Optimized tag part

Abstract

The present disclosure provides improved peptide tag moieties with affinity for binding molecules. The peptide tag moieties are useful, for example, as part of tag constructs, together with cargo moieties. They offer unexpected advantages as part of biopharmaceutical product conjugates compared to known tag moieties with similar amino acid sequences. Also disclosed are binding molecules and bispecific conjugates with affinity for the peptide tag moieties, as well as conjugates comprising a peptide tag moiety non-covalently bound to such binding molecules or bispecific conjugates. In such conjugates, the tag moiety can form part of a tag construct that further comprises an antigen as a cargo moiety, for example, for antigen delivery to immune cells. Medical uses of the tag moieties, tag constructs, and conjugates of the present disclosure are also provided.

Description

[Technical Field]

[0001] The present disclosure relates to improved peptide tag moieties that have affinity for binding molecules. The peptide tag moieties are useful, for example, as part of a tag construct, together with a cargo moiety. They offer unexpected advantages as part of a biopharmaceutical product conjugate when compared with known tag moieties with similar amino acid sequences. Also disclosed are binding molecules and bispecific conjugates that have affinity for the peptide tag moiety, as well as conjugates comprising a peptide tag moiety non-covalently bound to such binding molecules or bispecific conjugates. In such conjugates, the tag moiety can form part of a tag construct that further comprises an antigen as a cargo moiety, for example, for antigen delivery to immune cells. Medical uses of the tag moieties, tag constructs, and conjugates of the present disclosure are also provided. [Background technology]

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

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

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

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

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

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

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

[0009] WO2011 / 115483 relates to conjugates containing tetanus toxoid peptide ("MTTE") for the induction of an immune response when administered to a subject with pre-existing antibodies to tetanus toxoid. The PCT application defines the peptide as containing at least 10 amino acids. A large number of peptides were tested, and in section 2.5 on page 42 it is stated that best results were achieved with 22-mer peptides, although the shortest peptide bound by the test reagent contained 10 amino acids. The goal of this disclosure is to optimize binding to polyclonal antibodies pre-existing in the host and directed against pan-B cell epitopes.

[0010] WO2020 / 104690 and WO2021 / 239968 propose and test a number of peptide tag subsequences, some of which are derived from tetanus toxin, as candidates for conjugation with bispecific conjugates.

[0011] On page 19, lines 3-20 of WO2020 / 104690, the MTTE peptide is presented as a candidate, along with several other peptides derived from tetanus toxin. The MTTE peptide has 18 amino acids and is designated "SEQ ID NO: 6," while other candidate tag peptides are presented as "SEQ ID NOs: 10-14." Specific tag peptides consisting of fewer than 18 amino acids are not disclosed, let alone created and tested. While this section speculatively discusses the use of shorter fragments of MTTE, this discussion does not point to any individual portions of the sequence that would be useful as fragments, and importantly, there is no experimental data to the effect that any particular length or sequence of such fragments could actually be effectively used in the disclosed conjugates.

[0012] WO2021 / 239968 also discusses the MTTE peptide, for example, on page 24, lines 23-31. There, the authors describe trimming the MTTE sequence to a peptide consisting of the 12 N-terminal amino acids of the sequence, with the resulting sequence presented as "SEQ ID NO: 16." The next section, from page 24, line 32 to page 25, line 4, teaches various sequence variants of the 12-mer sequence and longer sequences containing it. Importantly, the present disclosure is consistent with experiments in WO2021 / 239968 showing that the peptide can indeed be shortened to an N-terminal fragment consisting of 12-13 amino acids (see Example 17 on pages 67-68 of WO2021 / 239968). As is clear from the heading of Example 17 in WO2021 / 239968—“Determination of the Minimal Epitope Recognized by 14GIIICII and IBIIICI” (emphasis added), the present disclosure teaches those skilled in the art that the MTTE peptide cannot be further shortened. This message is made clearer by the disclosure of other potentially useful peptide sequences on page 25, lines 14-33 of WO2021 / 239968, which discusses unrelated tetanus toxin sequences presented as “SEQ ID NOS: 11-15” (incidentally, these are the same sequences listed as “SEQ ID NOS: 10-14” in WO2020 / 104690). Only in this section, unlike the truncated MTTE variants discussed on the previous page, is there a proposal to create shorter fragments, albeit only with respect to SEQ ID NOS: 11-15. There is no disclosure of fragments of the MTTE peptide shorter than 12 amino acids. In other words, there is no disclosure of MTTE peptide fragments consisting of less than 12 amino acids.

