ID NO:54 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity. The present disclosure also encompasses any independent scFv sequence or sequence subset, similar to the independent VH and VL sequence pairs (see 2nd to 63rd items) listed in "detailed list of embodiments". In a specific embodiment, the binding molecules of the present disclosure are scFv comprising amino acid sequence SEQ ID NO:49, which is expressed as "SG" or "IBIIICI CDR grafting SG".

[0053] In one embodiment, the binding molecule is a humanized protein, particularly a humanized scFv. A humanized binding molecule may comprise a VH region and a VL region, wherein the framework is derived from human germline immunoglobulin sequences. However, such a protein may contain amino acids not encoded by human germline Ig sequences, for example, mutations introduced by random or site-specific mutagenesis or amino acids derived from ancestral binding molecules produced in other species (e.g., mice).

[0054] In one embodiment of the binding molecule of the present disclosure, the binding molecule is capable of selectively binding to the peptide tag portion contained in the tag construct. "Selective binding" refers to the binding molecule binding to its target in a manner different from that of binding to non-target molecules, and more particularly refers to the binding molecule binding to its target with a greater affinity than that of its binding to other molecules. That is, the binding molecule does not bind to other non-target molecules, or does not bind to a significant or significant degree, or binds to these other molecules with an affinity lower than that of its binding to the tag portion. The binding molecule that "selectively binds" to the tag portion can alternatively be referred to as "directed against" or "recognizing" the tag portion. In other words, the tag portion 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 portion as its antigen. Those skilled in the art will recognize that the terms "antigen" and "antigen binding protein" in the preceding sentence are used in a metaphorical context to attempt to illustrate the binding relationship between the tag portion and the binding molecule of the present disclosure. Those skilled in the art will also recognize that the preceding sentence does not mean that the tag moiety itself is the antigen that induces a nonspecific or specific immune response against it, but rather that the tag moiety is a customized binding partner of the binding molecule of the present disclosure.

[0055] In one embodiment of the present disclosure, the tag portion to which the binding molecule is specifically bound comprises the amino acid sequence FIGITELK (SEQ ID NO: 43). Examples of such tag portions comprising SEQ ID NO: 43 were tested in the following experimental section and formed a group consisting of SEQ ID NO: 26 to SEQ ID NO: 35, SEQ ID NO: 39 to SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47. The specific tag portion to which the binding molecule described herein is specifically bound consists of SEQ ID NO: 42 (i.e., FIGITELKK).

[0056] In another embodiment, the tag portion comprises the amino acid sequence FIGITELL (SEQ ID NO: 74), for example, selected from the group consisting of SEQ ID NO: 44 and SEQ ID NO: 74. In another embodiment, the tag portion comprises the amino acid sequence FIGITELH (SEQ ID NO: 75), for example, selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 46, and SEQ ID NO: 75. In another embodiment, the tag portion comprises the amino acid sequence FIGISELK (SEQ ID NO: 76), for example, selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 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).

[0057] As determined in WO2020 / 104690 and WO2021 / 239968, the tag portion to which the second binding protein is bound can advantageously form a part of a tag construct. In one embodiment, the tag construct comprising a peptide tag portion further comprises at least one cargo domain. At least one cargo domain may, for example, be an antigen portion, and it may be meaningful to trigger an immune response against the antigen portion. The at least one cargo domain may, for example, be a peptide portion (such as an antigen peptide portion). For the sake of clarity, although the tag portion can be considered to be an antigen that the binding molecule of the present disclosure can selectively bind to, the "antigen portion" (an example of a class of "cargo domain") present in one embodiment of the tag construct is different from the "tag portion" contained in the construct. In an embodiment in which the tag construct comprises a peptide tag portion and a peptide cargo domain (e.g., a peptide antigen or "antigen portion"), the C-terminus of the tag portion can be covalently bound to the N-terminus of the cargo domain, and vice versa. In another embodiment, the cargo domain is a nucleic acid portion.

[0058] In embodiments where at least one cargo domain is a nucleic acid moiety, the tag moiety and the cargo domain can be arranged in any order in the polypeptide-nucleic acid combination. In one such embodiment, the cargo domain is an siRNA molecule.

[0059] In one embodiment of the binding molecules of the present disclosure, the binding molecules are capable of binding to a tag moiety such that the interacting K D The value is up to 5×10 -9 M, for example, up to 1×10 -10 M, for example, up to 1×10 -11M. Those skilled in the art will appreciate that the binding interaction occurs between the binding protein of the present disclosure and the tag moiety.

[0060] In a second aspect, the present disclosure provides a bispecific conjugate comprising:

[0061] - at least one first moiety which is a binding molecule of the present disclosure; and

[0062] - at least one second moiety which is an antibody or an antigen-binding fragment thereof.

[0063] The design considerations for preparing the bispecific conjugates of this aspect of the present disclosure are similar to those described for similar conjugates in WO2020 / 104690 and WO2021 / 239968. However, the first part of the bispecific conjugate herein is a binding molecule according to the first aspect, and in one embodiment, advantageously, the first part is capable of binding to a tag portion. In one embodiment herein, the second part is an antibody or antigen-binding fragment thereof having binding affinity for an immunologically relevant target.

[0064] In one embodiment of the bispecific conjugate of the present disclosure, the second portion is an antigen-binding fragment of an antibody, said 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, (scFv)2 and a region antibody.

[0065] In one embodiment of the bispecific conjugate, the second moiety is an antibody, optionally an antibody of the IgG2 subtype.

[0066] In one embodiment of the bispecific conjugate, the first portion is optionally covalently linked to the second portion via a peptide linker (e.g., selected from the linkers discussed above in conjunction with the first aspect). Thus, the linker, if present, may comprise 1 to 20 amino acids and may, for example, be formed by any generally convenient amino acid residues (e.g., glycine and / or serine). An example of a suitable linker is G4S (SEQ ID NO: 70) and multimers thereof, such as (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).

[0067] In one embodiment of the bispecific conjugate, said first moiety is covalently linked to said second moiety via a linker peptide.

[0068] In an alternative embodiment of the bispecific conjugate, said first moiety is directly linked to said second moiety.

[0069] In one embodiment of the bispecific conjugate, the first moiety is covalently linked (directly or via a linker) to the C-terminal end or N-terminal end of the light or heavy chain of the second moiety.

[0070] In one embodiment of the bispecific conjugate, the bispecific conjugate comprises two first moieties and an antibody as a second moiety, and one first moiety is conjugated to the CH3 domain of each heavy chain of the second antibody moiety.

[0071] In another embodiment of the bispecific conjugate, the bispecific conjugate comprises two first moieties and an antibody as a second moiety, and one first moiety is conjugated to the CL domain of each light chain of the second antibody moiety.

[0072] In one particularly interesting embodiment of the bispecific conjugate, the second moiety is an anti-CD40 antibody or antigen-binding fragment thereof.

[0073] In such an embodiment, the second moiety can, for example, be selected from the group consisting of CP-870,893, APX005M, ADC-1013, ChiLob7 / 4, SEA-CD40 and ABS-1150 / 1151 and an antibody comprising an antigen-binding fragment derived from any one or more of said antibodies.

[0074] Alternatively, the second portion can be an anti-CD40 binding antibody or antigen-binding fragment thereof as described in WO2021 / 239968, for example, the antibody designated as "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 embodiment of this invention, the light chain variable region (VL) of the anti-CD40 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 21; and the heavy chain variable region (VH) of the anti-CD40 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 22. For example, such an anti-CD40 antibody may comprise a light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and an amino acid sequence having at least 90% sequence identity thereto, and a heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and an amino acid sequence having at least 90% sequence identity thereto. Similar to the sequences of the binding molecules disclosed according to the first aspect of the present invention, in specific embodiments, the VH sequence, VL sequence, HC sequence, or LC sequence of the anti-CD40 antibody may be 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% identical to any one of the respective listed sequences SEQ ID NO: 21 to SEQ ID NO: 24.

[0075] Exemplary of individual bispecific conjugates of the present invention that have been tested in the experimental section herein and found to exhibit the desired properties are the bispecific conjugates denoted herein as SP007, SP019, and SP027. Each of these bispecific conjugates is composed of two polypeptide chains comprising a first portion, which is an scFv embodiment of the binding molecule of the first aspect (see Table 4 in Example 6), and an antibody heavy chain of an anti-CD40 antibody, and two polypeptide chains which are antibody light chains of the same anti-CD40 antibody.

[0076] In a third aspect of the present disclosure, polynucleotides encoding binding molecules or bispecific conjugates as described herein, expression vectors comprising the polynucleotides, and host cells comprising the expression vectors are provided. The present disclosure also encompasses methods for preparing the binding molecules or bispecific conjugates of the present disclosure, the methods comprising: culturing the host cell under conditions that allow expression of the binding molecule or bispecific conjugate from its expression vector; and isolating the binding molecule or bispecific conjugate.

[0077] In a fourth aspect, the present disclosure provides a complex, such as a non-covalent complex, comprising a bispecific conjugate of the present disclosure and a tag construct of the present disclosure, the tag construct comprising a tag portion, and the binding molecule of the first aspect is capable of binding to the tag portion. In embodiments of the complex, the tag construct is as defined above in conjunction with 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 through the affinity of the binding molecule for the tag portion contained in the tag construct.

[0078] In another 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 further comprises at least one pharmaceutically acceptable carrier or excipient.

[0079] The present disclosure also provides kits and products comprising the bispecific conjugate and tag construct as described above, respectively. In such kits and products, the conjugate and tag construct can be provided in compositions containing pharmaceutically acceptable carriers or excipients, respectively.

[0080] As used herein, "pharmaceutically acceptable carriers or excipients" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0081] Preferably, the carrier or excipient is suitable for parenteral administration (e.g., by injection or infusion), such as intradermal, intravenous, intramuscular, or subcutaneous administration. 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 acids and other natural conditions that may inactivate or denature it.