[0013] In summary, WO2020 / 104690 and WO2021 / 239968 disclose promising platforms for the development of personalized biopharmaceuticals. However, the development and manufacture of candidate products places high demands on all components or parts contained in the product, and therefore there is a general need to optimize and improve the platform. DETAILED DESCRIPTION OF THE INVENTION

[0014] It is an object of the present disclosure to meet this need by improving upon previously tested peptide tags and providing peptide tag moieties that present smaller (shorter) epitopes for binding by bispecific conjugates than previously thought possible, while maintaining efficacy.

[0015] It is another object of this disclosure to identify a narrow selection of peptide tag subsequences that are particularly useful as binding partners for bispecific conjugates or binding molecules.

[0016] It is a further object to enable the use of peptide tag subsequences that are unlikely to provoke an immune response against themselves and / or that are unlikely to have pre-existing antibodies that would limit their use in subjects, in other words, to provide tag moieties that are less immunogenic in the host than previously known tag moieties.

[0017] Another object is to provide a peptide tag moiety that leaves the maximum amount of room for at least one cargo moiety when the peptide tag moiety and the at least one cargo moiety are included in a tag construct.

[0018] Yet another object is to provide tag moiety sequences that simplify recombinant production and / or chemical synthesis of the tag moiety as much as possible, for example, by being as short as possible.

[0019] A further object of the present disclosure is to provide tag moieties that are optimized for interaction with engineered binding molecules, as opposed to relying on existing endogenous binding entities in a subject.

[0020] Another object of the present disclosure is to provide alternative tag moieties that exhibit increased compatibility with binding molecules having affinity for the tag moiety, for example, when used in the context of the conjugates described in WO2020 / 104690 and WO2021 / 239968.

[0021] These and other objects apparent to those skilled in the art from this disclosure are met by the different aspects of the present invention as claimed in the appended claims and as generally disclosed herein.

[0022] Thus, in a first aspect, there is provided a tag moiety consisting of the amino acid sequence FIGITELX8X9 (SEQ ID NO: 1), wherein X8 is selected from K, L and H, and X9 is K or absent, and which tag moiety comprises an epitope for binding by a specific binding molecule thereto.

[0023] In one embodiment, X8 is selected from K and L.

[0024] In one embodiment, X8 is selected from L and H.

[0025] In one embodiment, X8 is selected from K and H.

[0026] In one embodiment, X8 is K. In yet another embodiment, X8 is L. In yet another embodiment, X8 is H.

[0027] In one embodiment, X9 is K. In another embodiment, X9 is absent.

[0028] The alternative embodiments of the amino acids at positions X8 and X9 can be freely combined in any subgroup. For example, in one embodiment, the tag portion consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 2 to 4. In another embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 5 to 7.

[0029] The tag moiety in this embodiment is useful as a minimal binding target or epitope for a binding molecule specific thereto. This property makes it useful in the context of drug platforms, such as those described in WO2020 / 104690 and WO2021 / 239968, insofar as the platform incorporates a binding molecule specific for the tag moiety in the bispecific conjugate molecule.

[0030] In this and other contexts, it may be of interest to incorporate the tag moiety into a tag construct that further comprises at least one cargo moiety, which may be any compound of interest for transport or delivery by virtue of the affinity between the tag moiety and a specific binding molecule.

[0031] Thus, in a second aspect, the present disclosure provides a tag construct comprising a tag moiety according to the first aspect and at least one cargo moiety. The tag moiety of the tag construct is designed to interact with a binding molecule specific thereto, while the cargo moiety is a moiety that exists to achieve some other function, for example, eliciting a specific immune response against a cargo representing an antigen. In one embodiment, the tag moiety and cargo moiety(s) in the tag construct are covalently linked. In the context of the tag constructs disclosed herein, the tag moiety and cargo moiety(s) do not overlap. An example is illustrated by the case where the cargo moiety is a peptide moiety, and the amino acid sequence of the tag moiety is structurally and functionally different from that of the cargo moiety. Those skilled in the art will therefore understand that the cargo moiety is not part of the tag moiety. In one embodiment, the cargo moiety is a peptide moiety. In another embodiment, the cargo moiety is a nucleic acid moiety.

[0032] In embodiments in which at least one cargo moiety is a nucleic acid moiety, the tag moiety and cargo moiety can be arranged in any order in the polypeptide-nucleic acid combination. In one such embodiment, the cargo moiety is an siRNA molecule.

[0033] In embodiments in which at least one cargo moiety is a peptide, the tag moiety and cargo moiety can be arranged in any order in the polypeptide chain. Thus, in one embodiment, the C-terminus of the tag moiety is covalently linked to the N-terminus of at least one cargo moiety. In another embodiment, the N-terminus of the tag moiety is covalently linked to the C-terminus of at least one cargo moiety.

[0034] In one embodiment, the cargo moiety is a small molecule compound, such as a small molecule drug, such as a drug selected from the group consisting of a cytotoxic agent, an anti-inflammatory agent, a kinase inhibitor, a receptor kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a serine / threonine kinase inhibitor, an epigenetic inhibitor, a BCL2 inhibitor, a hedgehog pathway inhibitor, a proteasome inhibitor, a PART inhibitor, and an indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor.

[0035] In alternative embodiments, at least one cargo moiety is an antigen. In more specific embodiments, at least one cargo moiety is a peptide antigen. In such embodiments, the peptide antigen comprises a target antigen amino acid sequence that may be of interest for generating an immune response. It should be understood that the at least one cargo moiety included in the tag construct can be two or more cargo moieties, such as a plurality of cargo moieties. In specific embodiments, the at least one cargo moiety is a plurality of cargo moieties, such as at least two cargo moieties, such as at least three cargo moieties, or at least five cargo moieties. It should be understood that the number of cargo moieties included in the tag construct is not limited to a specific number of moieties. In embodiments in which the at least one cargo moiety is a peptide antigen, it preferably comprises the target antigen amino acid sequence. In such embodiments, the target antigen amino acid sequence may constitute the entire at least one cargo moiety, or the at least one cargo moiety may comprise additional amino acids that are not part of the target antigen amino acid sequence but are present for another purpose, for example, as a linker between the tag moiety and the target antigen sequence within the cargo moiety. It should be further understood that the target antigen amino acid sequence may then comprise amino acid sequences corresponding to multiple peptide antigens. The multiple peptide antigens contained in the cargo moiety or contained in the target amino acid sequence may be fused to each other directly or indirectly. Furthermore, as described above, the at least one cargo moiety may comprise additional amino acids that are not part of the target antigen amino acid sequence but are present for another purpose, for example, as a linker between a tag moiety and the target antigen sequence within the cargo moiety, or as a linker between multiple cargo moieties or multiple peptide antigen sequences.

[0036] In embodiments in which at least one cargo moiety is a peptide antigen moiety, the target antigen amino acid sequence may be selected from the group consisting of, for example, a cancer antigen, an autoantigen, and an antigen derived from a pathogen. It should be understood that a tag construct comprising at least one cargo moiety may comprise two or more cargo moieties. In one such embodiment, it is an autoantigen. In another such embodiment, it is an antigen derived from a pathogen. In yet another such embodiment, the target antigen amino acid sequence is a cancer antigen.