[0082] The preferred pharmaceutically acceptable carrier comprises an aqueous carrier or a diluent. The example of the suitable aqueous carrier that can be used for pharmaceutical composition, test kit and product comprises water, buffered water and normal saline. The example of other carriers comprises ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol etc.) and suitable mixture thereof, vegetable oil and injectable organic ester, such as ethyl oleate. Can for example, by using a coating material such as lecithin, by maintaining required granularity and by using a surfactant to maintain suitable fluidity in the case of dispersion. In many cases, preferably comprise isotonic agent, such as sugar, polyol (such as mannitol, sorbitol), sodium chloride etc.

[0083] Pharmaceutical compositions, products or kits may also include pharmaceutically acceptable antioxidants. They may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Sterilization procedures and the addition of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. can ensure protection against the presence of microorganisms. In addition, prolonged absorption of injectable drug forms can be achieved by adding agents that delay absorption such as aluminum monostearate and gelatin.

[0084] 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.

[0085] Sterile injectable solutions can be prepared by adding the desired amount of the active agent (e.g., complex) and one or a combination of the above-mentioned ingredients to an appropriate solvent as needed, followed by sterile microfiltration. Typically, dispersions are prepared by adding the active agent to a sterile carrier containing a basic dispersion medium and the above-mentioned required other ingredients. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which obtain a powder of the active agent plus any other required ingredients from a previously sterile-filtered solution thereof.

[0086] Pharmaceutical compositions, products, and kits can contain additional active ingredients as well as binding proteins, bispecific conjugates, complexes, or components thereof, for example, they can contain additional therapeutic or prophylactic agents. Thus, the complexes can be used as a monotherapy or as part of a combination therapy, for example, for the treatment of cancer. The kits or combination products described herein can further include instructions for use.

[0087] The binding proteins of the present disclosure, the bispecific conjugates of the present disclosure, the complexes of the present disclosure, the pharmaceutical compositions of the present disclosure, the kits of the present disclosure, and the combination products of the present disclosure can be used for treatment. Therefore, the present disclosure provides binding proteins of the present disclosure, bispecific conjugates, complexes, pharmaceutical compositions, or kits for treatment. Treatment refers to the treatment of a subject. As used herein, "treatment" refers to the treatment of any medical condition. Such treatment can be prophylactic (i.e., preventative), curative (or intended to be curative) or palliative (i.e., a treatment designed only to limit, alleviate, or improve the symptoms of a condition). In curative and palliative applications, the complex or composition is administered to a subject already suffering from a disease or condition in an amount sufficient to cure, alleviate, or partially inhibit one or more of the condition or its symptoms. This treatment method can 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 achieve these is defined as a "therapeutically effective amount." The effective amount for a given purpose will depend on the disease or condition to be treated, its severity, and the size / weight and general condition of the subject.

[0088] Prophylactic treatment can include the prevention of a condition or the delay of the development or onset of a condition. For example, the complex can be used to prevent infection, or reduce the extent to which an infection may develop, or to prevent, delay or reduce the extent to which cancer develops or recurs, or, for example, to prevent or reduce the extent of metastasis.

[0089] A subject as defined herein refers to any mammal, such as a farm animal (e.g., a cow, horse, sheep, pig, or goat), a pet animal (e.g., a rabbit, cat, or dog), or a primate (e.g., a monkey, chimpanzee, gorilla, or human). Most preferably, the subject is a human.

[0090] The combination products of the present disclosure include bispecific conjugates as defined herein and tag constructs as defined herein, as combined preparations for simultaneous or sequential treatment. That is, when the combination products disclosed herein are used according to the present disclosure, i.e., in treatment, the conjugate and the tag construct are administered to the subject simultaneously or sequentially. Similarly, when the kit of the present disclosure is used for treatment, the bispecific conjugate and the tag construct are administered to the subject simultaneously or sequentially. As used herein, "simultaneous" administration means that the two components are administered to the subject at the same time or at least substantially the same time, by the same route of administration and at substantially the same location. As used herein, "sequential" administration refers to administering the two components to the subject at different times. In particular, the administration of the first component is completed before the administration of the second component begins.

[0091] Due to the nature of the disclosure, the sequential administration of the bispecific conjugate and the tag construct requires that both are administered through the same route and at essentially the same location. Furthermore, while the administration of the conjugate and the tag construct can be temporally spaced, the interval between the administrations should be such that the complex is able to form when both components are present after administration. Thus, for example, the two components can be administered within 1 hour of each other, or more particularly within 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes or 1 minute of each other, or less than 1 minute of each other.

[0092] When referring to administering a complex of the disclosure (or a pharmaceutical composition comprising such a complex) to a subject for therapeutic purposes, it is understood to refer to a pre-mixed composition (e.g. solution) comprising both components of the complex (i.e. the bispecific conjugate of the disclosure and the tag construct as described above) which can be present in a dynamic equilibrium comprising the complex and its two individual components.

[0093] In particular, the binding proteins, bispecific conjugates, complexes, compositions, kits or combination products of the disclosure are useful for the treatment or prevention of cancer. Cancer refers to any malignant or precancerous neoplastic condition. Thus, the cancer can be any cancer of any organ, tissue or cell type. Cancers including those manifesting as solid tumors and non-solid tumors are included. Thus, hematopoietic cancers are also included.

[0094] The cancer can 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. The cancer can also be adrenal cancer, bone cancer, brain cancer, esophageal cancer, eye cancer, gastric cancer, oral cancer, penile cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer. Mast cell tumors and angiosarcomas can also be treated according to the disclosure. The cancer can be de novo and untreated, or can be relapsed or refractory, or relapsed and refractory, primary or metastatic.

[0095] When the CD40-specific second portion is present in the bispecific conjugate of the present invention, it activates the immune system by exciting or stimulating CD40 on APCs, in particular on 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 without taking into account CD40 expression by the tumor. Therefore, the bispecific conjugate of this embodiment or a complex containing the conjugate can be effective against both CD40-positive cancers and CD40-negative cancers. Such a bispecific conjugate can also play a role as an adjuvant in vaccination programs against pathogens. If the vaccine platform (e.g., attenuated virus or DNA / RNA-based vaccine) itself is unable to stimulate a sufficient immune response, CD40 activation may be required to stimulate an effective anti-pathogen immune response, thereby generating neutralizing antibodies or T cell responses.

[0096] In embodiments where a CD40-specific second moiety is present, in addition to the agonistic immune activation provided thereby, the disclosed complexes also provide antigens that can be processed and presented to activated T cells, and thus T cells can be primed to target cancer cells or virally infected cells expressing the antigen. This can be particularly beneficial in situations where the amount of cancer antigen present is low or reduced, such as when a tumor has been surgically resected, when anti-CD40 therapy cannot be delivered intratumorally, or when antigen presentation at the tumor site is suboptimal due to inhibitory factors secreted by the tumor. The complexes according to this embodiment provide a means for providing cancer antigens in the vicinity of agonist activation signals, for example, at non-tumor sites, to ensure T cell sensitization and activation, enabling subsequent migration to the tumor and exerting their function.

[0097] In some embodiments, the cancer antigen delivered by the complex can be selected based on the subject and the specific cancer, thereby allowing personalized medicine. For example, a genetic profile of the subject's cancer can be analyzed, thereby allowing the selection of an appropriate antigen. A library of antigens or antigen libraries, or tag constructs containing antigens in the form of cargo domains, can be provided, from which an appropriate tag construct can be prepared or selected based on the subject's cancer type.

[0098] The bispecific conjugates and complexes (and kits and combination products) of the present disclosure may also be used for the treatment or prevention of infection. The present disclosure is particularly useful for the treatment of (e.g., vaccinations) against viral infections, particularly infections caused by RNA viruses. According to the present invention, infections caused by RNA viruses that may be treated or prevented (by vaccination) include infections caused by coronaviruses (e.g., SARS-CoV 1 (the pathogen of SARS), SARS CoV 2 (the pathogen of COVID 19), and MERS-CoV), influenza virus, Ebola virus, hepatitis C virus (HCV), hepatitis E virus (HEV), rabies virus, polio virus, Ross River virus, and measles virus.

[0099] Although in principle any viral infection can be treated or prevented according to the present disclosure, the present disclosure can also be used to treat or vaccinate against infections caused by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesviruses (e.g. HHV6), parvovirus B19 and human papillomavirus (HPV).

[0100] According to the present invention, intracellular bacterial infections can also be treated, such as brucellosis (caused by bacterial species of the genus Brucella), Q fever (caused by Coxiella burnetii), diseases caused by Chlamydia species (e.g., 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)). The present disclosure can also treat intracellular fungal or protozoan infections, including leishmaniasis (caused by trypanosomes of the genus Leishmania) and toxoplasmosis (caused by Toxoplasma gondii). The antigen may therefore be derived from any of the above pathogens.

[0101] For bispecific conjugates comprising an anti-CD40 antibody or fragment thereof, agonism of CD40 can activate the immune system to fight infection (similar in principle to the use for cancer therapy). For the complexes of the disclosure, as described above, it can provide an antigen from a target pathogen, thereby inducing a specific immune response against the pathogen. The vaccine platform is beneficially applicable, for example, in the context of an epidemic. This adaptability can be provided, in particular, by using a tag construct comprising at least one cargo domain that is an antigen that can be modified for viral diversity and antigenic drift. Viral antigens can be selected based on the HLA prevalence in a particular region as well as viral serotype determinants.

[0102] In one embodiment, the bispecific conjugates of the disclosure can comprise an antagonistic anti-CD40 antibody or antigen-binding fragment thereof. In such an embodiment, the bispecific conjugates can be used to tolerize the immune system to a target that drives an autoimmune disease. In one embodiment of the complexes of the disclosure, the complex comprises a bispecific conjugate whose second moiety is an antagonist of CD40 and a tag construct comprising at least one cargo domain that is an antigen known to be a driver of an autoimmune disease, such as one caused by T cells. In this case, the purpose of the complex of the bispecific conjugate with the tag construct comprising at least one cargo domain as an antigen is to retrain the immune system to be tolerant to the antigen that causes the disease, and the complex can thereby prevent over destruction of healthy tissue. According to this embodiment, examples of antigens that can be used in the tag construct are selected from the group consisting of antigens associated with SLE, Type I diabetes, rheumatoid arthritis, vasculitis, myositis, multiple sclerosis, psoriasis, and allergies.