[0037] In embodiments where the target antigen amino acid sequence is a cancer antigen, it may be suitably selected from the group consisting of neoantigens, tumor-associated antigens, and antigens derived from oncoviruses. In another embodiment, the cancer antigen is a plurality of cancer antigens, such as a plurality of neoantigens, a plurality of tumor-associated antigens, or a plurality of oncovirus-derived antigens.

[0038] Specific, but non-limiting, examples of amino acid sequences that may constitute the target antigen amino acid sequence in a tag construct according to the present disclosure and that have been tested in the examples below are those selected from the group consisting of SEQ ID NOs: 17-23, 25, 116, and 140. One of skill in the art will recognize many other antigen sequences that may be of interest for generating an immune response, e.g., a T cell immune response. In specific embodiments, the target antigen amino acid sequence included in a tag construct according to the present disclosure is selected from the group consisting of SEQ ID NOs: 21-23, 25, and 116. In one such embodiment, the sequence is SEQ ID NO: 21. In another such embodiment, the sequence is SEQ ID NO: 22. In another such embodiment, the sequence is SEQ ID NO: 23. In another such embodiment, the sequence is SEQ ID NO: 25. In another such embodiment, the sequence is SEQ ID NO: 116.

[0039] When combining non-limiting examples of alternative sequences for at least one cargo moiety with embodiments of tag moiety sequences (SEQ ID NOS: 2-7), it is clear that in one embodiment of a tag construct according to the present disclosure, the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NOS: 24, 26-67, 88, 90-91, 98-99, 103, 106, 117-123, and 141-146. In a more specific embodiment, the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NOS: 24, 26-40, 88, 98-99, 103, 106, 117, and 141-143. In an alternative specific embodiment, the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NOS: 41-67, 90-91, 118-123, and 144-146.

[0040] As described herein, the tag moiety of the present disclosure consists of an amino acid sequence described herein and comprises an epitope for binding by a specific binding molecule. In turn, the tag construct of the present disclosure comprising the tag moiety comprises an epitope for binding by a specific binding molecule. In one embodiment, the tag moiety, or a tag construct comprising the tag moiety, comprises an epitope for binding by a specific binding molecule. D The value is up to 1×10 -9 M, e.g., up to 1 x 10 -10 M, e.g., up to 1 x 10 -11 M is capable of binding to a binding molecule.

[0041] In one embodiment, the binding molecule is an antibody construct, such as an antibody or an antigen-binding fragment thereof. In a more specific embodiment, the binding molecule is an scFv.

[0042] In one embodiment, the binding molecule specific for the tag moiety is an immunoglobulin heavy chain variable region (VH) comprising three complementarity-determining domains (CDRs), VHCDR1 has the sequence set forth in SEQ ID NO: 68, VHCDR2 has the sequence IGRIDPEX a X b DAEYVP (SEQ ID NO: 69) a Xb is selected from the group consisting of SG, GG, QG, DG, NA, and NG; VH, wherein VHCDR3 has the sequence set forth in SEQ ID NO: 70; an immunoglobulin light chain variable region (VL) comprising three complementarity-determining domains (CDRs), VLCDR1 has the sequence set forth in SEQ ID NO: 71; VLCDR2 has the sequence set forth in SEQ ID NO: 72; and VL, wherein VLCDR3 has the sequence set forth in SEQ ID NO:73.

[0043] In other words, X in VHCDR2 a X b means that the VHCDR2 amino acid sequence is selected from the group consisting of SEQ ID NOs: 124-129.

[0044] In an alternative embodiment, the binding molecule specific for the tag moiety is an immunoglobulin heavy chain variable region (VH) comprising three complementarity-determining domains (CDRs), VHCDR1 has the sequence set forth in SEQ ID NO: 134, VHCDR2 has the sequence set forth in SEQ ID NO: 135, VH, wherein VHCDR3 has the sequence set forth in SEQ ID NO: 136; an immunoglobulin light chain variable region (VL) comprising three complementarity-determining domains (CDRs), VLCDR1 has the sequence set forth in SEQ ID NO: 137; VLCDR2 has the sequence set forth in SEQ ID NO: 138; and VL, wherein VLCDR3 has the sequence set forth in SEQ ID NO: 139.

[0045] In one embodiment, the binding molecule comprises an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13, 130, and 132, and amino acid sequences having at least 90% identity thereto, and an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 131, and 133, and amino acid sequences having at least 90% identity thereto. In a specific embodiment, one or more of these VH and VL sequences have at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% identity thereto, to any one of the specifically recited sequences.

[0046] As described herein, binding molecules having amino acid sequences with 90% or greater identity to the listed sequences are also contemplated as falling within the scope of the present disclosure. Such variant sequences may be modified compared to the listed sequences by one or more amino acid substitutions, insertions, and / or deletions.

[0047] Amino acid substitutions to the recited sequences may be conservative amino acid substitutions. The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, and therefore, conservative substitutions of amino acids important for the structure or function of a polypeptide may be expected to have less of an effect on polypeptide structure / function than non-conservative amino acid substitutions at the same position. Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, conservative amino acid substitutions can be considered substitutions in which a particular amino acid residue is replaced with a different amino acid within the same family. However, amino acid substitutions can also be non-conservative, in which one amino acid is replaced with another amino acid with a side chain belonging to a different family.

[0048] As detailed above, according to the present disclosure, variants of the listed sequences share at least 90% sequence identity with each listed sequence. Sequence identity can be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform pairwise or multiple alignment of sequences are useful; for example, EMBOSS Needle or EMBOSS Stretcher (both Rice et al. (2000), Trends Genet., 16(6):276-277) can be used for pairwise sequence alignment, while Clustal Omega (Sievers et al. (2011), Mol. Syst. Biol. 7:539) or MUSCLE (Edgar (2004), Nucleic Acids Res. 32(5):1792-1797) can be used for multiple sequence alignment, although any other suitable program can also be used. Whether the alignment is pairwise or multiple, it must be done globally (i.e., across the entire reference sequence) rather than locally.

[0049] Sequence alignments and percent identity calculations may be determined, for example, using standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1. Alternatively, standard EMBOSS Needle parameters may be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters may alternatively be used.

[0050] As explained above, the binding molecule, in some embodiments, may be an scFv molecule, in which the VH and VL domains are linked together in a single polypeptide chain by means well known to those of skill in the art, for example, via an amino acid linker. In one embodiment, the binding molecule is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16 and 107-112. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 107-112. In a more specific embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 107 and 112. In an even more specific embodiment, the amino acid sequence is SEQ ID NO: 107.

[0051] As described above, the tag construct is useful as a component within the biopharmaceutical platform described in WO2020 / 104690 and WO2021 / 239968. Accordingly, the present disclosure also provides a complex, such as a noncovalent complex, formed between one tag construct (including a tag portion) and another binding molecule specific for the tag portion. In other words, a third aspect of the present disclosure provides a complex, such as a noncovalent complex, comprising a tag construct comprising a tag portion according to the first aspect and a binding molecule specific for the tag portion. With regard to the complex embodiment, all of the embodiments described above in relation to aspects of the tag portion, aspects of the tag construct, and binding molecules specific for the tag portion equally apply to the complex according to this aspect and will not be repeated here for brevity.

[0052] In a conjugate according to this aspect, it may be advantageous for the binding molecule to be, in turn, comprised in and covalently linked to a second moiety which is an antibody or an antigen-binding fragment thereof, e.g., a fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fc fragment, an Fv fragment, a single-chain (scFv) fragment, an (scFv)2, and a domain antibody. In one embodiment, the second moiety is an antibody, such as an antibody of the IgG2 subtype.