[0103] The at least one cargo domain as 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 recognition by T cells used in adoptive cell therapy of the subject to be treated.

[0104] For example, in adoptive cell therapy, T cells can be obtained from a subject and T cells that recognize a target antigen are isolated. The isolated T cells can then be expanded and / or otherwise treated to stimulate their effector function, and then reinfused into the subject to be treated. In this context, the antigen recognized by the reinfused T cells can be used in the tag construct by being comprised as the at least one cargo domain in the tag construct. The complex of the disclosure can then be administered to the subject such that the antigen activates the reinfused T cells.

[0105] Alternatively, T cells can be obtained from a subject to be treated or a donor and genetically modified to express a TCR that recognizes the target antigen. The genetically modified T cells can then be expanded and / or otherwise processed to stimulate their effector functions 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 the tag construct by being included in the tag construct as at least one cargo domain. The complex of the present disclosure can then be administered to the subject so that the antigen activates the infused T cells. The method of combining the administration of the complex of the present disclosure with adoptive cell therapy is particularly useful in the treatment of cancer, in which case the antigen used in the tag construct becomes a cancer antigen by being included in the tag construct as the at least one cargo domain.

[0106] Therefore, the present disclosure provides a method of treating or preventing cancer, comprising administering to a subject a binding molecule of the present disclosure, a bispecific conjugate of the present disclosure, a complex of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0107] In a specific embodiment, the present disclosure provides a method of treating cancer in a subject, the method comprising:

[0108] (i) obtaining T cells from a subject;

[0109] (ii) isolating T cells that recognize the target cancer antigen, and optionally expanding the isolated T cells;

[0110] (iii) returning the isolated T cells to the subject; and

[0111] (iv) administering the complex of the present disclosure to the subject, wherein the tag construct comprises the target cancer antigen. Equivalently, in step (iv), the bispecific conjugate of the present disclosure and the tag construct comprising the target cancer antigen may alternatively be administered to the subject separately.

[0112] In another embodiment, the present disclosure provides a method of treating cancer in a subject, the method comprising:

[0113] (i) obtaining T cells from a subject or a donor;

[0114] (ii) genetically engineering the T cells to express a TCR that recognizes a target cancer antigen, and optionally expanding the T cells before or after the genetic engineering;

[0115] (iii) infusing the genetically modified T cells into the subject; and

[0116] (iv) administering to the subject a complex of the disclosure, wherein the tag construct comprises the target cancer antigen. Equivalently, in step (iv), the subject can alternatively be administered separately the conjugate of the disclosure and the tag construct comprising the target cancer antigen.

[0117] The disclosure similarly provides the use of a binding molecule of the disclosure, a bispecific conjugate of the disclosure, or a complex of the disclosure in the manufacture of a medicament for the treatment or prevention of cancer.

[0118] Similarly, the disclosure provides a method of treating or preventing an infection, the method comprising: administering to the subject a binding molecule of the disclosure, a bispecific conjugate of the disclosure, a complex of the disclosure, or a pharmaceutical composition of the disclosure.

[0119] The disclosure also provides the use of a binding molecule of the disclosure, a bispecific conjugate of the disclosure, or a complex of the disclosure in the manufacture of a medicament for the treatment or prevention of an infection.

[0120] Further, the disclosure provides a method of treating or preventing an autoimmune disease, the method comprising administering to the subject a binding molecule of the disclosure, a bispecific conjugate of the disclosure, a complex of the disclosure, or a pharmaceutical composition of the disclosure.

[0121] The disclosure also provides the use of a binding molecule of the disclosure, a bispecific conjugate of the disclosure, or a complex of the disclosure in the manufacture of a medicament for the treatment or prevention of an autoimmune disease.

[0122] Throughout the above embodiments, reference to the use of a complex of the disclosure includes the use of a bispecific conjugate of the disclosure in combination with a tag construct, wherein the bispecific conjugate and the tag construct are administered separately or sequentially.

[0123] In alternative embodiments, the complex of the disclosure can be used for gene therapy. In this embodiment, a gene therapy vector or delivery system encoding a bispecific conjugate of the disclosure and a corresponding tag construct can be administered to the subject. Upon uptake by the cells of the subject, the conjugate and the tag construct are expressed and secreted, and the complex is formed in vivo.

[0124] As described above, the binding molecules of the present invention, the bispecific conjugates of the present invention or the complexes of the present invention can be used as a monotherapy or in combination with other therapeutic agents. Thus, in the treatment of cancer, the other therapeutic agents may be: anticancer agents (e.g., chemotherapeutics, a variety of which are known in the art) or immune agents (including, for example, interferons, immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies)); and other immunopotentiators (e.g., anti-OX40 agonist antibodies). Other therapeutic agents may be beneficial for the treatment of cancer or infection, such as antiproliferative or anti-inflammatory cytokines, as well as antiproliferative agents, immunomodulators, or factors that affect blood coagulation, or angiogenesis inhibitors. To treat infection, another (or second) therapeutic agent may be an antimicrobial agent, such as an antibiotic, an antifungal agent, or an antiviral agent.

[0125] One or more of many methods known in the art can be used to administer binding molecules, bispecific conjugates, complexes or pharmaceutical compositions comprising binding molecules, bispecific conjugates or complexes (or their conjugates and label construct components) via one or more routes of administration. Similarly, conjugates and label constructs can be administered separately by these same methods. It will be appreciated by those skilled in the art that routes of administration and / or modes will vary depending on the desired result. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral, such as by injection or infusion, for example, directly to the tumor site.

[0126] As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, typically by injection. Alternatively, non-parenteral routes, such as topical, epidermal, or mucosal routes of administration, may be used. Topical administration is preferred, including peritumoral, proximal, intratumoral, intralesional, perilesional, intracavitary infusion, intrathecal administration, and inhalation. However, the binding molecules, bispecific conjugates, complexes, or compositions may also be administered systemically.

[0127] In the embodiment where the bispecific conjugate and the tag construct are administered separately, i.e., they are not pre-mixed to form a complex, the conjugate and the tag construct must be administered via the same route. Preferably, they are all administered topically at the same (or substantially the same) site, such as intradermal administration, so that the two components mix rapidly after administration and thus combine to form a complex. In these embodiments, the two components must be administered to the subject simultaneously or quickly in succession, avoiding a delay between the administration of the first component and the administration of the second component. This ensures that the second component is administered before the first component degrades or diffuses excessively from the administration site, and enables the complex to form.

[0128] The appropriate dosage of the specific binding molecules, bispecific conjugates or complexes disclosed herein can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical compositions disclosed herein and the products can vary to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response of a specific subject (i.e., patient) and is nontoxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the special complex / conjugate employed, route of administration, time of administration, excretion rate of the complex, duration of treatment, other drugs, compounds and / or materials used in combination with the special composition employed, age, sex, weight, illness, overall health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0129] Suitable dosages of the binding molecules, bispecific conjugates, or complexes of the present disclosure can be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg of the body weight of the patient to be treated. For example, a suitable dosage can be about 0.1 μg / kg to about 10 mg / kg per kilogram of body weight per day or about 10 μg / kg to about 5 mg / kg per kilogram of body weight per day.

[0130] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form to facilitate administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier.

[0131] Binding molecules, bispecific conjugates or complexes (or a combination of conjugates and tag constructs) can be administered in single doses or multiple doses. Multiple doses can be administered via the same or different routes and at the same or different locations. Alternatively, the complex can be administered as a sustained-release formulation, in which case the required administration frequency is lower. Dosage and frequency may depend on the half-life of the administered substance in the patient's body and the duration of the desired treatment. Dosage and frequency may also depend on whether the treatment is preventive or therapeutic. In preventive applications, a relatively low dose can be administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dose can be administered, for example, until the patient shows partial or complete relief of symptoms of the disease. In an exemplary dosage regimen, the complex (or a combination of conjugates and tag constructs) is administered to the subject once a week, once every two weeks, or once every three weeks, and repeated 2 to 10 times is a cycle.

[0132] The combined administration of two or more agents can be achieved in a variety of different ways. In one embodiment, the complex and other agents can be co-administered in a single composition. In another embodiment, the complex and other agents can be administered in the form of independent compositions as part of a combination therapy. For example, the complex can be administered before, after, or simultaneously with other agents. The complex disclosed herein can be combined with tumor-targeted antibodies, targeted therapies, pathway inhibitors, or other immunomodulatory antibodies (e.g., PD-1, PD-L1, CD137, GITR, OX40, CTLA-4, CD27, HVEM, LTβR, and LAG3) or administered sequentially. In addition, the complex can also be used in combination with local radiotherapy. Similarly, when the conjugate and the label construct are administered to the subject separately, this additional therapy can be administered together.

[0133] The present disclosure also provides an in vitro or ex vivo method for activating T cells expressing a TCR that recognizes an antigen, the method comprising contacting an antigen-presenting cell with:

[0134] i) a bispecific conjugate of the present disclosure and a tagging construct as defined above, wherein the tagging construct comprises an antigen recognized by the TCR; or

[0135] ii) The complex of the present disclosure, wherein the tag construct of the complex comprises an antigen recognized by the TCR.

[0136] Thus, the complexes or bispecific conjugates disclosed herein can be used to activate APCs in vitro or ex vivo as well as in vivo. Thus, the complexes and bispecific conjugates (in combination with the tag peptide contained in the tag construct) have medical and non-medical uses, and all such uses are encompassed herein. For example, isolated or cultured APCs can be contacted with the complex, for example, in a laboratory environment, for example, for research, development or testing purposes. This can be achieved by pre-mixing the conjugate and the tag construct to form a complex, and then applying the complex to the APCs. Alternatively, the conjugate and the tag construct can be applied separately to the APCs so that the complex is formed within the APC culture.