[0053] In certain embodiments of the conjugate in which the binding molecule is covalently linked to a second moiety, the second moiety is an anti-CD40 antibody or antigen-binding fragment thereof. In one such embodiment, 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 antibodies comprising an antigen-binding fragment derived from any one or more of the antibodies. In a more preferred embodiment, the anti-CD40 antibody or antigen-binding fragment thereof comprises six CDRs: VLCDR1 has the sequence set forth in SEQ ID NO: 74; VLCDR2 has the sequence set forth in SEQ ID NO: 75; VLCDR3 has the sequence set forth in SEQ ID NO: 76; VHCDR1 has the sequence set forth in SEQ ID NO: 77, VHCDR2 has the sequence set forth in SEQ ID NO: 78, VHCDR3 has the sequence set forth in SEQ ID NO:79.

[0054] In one embodiment, such an anti-CD40 antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) comprising an amino acid sequence selected from SEQ ID NO: 80 and amino acid sequences having at least 90% sequence identity thereto, and a heavy chain variable domain (VH) comprising an amino acid sequence selected from SEQ ID NO: 81 and amino acid sequences having at least 90% sequence identity thereto. In embodiments where the anti-CD40 antibody or antigen-binding fragment thereof is a full-length antibody, it may comprise, for example, a light chain (LC) comprising an amino acid sequence selected from SEQ ID NO: 82 and amino acid sequences having at least 90% sequence identity thereto, and a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO: 83 and amino acid sequences having at least 90% sequence identity thereto.

[0055] As described above in relation to binding molecules that are specific for a tag moiety, one or more of these VH, VL, LC, and HC sequences have at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identity to any one of the specifically listed sequences.

[0056] In a fourth aspect of the present disclosure, there are provided polypeptides encoding the tag moieties or tag constructs described herein, expression vectors comprising the polynucleotides, and host cells comprising the expression vectors. Also encompassed by the present disclosure is a method of producing the tag moieties or tag constructs of the present disclosure, the method comprising culturing the host cell under conditions that allow expression of the tag moiety or tag construct from the expression vector, and isolating the tag moiety or tag construct.

[0057] In alternative aspects, tag moieties or tag constructs according to the present disclosure are produced de novo using peptide synthesis, using methods available to those of skill in the art. Thus, the present disclosure provides methods of producing tag moieties or tag constructs according to the present disclosure, which methods involve the stepwise addition of amino acid monomers to a growing polypeptide chain until the final tag moiety or tag construct described herein is formed.

[0058] In a further aspect, the present disclosure provides a pharmaceutical composition comprising (i) a tag moiety as described above, (ii) a tag construct as described above, or (iii) a conjugate as described above. In addition to the tag moiety, tag construct, or conjugate, the pharmaceutical composition also comprises at least one pharmaceutically acceptable carrier or excipient.

[0059] Also provided by the present disclosure are kits and articles of manufacture comprising, separately, a binding molecule (e.g., comprised in a bispecific conjugate) and a tag moiety or tag construct as described above. In such kits and articles of manufacture, the binding molecule and tag moiety or tag construct may be provided separately in a composition containing a pharmaceutically acceptable carrier or excipient.

[0060] As used herein, "pharmaceutically acceptable carrier or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0061] Preferably, the carrier or excipient is suitable for parenteral, e.g., intradermal, intravenous, intramuscular, or subcutaneous, administration (e.g., by injection or infusion). Depending on the route of administration, the tag moiety, tag construct, conjugate, or component thereof may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate or denature it.

[0062] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in pharmaceutical compositions, kits, and products include water, buffered water, and physiological saline. Other examples of carriers include ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyols such as mannitol and sorbitol, sodium chloride, etc.

[0063] The pharmaceutical compositions, products, or kits may also contain pharmaceutically acceptable antioxidants. They may also contain auxiliary substances such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that prolong absorption, such as aluminum monostearate and gelatin.

[0064] 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.