[0137] The complex can be used to activate T cells expressing TCRs that recognize antigens contained in the tag construct. Specifically, when the antigen is presented by APC (i.e., in the context of MHC), TCR will recognize the antigen. Therefore, APCs activated by the complex or APCs for activation by the complex can contact T cells. Therefore, for example, APCs can be cultured or incubated in the presence of the complex (or its components), after which APCs can be contacted with T cells, such as co-cultured or further incubated in the presence of T cells. Alternatively, the complex (or its components), APCs and T cells can be incubated or co-cultured together. Therefore, antigens are delivered to APCs and presented to T cells, resulting in T cell activation.

[0138] The present invention is further illustrated by the following non-limiting figures and examples: BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 Shown is a sequence alignment showing the design and construction of humanized versions of mouse scFv IBIIICI as described in Example 1. From top to bottom: VH and VL sequences from wild-type mouse scFv IBIIICI ("VH_Murine": SEQ ID NO: 55; "VL_Murine": SEQ ID NO: 56); selected human germline framework sequences; sequences resulting from grafting mouse scFv CDR:s into human germline frameworks, respectively (VH: SEQ ID NO: 6) and VL (SEQ ID NO: 10).

[0140] Figure 2 Shown is an SDS-PAGE gel of scFv expressed and purified 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).

[0141] Figure 3 shows sensorgrams obtained by affinity measurement of the interaction of immobilized IBIIICI mouse scFv (left; A, C, E; SEQ ID NO: 25) or IBIIICI CDR-grafted scFv (right; B, D, F; SEQ ID NO: 54) with a panel of 14 peptides (A to B: UU0070 to UU0074, SEQ ID NO: 27 to SEQ ID NO: 31; C to D: UU0075 to UU0078, SEQ ID NO: 32 to SEQ ID NO: 35; and E to F: UU0024, UU0080-UU0083, SEQ ID NO: 26, SEQ ID NO: 36 to SEQ ID NO: 39) by SPR as described in Example 3.

[0142] Figure 4 Shown is a sequence alignment of mouse IBIIICI (SEQ ID NO: 55), the original IBIIICI CDR-grafted VH (SEQ ID NO: 6), and five CDR-grafted variant VHs mutated at putative deamidation sites and designated as 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). The boundaries and residue numbering of the CDRs are defined by the IMGT nomenclature (Lefranc et al., 2003, Dev Comp Immunol 27(1): 55-77).

[0143] Figure 5 Shown is an SDS-PAGE gel of the indicated scFv variants expressed and purified as described in Example 4.

[0144] Figure 6 shows sensorgrams obtained from the analysis of the six IBIIICI CDR-grafted scFv variants described in Example 5 when analyzing their interactions with five peptides. The scFv variants analyzed are represented as follows: 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 are: 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).

[0145] Figure 7 shows SPR sensorgrams obtained from the six IBIIICI CDR-grafted scFv variants described in Example 5 when analyzed for interaction with four peptides. The scFv variants analyzed are represented as: 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 are: A) UU0090 (SEQ ID NO: 44); B) UU0091 (SEQ ID NO: 45); C) UU0092 (SEQ ID NO: 46); D) negative control p003 (SEQ ID NO: 48).

[0146] Figure 8 Shown are the results of Western blot experiments performed on the indicated bispecific conjugate constructs SP031 and SP007 as described in Example 6. 2.5 ml of supernatant from expression cultures collected 5 days after transfection were analyzed.

[0147] Figure 9 Shown are the results of biolayer interferometry experiments performed as described in Example 6 for the indicated bispecific conjugate constructs SP031 and SP007.

[0148] Figure 10 This is a survival curve graph showing the survival probability of the three groups of mice shown in the experiment described in Example 7. HD: high dose. LD: low dose. Specific embodiments

[0149] The following examples disclose the development of novel binding molecules IBIIICI CDR-grafted scFvs constructed by grafting the complementarity determining regions (CDR:s) of a known mouse scFv IBIIICI into human germline variable heavy (VH) and variable light (VL) frameworks and introducing additional point mutations. The amino acid sequences of the designed humanized VH and VL regions as described herein are listed in the sequence listing as SEQ ID NO: 1 to SEQ ID NO: 6 (VH sequences) and SEQ ID NO: 10 (VL sequence), and the complete scFv sequences are listed as SEQ ID NO: 49 to SEQ ID NO: 54. The examples further describe the expression and characterization of the scFv polypeptides and variants thereof and bispecific conjugates and complexes comprising them, and demonstrate their surprisingly superior properties in terms of expression and function in bacterial and mammalian expression environments.

[0150] Example 1

[0151] Design and construction of a humanized version of the IBIIICI mouse scFv

[0152] Mangsbo et al. (Mol Immunol 93:115-124, 2018) describe the creation of the mouse scFv IBIIICI, which was further investigated in the context of bispecific conjugates in WO2020 / 104690 and WO2021 / 239968. The complete amino acid sequence of the mouse scFv IBIIICI is listed herein as SEQ ID NO: 25 (SEQ ID NO: 48 in WO2020 / 104690). In the present disclosure, the humanization of this ancestral mouse scFv was performed via grafting of the CDRs into human germline variable heavy and variable light (VH / VL) frameworks, as described below.

[0153] Materials and methods

[0154] Design of IBIIICI CDR-grafted scFvs

[0155] The human germline VH sequences IGHV1 to IGHV46, which share 59.4% sequence homology with the IBIIICI mouse VH (residues 1-104; IMGT / DomainGapAlign), were selected as the VH framework. For the selection of the VL, not only sequence homology but also VH / VL pairings with favorable biophysical properties were considered (Tiller et al, 2013, mAbs 5(3):445–470). Taken together, this led to the selection of human germline genes IGKV1 to IGKV39 for the VL graft. Based on IMGT / DomainGapAlign domain searches, IGHJ4 and IKVJ2 were selected as the connecting segments for the VH and VL, respectively. Construction of the IBIIICI CDR-grafted scFv

[0156] IBIIICI CDR grafts were created by grafting six antigen-binding loops from mouse sequences onto the human IGHV1-46 / IGKV1-39 scaffold. Figure 1 ). A gene encoding VH fused to VL via a glycine-serine linker was designed ((G4S)4; SEQ ID NO: 14). Nucleotides encoding two additional amino acids (R and T, both of which are part of the light chain constant region) were added to the ends of the VL gene to include a BsiWI restriction site. Synthesis and subcloning of scFv genes were performed without codon optimization (GenScript, Piscataway, USA). For the framework region, nucleotide sequences found in human germline genes were used. In CDRs, codons found in original mouse antibodies were used. After synthesis, the scFv gene was cloned into an internal vector using restriction enzymes SfiI and BsiWII so that the scFv carried a triple FLAG tag and a six-histidine tag at the C-terminus when expressed.

[0157] result

[0158] Design of IBIIICI CDR-grafted scFv

[0159] The degree of humanization introduced during the humanization of the IBIIICI mouse scFv was calculated using the T20 score analyzer (Gao et al., 2013, BMC Biotechnology 13:55). Briefly, the T20 score ranges from 0 to 100, with higher scores representing closer humanization. The T20 scores obtained for the original IBIIICI mouse scFv and the IBIIICI CDR-grafted scFv are shown in Table 1.

[0160] Table 1:

[0161] area IBIIICI mouse scFvT20 scoring IBIIICI CDR scFv transplantation T20 score VH, frame + CDR 58.7 77.0 VH, frame only 66.0 90.4 VL, framework + CDR 76.1 86.0 VL, frame only 84.6 99.0

[0162] The T20 scores for both the VH and VL sequences of the humanized IBIIICI CDR-grafted scFv demonstrated significantly improved humanization compared to the T20 scores of their corresponding mouse sequences. Both the IBIIICI CDR-grafted VH and VL frameworks had T20 scores >90, exceeding the standard of a T20 score above 85 to be considered "human-like." The humanization procedure was successful based on a comparison of the T20 scores between the original IBIIICI mouse scFv and the resulting IBIIICI CDR-grafted scFv.

[0163] Construction of IBIIICI CDR-grafted scFv

[0164] After gene synthesis and cloning, 4 μg of vector encoding IBIIICI CDR-grafted scFv was obtained. DNA sequencing verified the sequence accuracy.

[0165] Example 2

[0166] Small-scale expression and purification of IBIIICI mouse scFv and IBIIICI CDR-grafted scFv

[0167] This example describes the small-scale expression and purification of the known IBIIICI mouse scFv (SEQ ID NO: 25) and a newly designed and constructed humanized version, IBIIICI CDR-grafted scFv (SEQ ID NO: 54). Expression and purification were performed and the results were compared to determine whether the humanization process affected the developability of the scFv construct.

[0168] Materials and methods

[0169] The vectors encoding the scFv variants described in Example 1 were transformed into Top10 E. coli, and the expected sequences were confirmed by sequencing.

[0170] The 50ml culture overnight culture containing the corresponding scFv vector was centrifuged and the precipitate was resuspended in B-Per protein extraction buffer (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 scFv protein expressed was eluted with the buffer containing imidazoles, and the sample was subjected to buffer exchange and concentrated to about 0.5ml in PBS. Protein concentration was measured using Implen Nanophotometer, and purity and integrity were verified by SDS-PAGE.

[0171] result

[0172] When evaluated by SDS-PAGE, the IBIIICI CDR-grafted scFv showed satisfactory purity, as evidenced by the presence of a major band consistent with the expected molecular weight of the scFv (approximately 30 kDa; Figure 2 ). Furthermore, 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 lower sample purity and was accompanied by multiple protein bands derived from E. coli. Furthermore, the protein band corresponding to the scFv (approximately 30 kDa) of the IBIIICI mouse scFv was very weak compared to the other bands on the gel, indicating that the concentration of this product was much lower than the measured value (0.2 mg / ml).

[0173] in conclusion

[0174] Small-scale expression experiments demonstrated that the IBIIICI CDR-grafted scFv had a superior expression profile compared to its mouse progenitor.

[0175] Example 3

[0176] Binding properties of mouse IBIIICI scFv and IBIIICI CDR-grafted scFv using surface plasmon resonance Taking measurements

[0177] The binding kinetics of the novel IBIIICI CDR-grafted scFv were compared with the binding kinetics of the IBIIICI mouse scFv to a panel of peptides using surface plasmon resonance (SPR).

[0178] Materials and methods

[0179] Affinity measurements by SPR were performed on the IBIIICI mouse scFv and IBIIICI CDR-grafted scFv variants obtained as described in examples 1 to 2 using a Biacore T200 instrument (GE Healthcare) and single-cycle kinetics. According to the manufacturer's instructions, the FLAG M2 antibody (Sigma-Aldrich) was immobilized on a Series S CM5 chip by primary amine coupling using NHS-EDC chemistry, allowing the capture of the respective scFv by the FLAG tag provided by the expression vector. A 5-fold dilution series consisting of five concentrations (0.16 nM to 100 nM) of different peptides (Table 2) was sequentially injected into the flow cell, allowing the binding to the captured scFv. After a dissociation phase, regeneration of the surface was accomplished using 10 mM glycine-HCl, pH 2.1, under acidic conditions. Response unit sensorgrams for all peptides were obtained by subtracting the response curve of the anti-FLAG antibody-immobilized surface (reference surface). Data were analyzed using BIAeval v.3.1 (GE Healthcare).

[0180] Table 2: A set of peptides used in Examples 3 and 5, where the tag portion is in bold

[0181]

[0182]

[0183] result

[0184] Results are shown in Figure 3. Overall, the binding characteristics of the IBIIICI mouse scFv (Figure 3A, 3C and 3E) and the humanized IBIIICI CDR-grafted scFv (Figure 3B, 3D and 3F) were very similar, with low nanomolar binding affinities for most of the target peptides in the tested panel, indicating the success of humanization.

[0185] Example 4

[0186] Generation of five IBIIICI CDR-grafted scFv variants by point mutagenesis

[0187] There is a potential deamidation site at position 62 to 63 of CDR-H2 of the IBIIICI VH sequence. In order to make mutations at this site that do not negatively affect the binding characteristics or other important biophysical parameters of the humanized IBIIICI scFv, five point mutation variants were created.

[0188] Materials and methods

[0189] Design and construction of five IBIIICI CDR-grafted scFv variants

[0190] Five different versions of CDR-grafted VH sequences with mutated deamidation sites were designed. The diversity of the natural antibody repertoire found at these positions was taken into account (Kirik et al. 2017, Front Immunol 8: 1433; Persson et al. 2018, Front Immunol 9: 1391). Thereafter, conservative substitutions were performed, i.e., amino acids were replaced with different amino acids with similar biochemical properties. The VH sequence alignment of the original mouse scFv, the humanized scFv described in Examples 1 to 3, and the five point mutation variants is shown in Figure 3. The amino acid sequences of the corresponding VH regions of the five point mutation variants and the original humanized variants are represented by SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence table. The complete scFv sequences of the five point mutation variants are represented as: 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) is represented as "IB CDR-grafted scFv NG" or "IB CDR-grafted scFv WT" and contains the original amino acids NG at positions 62 to 63 of CDR-H2 of the VH sequence ( FIG. 3 ).

[0191] Genes encoding scFv variants were constructed by introducing point mutations into the IBIIICI CDR-grafted scFv vector designed as described in Example 2. This vector provides a triple FLAG tag and a hexahistidine tag at the C-terminus for scFv cloning.

[0192] Small-scale expression and purification

[0193] Vectors encoding the designed scFv constructs were transformed into Top10 E. coli, and the expected sequences were confirmed by sequencing.

[0194] Six 50 ml cultures of the different humanized scFv variants and the original IBIIICI mouse 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 clear supernatant. After incubation, the mixture was transferred to empty gravity flow columns. After washing, the polypeptides were eluted with imidazole containing buffer and the samples were buffer exchanged and concentrated to about 0.4 ml in PBS. Protein concentration was determined using an Implen Nanophotometer and purity and integrity were verified by SDS-PAGE.

[0195] result

[0196] Bacterial expression levels were relatively high and very similar between the five different point mutant IBIIICI CDR grafted scFv variants and the IBIIICI CDR grafted WT scFv (“variant NG”). This is in contrast to the IBIIICI mouse scFv, which showed a relatively low expression (as shown previously in Example 2).

[0197] SDS-PAGE gels are shown in Figure 5 The IBIIICI mouse scFv showed a lower sample purity with multiple E. coli derived protein bands. In addition, the protein band in the IBIIICI mouse scFv lane corresponding to the size of the scFv (about 30 kDa) was very weak compared to the other bands on the gel, indicating that the concentration of this clone was much lower. All point mutant variants showed a satisfactory purity as evidenced by one major band corresponding to the expected molecular weight of the scFv (about 30 kDa). In addition, the protein concentrations measured for these samples were relatively high (2.3 mg / ml to 3.3 mg / ml). The expression and purification of the variants was not affected compared to the IBIIICI CDR grafted WT scFv (“variant NG”), thus the creation of the point mutant variants was considered successful in terms of expression and purification.

[0198] Example 5

[0199] Biophysical characterization of IBIIICI CDR-grafted scFv variants

[0200] The five IBIIICI CDR grafted scFv point mutant variants created as described in Example 4 and the IBIIICI CDR grafted WT scFv as described in Examples 1 to 3 were characterized using three different biophysical characterization methods: SPR, nanoDSF and size exclusion chromatography (SEC).

[0201] Materials and methods

[0202] Surface plasmon resonance

[0203] Affinity measurements by SPR were performed on IBIIICICDR grafted WT scFv and IBIIICICDR grafted scFv point mutant variants 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 the capture of the corresponding scFv through its FLAG tag. A five-fold dilution series consisting of a panel of five concentrations (0.16 nM to 100 nM) of the peptides tested (Table 2) was sequentially injected into the flow cell, allowing the binding to the respective captured scFv. After the dissociation phase, surface regeneration was performed under acidic conditions using 10 mM glycine-HCl at pH 2.1. Response unit sensorgrams were obtained for all peptides by subtracting the response curve of the reference surface with immobilized anti-FLAG antibody. Data analysis was performed using BIAeval v.3.1 (GE Healthcare).

[0204] Determination of the melting temperature (Tm) by nanoDSF

[0205] To evaluate the stability of the purified scFv variants, the melting temperature was determined by nanoDSF using a Prometheus NT.48 instrument (NanoTemper Inc). The nanoDSF technology measures the intrinsic fluorescence of proteins during thermal denaturation, allowing the characterization of protein unfolding under native conditions.

[0206] Six purified IBIIICICDR grafted scFv variants were diluted to 1.0 mg / ml in PBS and loaded by capillary force into high sensitivity capillaries (NanoTemper, #PR-C006). The melting temperature gradient was set between 20°C and 95°C, heating at 1°C / min. Tryptophan emission fluorescence was measured at 330 nm and 350 nm and the calculated fluorescence ratio was plotted against temperature to obtain the melting curve for each variant. The Tm value was deduced using the software PR ThermoControl (NanoTemper Inc).

[0207] Stability studies and SEC

[0208] To further evaluate any differences in stability between variants, stability evaluations were performed. Six purified IBIIICI CDR-grafted scFv variants were analyzed by SEC HPLC using a BioSEC-3 3 μm 300A column (Agilent). After incubation at 37°C, samples were analyzed at two time points: t = 0 h and t = 24 h.

[0209] result

[0210] surface plasmon resonance

[0211] The results are shown in Figures 6 and 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 binding properties of 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 to 6E The binding sensorgrams of different IBIIICI CDR-grafted scFv variants to peptide UU0024, peptide UU0084, peptide UU0085, peptide UU0086 and peptide UU0087 are shown, and 7A to 7C The corresponding binding to peptides UU0090, UU0091 and UU0092 is shown (see Table 2 for peptide sequences). None of the variants showed any binding to the negative control peptide p003 (Figure 7). NanoDSF measurements

[0212] The measured Tm values ​​(inflection point #1) are shown in Table 3 and are between 60°C and 65°C. The measured Tm values ​​can be used as an indicator of protein stability. The data show that variant IBIIICI CDR-grafted scFv SG (64°C) has slightly higher stability than the other scFv variants analyzed. In contrast, variant IBIIICI CDR-grafted scFv NA has the lowest Tm value in the group (61°C).

[0213] Table 3: Melting temperature (Tm) values ​​obtained by nanoDSF

[0214] scFv SEQ ID NO: Temperature (℃) IBIIICI CDR-grafted scFv variant DG 52 62.4 IBIIICI CDR-grafted scFv variant GG 50 62.6 IBIIICI CDR-grafted scFv variant QG 51 62.2 IBIIICI CDR-grafted scFv variant SG 49 64.3 IBIIICI CDR-grafted scFv variant NA 53 60.8 IBIIICI CDR-grafted scFv variant NG (wt) 54 63.4

[0215] SEC HPLC analysis

[0216] To investigate the integrity and stability of the CDR-grafted variants, six IBIIICI CDR-grafted scFv variants were analyzed by SECHPLC before and after 24 hours of incubation at 37° C. After 24 hours, no detectable change in monomer content was observed in any of the IBIIICI CDR-grafted scFvs compared to the starting sample.

[0217] in conclusion

[0218] The point mutations introduced into CDR-H2 had little effect on the binding of the scFv variants to the peptides tested or on the stability of the scFv variants. All five CDR-grafted variants designed to remove potential deamidation sites showed very similar binding patterns to the first IBIIICI CDR-grafted scFv (variant "NG") and the original IBIIICI mouse scFv.

[0219] Example 6

[0220] Construction, expression, and characterization of bispecific conjugates of murine and humanized IBIIICI scFv with full-length antibodies

[0221] In this example, the humanized scFv according to the present disclosure is placed in the context of the bispecific conjugates described in WO2020 / 104690 and WO2021 / 239968, i.e., 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, such as a full-length antibody against CD40. For a schematic diagram of the construct design, see WO2020 / 104690. Figure 6A The anti-CD40 antibody used in this example is the antibody designated "A9" disclosed in WO2021 / 239968. Expression and purification were performed in the context of the IBIIICI mouse scFv and two humanized versions (IBIIICI CDR-grafted scFv NG and IBIIICI CDR-grafted scFv SG), and the results were compared.