[0065] Sterile injectable solutions can be prepared by incorporating the active agent (e.g., complex) in the required amount in a suitable solvent with one or a combination of the ingredients listed above, optionally followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which obtains a powder of the active agent plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0066] The pharmaceutical compositions, products, and kits may include additional active ingredients, as well as tag moieties, tag constructs, conjugates, or components thereof, e.g., they may include additional therapeutic or prophylactic agents. Thus, the conjugates may be used as monotherapy or as part of a combination therapy, e.g., in the treatment of cancer. The kits or combination products described herein may additionally include instructions for use.

[0067] The tag moieties, tag constructs, conjugates, pharmaceutical compositions, kits, and combination products of the present disclosure can be used in therapy. Accordingly, the present disclosure provides tag moieties, tag constructs, conjugates, pharmaceutical compositions, or kits of the present disclosure for use in therapy. The term "therapy" refers to the treatment of a subject. As used herein, "therapy" refers to the treatment of any medical condition. Such treatment can be preventative (i.e., preventative), curative (or treatment intended to be curative), or palliative (i.e., treatment designed to merely limit, alleviate, or improve the symptoms of a condition). In curative and palliative applications, the conjugate or composition is administered to a subject already suffering from a disease or condition in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount." An effective amount for a given purpose will depend on the disease or condition being treated, its severity, and the size / weight and general condition of the subject.

[0068] Prophylactic treatment can include preventing a condition or delaying the onset or development of a condition. For example, the conjugates can be used to prevent infection or reduce the extent to which an infection may occur, or to prevent, delay, or reduce the extent of cancer onset or recurrence, or to prevent or reduce the extent of metastasis, for example.

[0069] As defined herein, a subject refers to any mammal, for example, a livestock animal such as a cow, horse, sheep, pig, or goat, a pet animal such as a rabbit, cat, or dog, or a primate such as a monkey, chimpanzee, gorilla, or human. Most preferably, the subject is a human.

[0070] The combination product of the present disclosure comprises a binding molecule (e.g., contained in a bispecific conjugate) as defined herein and a tag moiety or tag construct as defined herein as a combined preparation for simultaneous or sequential use in therapy. That is, when the combination product disclosed herein is used according to the present disclosure, i.e., in therapy, the binding molecule and tag moiety or tag construct are administered to a subject simultaneously or sequentially. Similarly, when the kit of the present disclosure is used in therapy, the binding molecule and tag moiety or tag construct are administered to a subject simultaneously or sequentially. "Concurrent" administration, as used herein, means that the two components are administered to a subject simultaneously, or at least substantially simultaneously, by the same route of administration and at substantially the same site. "Sequential" administration, as used herein, means that the two components are administered to a subject at different times. In particular, the administration of the first component is completed before the administration of the second component is initiated.

[0071] Due to the nature of the present disclosure, sequential administration of a binding molecule and a tag moiety or tag construct requires that both be administered by the same route and at substantially the same site. Furthermore, the administration of a binding molecule and a tag moiety or tag construct can be separated in time, but the interval between administrations should be such that a complex can be formed when both components are administered. Thus, for example, both components can be administered within 1 hour of each other, or more specifically, within 40, 30, 20, 15, 10, 8, 7, 6, 5, 4, 3, 2, or 1 minute of each other, or less than 1 minute.

[0072] Reference to a conjugate of the present disclosure (or a pharmaceutical composition comprising such a conjugate) administered to a subject for therapeutic purposes should be understood to refer to a pre-mixed composition (e.g., a solution) comprising both components of the conjugate (i.e., the binding molecule and tag moiety or tag construct described above), which may exist in a dynamic equilibrium comprising the conjugate and its two individual components.

[0073] In particular, the tag moieties, tag constructs, binding molecules (e.g., contained in bispecific conjugates), complexes, compositions, kits, or combined products of the present disclosure can be used to treat or prevent cancer. Cancer refers to any malignant or pre-malignant neoplastic condition. Thus, cancer can be any cancer of any organ, tissue, or cell type. Included are cancers that present as solid tumors and cancers that do not present as solid tumors. Thus, included are hematopoietic cancers.