[0222] Materials and methods

[0223] Expression of murine and humanized scFv-A9 conjugates

[0224] According to the manufacturer's instructions, the TM Four bispecific binding constructs (Table 4) were expressed in 100 μL 100 μL 100 μL 100 μL 200 μL 400 μL 400 μL 2 ...

[0225] The conjugates were then transfected and expressed in cultures expanded to 25 ml, incubated at 37°C, 5% CO2, humidified air, and 120 rpm, and harvested 7 days after transfection for affinity purification.

[0226] Western blotting

[0227] A total of 20 μl of supernatant harvested on day 5 after transfection was mixed with 3× loading buffer (0.1 M Tris-HCl, 45% glycerol, 0.03% bromophenol blue, 0.3% SDS) under non-reducing conditions. The samples were washed with 4% to 20% Criterion TM TGX Stain-Free TM Electrophoresis was performed on a protein gel (Bio-Rad Laboratories, #5678095). The gel was transferred to a PVDF membrane (Bio-Rad Transfer-Blot Turbo Transfer System) for Western blotting analysis. The membrane was incubated in 5% TBST milk buffer for 1 hour, followed by incubation with goat anti-human HRP antibody (ThermoFisher) for 1 hour. The membrane was washed three times with TBST for 5 minutes each before incubation. Chemiluminescent substrate (Merck) was then added and Western blot analysis was performed using a Chemidoc XRS+ system (BioRad).

[0228] Biolayer Interferometry

[0229] According to the manufacturer's instructions, use RED96e System and Dip and Read TM Titers were measured in supernatants 5 days after transfection using a Protein A biosensor (Fortébio Biologics, a Molecular Devices company, #10819702) using biolayer interferometry. Supernatants were diluted 1:1 with equilibration buffer containing 20 mM citric acid (pH 4.0), 0.1% BSA (w / v), 0.1% Tween-20, and 0.5 M NaCl to a final volume of 200 μl in a 96-well black plate. A standard curve was prepared using IgG (IgG1 Protein A Standard, Sartorius #18-1118) with concentrations ranging from 1 μg / ml to 700 μg / ml.

[0230] Table 4: List of expressed bispecific constructs

[0231]

[0232] Purification of murine and humanized scFv-A9 conjugates

[0233] The conjugate was expressed by Protein A affinity chromatography on the system was performed using a mAbSelectSuRe column (GE Healthcare, #11003493) for purification. 20 mM sodium phosphate, 0.15 M sodium chloride (pH 7.3) buffer was used as the binding and wash buffers, 0.1 M glycine (pH 2.5) was used as the elution buffer, and 1 M Tris-HCl (pH 8.5) was used as the neutralization buffer.

[0234] Purity measurement of conjugates using size exclusion chromatography

[0235] A total of 25 μg of the corresponding conjugate in 100 μl was injected onto a Superdex Increase 200 10 / 30 GL gel filtration column (GE Healthcare, #28989336) coupled 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 absorption measurements at 280 nm. Data analysis and peak integration were performed using GraphPad Prism 8.0.

[0236] result

[0237] Western blotting and biolayer interferometry of expressed conjugates

[0238] Figure 8 The results of Western blotting of 2.5 ml cultures of SP007 and SP031 are shown. The intensity of SP007 is higher than that of SP031. Overall, Western blotting shows that the titer of construct SP007 containing humanized scFv is higher than that of construct SP031 containing mouse scFv.

[0239] Figure 9 The results of biolayer interferometry titer measurement of supernatants 5 days after transfection are shown. The titer of SP007 was approximately 3-fold higher than that of SP031, which is consistent with the results observed by Western blotting.

[0240] Purity measurement of the conjugate using size exclusion chromatography

[0241] Expression and purification of SP031 conjugate resulted in a significantly lower population of monomer than the quality control cutoff set at 95% monomer content as determined by SEC. Analysis of the chromatogram for SP031 after affinity purification showed a monomer content of 85.3% with a distinct peak representing a population of smaller, non-native low molecular species, accounting for 14.7% of the total sample. In comparison to the ancestral mouse version, all other conjugates tested containing humanized scFv:s (SP007, SP019, and SP027) showed superior SEC characteristics after purification (Table 5).

[0242] Table 5: Monomer content of conjugates

[0243] structure Monomer content (%) SP031 85.3 SP007 95.0 SP019 99.2 SP027 95.6

[0244] in conclusion

[0245] Constructs containing mouse scFv IBIIICI (variant SP031) exhibited significantly lower expression levels compared to constructs containing humanized scFv:s. In addition, the SEC profile of SP031 showed a considerable amount of non-anticipated species with a total monomer population of 85.3%. From a developability perspective, all conjugates containing humanized scFv:s were observed to have improved profiles: SP007, SP019, and SP027 observed >95% monomer population in SEC results. Therefore, from a developability perspective, conjugates containing humanized scFv:s and anti-CD40 antibodies are considered superior compared to conjugates containing non-humanized scFv:s.

[0246] Example 7

[0247] The efficacy of tumor treatment

[0248] The anti-tumor response induced by repeated vaccination of peptides in combination with bispecific binding conjugate SP027 in the TC-1 tumor model was investigated. By local vaccination at non-tumor sites, the tag construct containing the antigen associated with the tumor (viral or neoantigenic origin) is delivered to dendritic cells and internalized for release of the antigenic peptide by the bispecific conjugate binding to CD40. The antigenic peptide is processed and presented to T cells. By repeated injections, the antigen-specific T cells expand, migrate, and target tumor cells. The anti-tumor effect of the complex of bispecific conjugate and tag construct was compared to peptide alone and / or bispecific conjugate alone, and also injected at non-tumor sites.

[0249] Materials and methods

[0250] Mice (strain B-hCD40 (Biocytogen) (9-12 weeks)) were divided into the following three treatment groups (6-7 mice per group): Group 1: high dose of 30 μg antigenic peptide UU0171 (SEQ ID NO: 77; immunogenic E7-derived synthetic long peptide HPV16 E7 [HPV16 E7 44 to HPV16 E7 62]); Group 2: bispecific binding conjugate SP027 alone, with an initial injection of 50 μg on day 5 and 30 μg on days 10 and 15; Group 3: bispecific binding conjugate SP027 (50 μg on day 5 and 30 μg on days 10 and 15) complexed with tag construct UU0169 (SEQ ID NO: 78), which contains a 9 amino acid tag portion linked to an HPV peptide and is administered at a low dose of 3 μg in the complex mixture at each dose. On day 0, approximately 5 × 10 5 The mice were treated with subcutaneous injections of 1000 tumor cells into the left hind footpad on days 5, 10, and 15, i.e., treatment was not given at the site of tumor growth. Tumor growth and survival were monitored three times a week. When the tumor size reached a maximum of 1000 mm, the mice were treated with 10000 mm 3 Mice were sacrificed at the experimental endpoint or if a humane endpoint (e.g., wound, body weight, and cosmetic health) was reached. Mice that did not reach the experimental endpoint but reached a humane endpoint were censored.

[0251] result

[0252] The results are as follows Figure 10 Even when injected at low doses at non-tumor sites, the TC-1 peptide derived from the oncogenic HPV protein E7, coupled to a 9-mer tag moiety (UU0169) and delivered in complex with the bispecific binding construct SP027, induced a potent immune response and significantly reduced tumor volume compared to controls given only the bispecific conjugate SP027 or a high dose of the antigenic peptide without the tag moiety (UU0171).

[0253] Informal Sequence Listing

[0254] SEQ ID NO: 1 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS

[0255] SEQ ID NO:2

[0256] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEGGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS

[0257] SE Q I D NO:3

[0258] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS

[0259] SEQ ID NO:4

[0260] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEDGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS

[0261] SE Q I D NO:5

[0262] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS

[0263] SEQ ID NO:6

[0264] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS

[0265] SE Q I D NO:7

[0266] FNIKDFNI

[0267] SE Q I D NO:8)

[0268] IGRIDPEX a X b DAEYVP

[0269] SE Q I D NO:9

[0270] TTGSYDLDVE

[0271] SE Q I D NO:10

[0272] DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0273] SEQ ID NO:11

[0274] HASQNINVWLS

[0275] SEQ ID NO:12

[0276] KASTLHT

[0277] SEQ ID NO:13

[0278] QQGQSYPLT

[0279] SE Q I D NO:14

[0280] GGGGSGGGGSGGGGSGGGGS

[0281] SE Q I D NO:15

[0282] QSISSY

[0283] SE Q I D NO:16

[0284] AAS

[0285] SE Q I D NO:17

[0286] QQGYPYPFT

[0287] SE Q I D NO:18

[0288] GFTFSSYA

[0289] SE Q I D NO:19

[0290] ISGYSGST

[0291] SE Q I D NO:20

[0292] ARYYSYYGYYYFDY

[0293] SE Q I D NO:21

[0294] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK

[0295] SEQ ID NO:22

[0296] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSS

[0297] SE Q I D NO:23

[0298] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0299] SEQ ID NO:24EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCARYYSYYGYYYFDYW GQGTLVTVSSASTKGPSVFPPLAPCSRSTSTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSNFGTQTYTCNVDHKPSNTKVDKTVVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNACTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLSPGK

[0300] SEQ ID NO:25

[0301] EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSSGGGG SGGGGSGGGGGGGSELQMTQSPSSLSASLGDTVTITCHASQNVWLSWYQQRPGNIPKLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLEK

[0302] SE QI D NO:26

[0303] FIGITELKKLESKINKVFC-Biotin

[0304] SE QI D NO:27

[0305] FIGITELKKLESKINK

[0306] SEQ I D NO:28

[0307] FIGITELKKLESKIN

[0308] SE Q I D NO:29

[0309] FIGITELKKLESKI

[0310] SE Q I D NO:30

[0311] FIGITELKKLESK

[0312] SE Q I D NO:31

[0313] FIGITELKKLES

[0314] SE Q I D NO:32

[0315] LEQLESIINFEKLLAAAAAKFIGITELKKLES

[0316] SE Q I D NO:33

[0317] FIGITELKKLESLEQLESIINFEKLLAAAAAK

[0318] SEQ ID NO:34

[0319] Biotin-(PEG3)FIGITELKKLES

[0320] SEQ ID NO:35

[0321] FIGITELKKLES(PEG3)K-Biotin

[0322] SEQ ID NO:36

[0323] FIGITELHHLESK-Biotin

[0324] SEQ ID NO:37

[0325] FIGISELKKLESK-Biotin

[0326] SEQ ID NO:38

[0327] FIGITELHKLESK-Biotin

[0328] SEQ ID NO:39

[0329] FIGITELKHLESK-Biotin

[0330] SEQ ID NO:40

[0331] FIGITELKKLE

[0332] SEQ ID NO:41

[0333] FIGITELKKL

[0334] SEQ ID NO:42

[0335] FIGITELKK

[0336] SEQ ID NO:43

[0337] FIGITELK

[0338] SEQ ID NO:44

[0339] FIGITELLEQLESIINFEKLAAAAAAK

[0340] SEQ ID NO:45

[0341] FIGITELKLEQLESIINFEKLAAAAK

[0342] SEQ ID NO:46

[0343] FIGITELHLEQLESIINFEKLAAAAK

[0344] SEQ ID NO:47

[0345] ASKFIGITELK

[0346] SEQ ID NO:48

[0347] Biotin-IDIKNDLYEKTLNDYKAIANKLSQV

[0348] SEQ ID NO:49

[0349] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0350] SEQ ID NO:50

[0351] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEGGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0352] SEQ ID NO:51

[0353] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0354] SEQ ID NO:52

[0355] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEDGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0356] SEQ ID NO:53

[0357] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0358] SEQ ID NO:54

[0359] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0360] SEQ ID NO:55

[0361] EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSS

[0362] SEQ ID NO:56

[0363] ELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK

[0364] SEQ ID NO:57

[0365] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSEVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK

[0366] SEQ ID NO:58

[0367] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0368] SEQ ID NO:59

[0369] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0370] SEQ ID NO:60

[0371] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0372] SEQ ID NO:61

[0373] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0374] SEQ ID NO:62

[0375] IGRIDPESGDAEYVP

[0376] SEQ ID NO:63

[0377] IGRIDPEGGDAEYVP

[0378] SEQ ID NO:64

[0379] IGRIDPEQGDAEYVP

[0380] SEQ ID NO:65

[0381] IGRIDPEDGDAEYVP

[0382] SEQ ID NO:66

[0383] IGRIDPENADAEYVP

[0384] SEQ ID NO:67

[0385] IGRIDPENGDAEYVP

[0386] SEQ ID NO:68

[0387] QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPSDNFSNLNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLTVSS

[0388] SEQ ID NO:69

[0389] SDIVMTQATPSVLVTPGEAVSISCRASRSLLHSNGITYLYWFLQRPGQSPQVLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEFPYTFGGGTKLEIK

[0390] SEQ ID NO:70

[0391] GGGGS

[0392] SEQ ID NO:71

[0393] GGGGSGGGGS

[0394] SEQ ID NO:72

[0395] GGGGSGGGGSGGGGS

[0396] SEQ ID NO:73

[0397] GGGGSGGGGSGGGGSGGGGSGGGGS

[0398] SEQ ID NO:74

[0399] FIGITELL

[0400] SEQ ID NO:75

[0401] FIGITELH

[0402] SEQ ID NO:76

[0403] FIGISELK

[0404] SEQ ID NO:77

[0405] QAEPDRAHYNIVTFCCKCD

[0406] SEQ ID NO:78

[0407] FIGITELKKQAEPDRAHYNIVTFCCKCD

[0408] SEQ ID NO:79

[0409] FIGITELLK

[0410] SEQ ID NO:80

[0411] FIGITELHK

[0412] 实施方式的详细列表

[0413] 1. A binding molecule comprising:

[0414] • an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 and an amino acid sequence having at least 90% identity to SEQ ID NO: 1 to SEQ ID NO: 6,

[0415] with the proviso that the heavy chain variable region (VH) comprises three complementarity determining regions (CDRs), wherein:

[0416] - VHCDR1 has a sequence as shown in SEQ ID NO: 7;

[0417] - 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; and

[0418] - VHCDR3 has a sequence as shown in SEQ ID NO: 9; and

[0419] • an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and an amino acid sequence having at least 90% identity to SEQ ID NO: 10,

[0420] with the proviso that the light chain variable region (VL) comprises three complementarity determining regions (CDRs), wherein:

[0421] - VLCDR1 has a sequence as shown in SEQ ID NO: 11 ;

[0422] - VLCDR2 has a sequence as shown in SEQ ID NO: 12; and

[0423] - VLCDR3 has a sequence as shown in SEQ ID NO: 13.

[0424] 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 NO: 2 to SEQ ID NO: 6 and an amino acid sequence having at least 90% identity to SEQ ID NO: 2 to SEQ ID NO: 6.

[0425] 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 NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and an amino acid sequence having at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0426] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0427] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6.

[0428] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6.

[0429] 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 NO: 1 to SEQ ID NO: 5 and amino acid sequences that are at least 90% identical to SEQ ID NO: 1 to SEQ ID NO: 5.

[0430] 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 NO: 3 to SEQ ID NO: 6 and amino acid sequences that are at least 90% identical to SEQ ID NO: 3 to SEQ ID NO: 6.

[0431] 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 NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0432] 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 NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6.

[0433] 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 NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6.

[0434] 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 NO: 2 to SEQ ID NO: 5 and amino acid sequences that are at least 90% identical to SEQ ID NO: 2 to SEQ ID NO: 5.

[0435] 13. The binding molecule according to 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 NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0436] 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 NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6.

[0437] 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 NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6.

[0438] 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 NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0439] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6.

[0440] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

[0441] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

[0442] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6.

[0443] 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 NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5.

[0444] 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 NO: 1 to SEQ ID NO: 4 and amino acid sequences that are at least 90% identical to SEQ ID NO: 1 to SEQ ID NO: 4.

[0445] 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 NO: 4 to SEQ ID NO: 6 and amino acid sequences that are at least 90% identical to SEQ ID NO: 4 to SEQ ID NO: 6.

[0446] 24. The binding molecule according to 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 NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6.

[0447] 25. The binding molecule according to 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 NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6.

[0448] 26. The binding molecule according to 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 NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0449] 27. The binding molecule according to 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 NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6.

[0450] 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 NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

[0451] 29. The binding molecule according to 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 NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

[0452] 30. The binding molecule of 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 NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6.

[0453] 31. The binding molecule according to 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 NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5.

[0454] 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 NO: 2 to SEQ ID NO: 4 and amino acid sequences that are at least 90% identical to SEQ ID NO: 2 to SEQ ID NO: 4.

[0455] 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 NO: 1, SEQ ID NO: 5 and SEQ ID NO: 6 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 6.

[0456] 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 NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6.

[0457] 35. The binding molecule of 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 NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5.

[0458] 36. The binding molecule of 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 NO: 1, SEQ ID NO: 3 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 6.

[0459] 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 NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5.

[0460] 38. The binding molecule of 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 NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4.

[0461] 39. The binding molecule of any one of claims 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 NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6, and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6.

[0462] 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 NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5 and an amino acid sequence that has at least 90% identity with SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5.

[0463] 41. The binding molecule of 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 NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4.

[0464] 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 NO: 1 to SEQ ID NO: 3 and amino acid sequences that are at least 90% identical to SEQ ID NO: 1 to SEQ ID NO: 3.

[0465] 43. The binding molecule of 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 NO: 5 to SEQ ID NO: 6 and amino acid sequences that are at least 90% identical to SEQ ID NO: 5 to SEQ ID NO: 6.

[0466] 44. The binding molecule of 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 NO: 4 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 4 and SEQ ID NO: 6.

[0467] 45. The binding molecule of 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 NO: 4 to SEQ ID NO: 5 and amino acid sequences that are at least 90% identical to SEQ ID NO: 4 to SEQ ID NO: 5.

[0468] 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 NO: 3 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and SEQ ID NO: 6.

[0469] 47. The binding molecule of 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 NO: 3 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and SEQ ID NO: 5.

[0470] 48. The binding molecule of 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 NO: 3 to SEQ ID NO: 4 and amino acid sequences that are at least 90% identical to SEQ ID NO: 3 to SEQ ID NO: 4.

[0471] 49. The binding molecule of 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 NO: 2 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 and SEQ ID NO: 6.

[0472] 50. The binding molecule of 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 NO: 2 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 and SEQ ID NO: 5.

[0473] 51. The binding molecule of 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 NO: 2 and SEQ ID NO: 4 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 and SEQ ID NO: 4.

[0474] 52. The binding molecule of 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 NO: 2 to SEQ ID NO: 3 and amino acid sequences that are at least 90% identical to SEQ ID NO: 2 to SEQ ID NO: 3.

[0475] 53. The binding molecule of 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 NO: 1 and SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and SEQ ID NO: 6.

[0476] 54. The binding molecule of 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 NO: 1 and SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and SEQ ID NO: 5.

[0477] 55. The binding molecule of 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 NO: 1 and SEQ ID NO: 4 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and SEQ ID NO: 4.

[0478] 56. The binding molecule of 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 NO: 1 and SEQ ID NO: 3 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and SEQ ID NO: 3.

[0479] 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 NO: 1 to SEQ ID NO: 2 and amino acid sequences that are at least 90% identical to SEQ ID NO: 1 to SEQ ID NO: 2.

[0480] 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 of SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 6.

[0481] 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 of SEQ ID NO: 5 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 5.

[0482] 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 of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity to SEQ ID NO: 4.

[0483] 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 of SEQ ID NO: 3 and an amino acid sequence having at least 90% identity to SEQ ID NO: 3.

[0484] 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 of SEQ ID NO: 2 and an amino acid sequence having at least 90% identity to SEQ ID NO: 2.

[0485] 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 of SEQ ID NO: 1 and an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

[0486] 64. The binding molecule according to any preceding item, wherein the binding molecule is an antibody construct.

[0487] 65. The binding molecule according to any preceding item, wherein the binding molecule is selected from the group consisting of an antibody and an antigen-binding fragment thereof, such as a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, an Fc fragment, a Fv fragment, a single-chain (scFv) fragment, a (scFv)2, and a domain antibody.

[0488] 66. The binding molecule according to any preceding item, wherein the binding molecule is a scFv.

[0489] 67. The binding molecule according to any preceding item, wherein the binding molecule further 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).

[0490] 68. The binding molecule according to item 67, wherein the peptide linker consists of the amino acid sequence of SEQ ID NO: 14.

[0491] 69. The binding molecule according to any preceding item, wherein the binding molecule is capable of selectively binding to a peptide tag moiety comprised in a tag construct.

[0492] 70. The binding molecule according to item 69, wherein the tag construct further comprises at least one cargo domain.

[0493] 71. The binding molecule according to item 70, wherein said at least one cargo domain is an antigenic portion.

[0494] 72. The binding molecule according to any one of items 70 to 71, wherein the at least one cargo domain is a peptide portion or a nucleic acid portion.

[0495] 73. The binding molecule of item 72, wherein the C-terminus of the tag portion is covalently bound to the N-terminus of the at least one peptide cargo domain.

[0496] 74. The binding molecule of item 72, wherein the N-terminus of the tag portion is covalently bound to the C-terminus of the at least one peptide cargo domain.

[0497] 75. The binding molecule according to any one of items 71 to 74, wherein the at least one cargo domain is a peptide antigen portion comprising the amino acid sequence of a target antigen.

[0498] 76. The binding molecule according to any one of items 69 to 75, wherein the binding molecule is capable of binding to the tag portion such that the interacting K D The value is up to 5×10 -9 M, for example, up to 1×10 -10 M, for example, up to 1×10 -11 M.

[0499] 77. A bispecific conjugate comprising:

[0500] - at least one first moiety, said first moiety being a binding molecule according to any of the preceding items; and

[0501] - at least one second moiety, said second moiety being an antibody or an antigen-binding fragment thereof.

[0502] 78. The bispecific conjugate of claim 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, (scFv)2, and a domain antibody.

[0503] 79. The bispecific conjugate according to claim 77, wherein the second part is an antibody, optionally an antibody of the IgG2 subtype.

[0504] 80. The bispecific conjugate according to any one of items 77 to 79, wherein the first part is covalently linked to the second part.

[0505] 81. The bispecific conjugate of claim 80, wherein the first portion is covalently linked to the second portion via a linker peptide.

[0506] 82. The bispecific conjugate of claim 80, wherein the first portion is directly linked to the second portion.

[0507] 83. The bispecific conjugate of any one of items 80 to 82, wherein the first part is covalently linked to the C-terminus of the light chain of the second part.

[0508] 84. The bispecific conjugate of any one of items 80 to 82, wherein the first part is covalently linked to the N-terminus of the light chain of the second part.

[0509] 85. The bispecific conjugate of any one of items 80 to 82, wherein the first part is covalently linked to the C-terminus of the heavy chain of the second part.

[0510] 86. The bispecific conjugate of any one of items 80 to 82, wherein the first part is covalently linked to the N-terminus of the heavy chain of the second part.

[0511] 87. The bispecific conjugate according to any one of items 77 to 86, wherein the conjugate comprises two first parts and an antibody as a second part, and one first part is conjugated to the CH3 domain of each heavy chain of the second part antibody.

[0512] 88. The bispecific conjugate of any one of items 77 to 86, wherein the conjugate comprises two first parts and an antibody as a second part, and one first part is conjugated to the CL domain of each light chain of the second part antibody.

[0513] 89. The bispecific conjugate according to any one of items 77 to 88, wherein the second part is an anti-CD40 antibody or an antigen-binding fragment thereof.

[0514] 90. The bispecific conjugate of claim 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 an antibody comprising an antigen-binding fragment derived from one or more of said antibodies.

[0515] 91. The bispecific conjugate of claim 89, wherein the anti-CD40 antibody or antigen-binding fragment thereof comprises six complementarity determining domains (CDRs), wherein:

[0516] VLCDR1 has the sequence shown in SEQ ID NO: 15;

[0517] VLCDR2 has the sequence shown in SEQ ID NO: 16;

[0518] VLCDR3 has the sequence shown in SEQ ID NO: 17;

[0519] VHCDR1 has the sequence shown in SEQ ID NO: 18;

[0520] VHCDR2 has the sequence shown in SEQ ID NO: 19; and

[0521] VHCDR3 has the sequence shown in SEQ ID NO:20.

[0522] 92. The bispecific conjugate of claim 91, wherein:

[0523] The light chain variable region of the anti-CD40 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21; and

[0524] The heavy chain variable region of the anti-CD40 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22.

[0525] 93. The bispecific conjugate of claim 92, wherein the anti-CD40 antibody comprises:

[0526] a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23; and

[0527] A heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24.

[0528] 94. A composite comprising:

[0529] - the bispecific conjugate according to any one of items 77 to 93; and

[0530] - A tagging construct comprising a peptide tag portion.

[0531] 95. The complex according to item 94, wherein the tag construct further comprises a cargo domain, wherein the cargo domain is a peptide antigen portion comprising the amino acid sequence of a target antigen, and wherein the C-terminus of the tag portion is covalently bound to the N-terminus of the antigen portion in the tag construct.

[0532] 96. A polynucleotide encoding the binding molecule or conjugate according to any one of items 1 to 93.

[0533] 97. An expression vector comprising the polynucleotide according to item 96.

[0534] 98. A host cell comprising the expression vector according to item 97.

[0535] 99. A method for preparing a binding molecule or conjugate according to any one of items 1 to 93, the method comprising:

[0536] - culturing the host cell according to item 98 under conditions allowing expression of the binding molecule or conjugate from the vector; and

[0537] - isolating said binding molecule or conjugate.

[0538] 100. A composition comprising the binding molecule according to any one of items 1 to 76 and at least one pharmaceutically acceptable excipient or carrier.

[0539] 101. A composition comprising the conjugate according to any one of items 77 to 93 and at least one pharmaceutically acceptable excipient or carrier.

[0540] 102. A composition comprising the complex according to any one of items 94 to 95 and at least one pharmaceutically acceptable excipient or carrier.

[0541] 103. A kit comprising

[0542] - the conjugate according to any one of items 77 to 93;

[0543] - a peptide tag construct comprising a peptide tag portion; and

[0544] - Instructions for forming the composite according to item 94.

[0545] 104. A kit according to item 103, wherein the tag construct further comprises a cargo domain, wherein the cargo domain is a peptide antigen portion comprising the amino acid sequence of a target antigen, wherein the C-terminus of the tag portion is covalently bound to the N-terminus of the antigen portion in the tag construct, and wherein the instructions are instructions for forming the complex according to item 95.

[0546] 105. The binding molecule according to any one of items 1 to 76, the conjugate according to any one of items 77 to 93, the complex according to any one of items 94 to 95, the composition according to any one of items 100 to 102, or the kit according to any one of items 103 to 104 for use in therapy.

[0547] 106. A binding molecule, conjugate, complex, composition or kit for use according to item 105 for the treatment or prevention of cancer, infection or autoimmune disease.

[0548] 107. A method for treating or preventing cancer, infection or autoimmune disease, the method 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.

[0549] 108. Use of the binding molecule according to any one of items 1 to 76, the conjugate according to any one of items 77 to 93, the complex according to any one of items 94 to 95 or the composition according to any one of items 100 to 102 in the preparation 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 NO: 1 to SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 to SEQ ID NO: 6, Provided that the heavy chain variable region (VH) comprises three complementarity determining regions (CDRs), wherein: - VHCDR1 has the sequence shown in SEQ ID NO: 7; -VHCDR2 has the sequence IGRIDPEX a X b DAEYVP (SEQ ID NO: 8), wherein X a X b Selected from the group consisting of SG, GG, QG, DG, NA and NG; and - VHCDR3 has the sequence shown in SEQ ID NO: 9; and an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, Provided that the light chain variable region (VL) comprises three complementarity determining regions (CDRs), wherein: - VLCDR1 has the sequence shown in SEQ ID NO: 11; - VLCDR2 has the sequence shown in SEQ ID NO: 12; and - VLCDR3 has the sequence shown in SEQ ID NO:

13.

2. The binding molecule according to claim 1, wherein The binding molecule is a scFv.

3. A binding molecule according to any one of the preceding claims, wherein The binding molecule is capable of selectively binding to the peptide tag portion contained in the tag construct, which optionally further comprises at least one cargo domain.

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

5. The binding molecule according to any one of claims 3 to 4, wherein The binding molecule is capable of binding to the tag portion, such that the interacting K D The value is up to 5×10 -9 M, for example, up to 1×10 -10 M, for example, up to 1×10 -11 M.

6. A bispecific conjugate comprising: - at least one first moiety, said first moiety being a binding molecule according to any one of the preceding claims; and - at least one second moiety, which is an antibody or an antigen-binding fragment thereof, such as an anti-D40 antibody or an antigen-binding fragment thereof.

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

20.

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

22.

9. The bispecific conjugate according to any one of claims 7 to 8, wherein the anti-CD40 antibody comprises: a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23; and A heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

24.

10. A composite comprising: The bispecific conjugate according to any one of claims 6 to 9; and A tagging construct comprising a peptide tag portion. The composite according to claim 10 , wherein: The tag construct further comprises a cargo domain, wherein the cargo domain is a peptide antigen portion comprising the amino acid sequence of a target antigen, and wherein the C-terminus of the tag portion is covalently bound to the N-terminus of the antigen portion in the tag construct.

12. A polynucleotide encoding the binding molecule or conjugate according to any one of claims 1 to 9.

13. A composition comprising: The binding molecule according to any one of claims 1 to 5, the conjugate according to any one of claims 6 to 9, or the complex according to any one of claims 10 to 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 portion; and - Instructions for forming a composite according to any one of claims 10 to 11.

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

16. A binding molecule, conjugate, complex, composition or kit for use according to claim 15 for the treatment or prevention of cancer, infection or autoimmune disease.

Images