[0074] The cancer may be prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, rhabdomyosarcoma, neuroblastoma, multiple myeloma, leukemia, acute lymphoblastic leukemia, melanoma, bladder cancer, head and neck cancer, lymphoma, glioblastoma, or skin cancer. It may also be adrenal gland cancer, bone cancer, brain cancer, esophageal cancer, eye cancer, stomach cancer, oral cancer, penile cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer. Mast cell tumors and angiosarcomas may also be treated according to the present disclosure. The cancer may be newly diagnosed and untreated, or it may be relapsed or refractory, or relapsed and refractory, primary, or metastatic.

[0075] When present in a bispecific conjugate together with the binding molecule of the present disclosure, the CD40-specific second moiety activates the immune system by agonizing or stimulating CD40 on APCs, particularly dendritic cells. In particular, this can lead to T cell activation. The subsequent immune response exerts an anti-cancer effect on adjacent or accessible tumor cells, regardless of CD40 expression by the tumor. Therefore, the bispecific conjugate of this embodiment, or a complex containing it, can be effective against both CD40-positive and CD40-negative cancers. Such bispecific conjugates can also be effective as adjuvants in vaccination regimens against pathogens. When a vaccine platform (e.g., an attenuated virus or DNA / RNA-based vaccine) does not stimulate a sufficient immune response by itself, CD40 activation may be required to stimulate an effective anti-pathogen immune response that results in neutralizing antibody or T cell responses.

[0076] In addition to the agonistic immunostimulatory effect provided by the CD40-specific second moiety, in embodiments where it is present, the conjugates of the present disclosure also provide an antigen that can be presented to treated and activated T cells, thus priming the T cells to target cancer cells or virus-infected cells that express the antigen. This can be particularly beneficial in situations where the presence of cancer antigens is low or reduced, e.g., where tumors have been surgically removed, where anti-CD40 therapeutic agents cannot be delivered intratumorally, or where antigen presentation is not ideal at the tumor site due to suppressive factors secreted by the tumor. The conjugates according to this embodiment provide a means for presenting cancer antigens in proximity to agonistic activation signals, e.g., at non-tumor sites, ensuring the priming and activation of T cells that can then migrate to the tumor and exert their function.

[0077] In some embodiments, the cancer antigen delivered by the conjugate can be selected based on the subject and the particular cancer, thus enabling personalized medicine. For example, the subject's cancer can be subjected to genetic profiling, allowing suitable antigens to be selected. Banks or libraries of antigens, or tag constructs containing antigens, can be provided from which suitable tag constructs can be prepared or selected depending on the type of cancer of the subject.

[0078] The tag moieties, tag constructs, binding molecules (e.g., contained in bispecific conjugates), complexes, compositions, kits, or combined products of the present disclosure may also be useful for treating or preventing infectious diseases. The present disclosure may be particularly useful in therapy for (e.g., vaccination against) viral infections, particularly infections caused by RNA viruses. Infections caused by RNA viruses that can be prevented (by vaccination) according to the present disclosure include infections caused by coronaviruses (such as SARS-CoV1 (the causative agent of SARS), SARS-CoV2 (the causative agent of COVID-19), and MERS-CoV), influenza virus, Ebola virus, hepatitis C virus (HCV), hepatitis E virus (HEV), rabies virus, poliovirus, Ross River virus, and measles virus.

[0079] The present disclosure may also be used to treat or vaccinate against infections caused by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (e.g., HHV6), parvovirus B19, and human papillomavirus (HPV), although in principle any viral infection can be treated or prevented in accordance with the present disclosure.

[0080] Intracellular bacterial infections, such as brucellosis (caused by bacterial species of the genus Brucella), Q fever (caused by Coxiella burnetii), diseases caused by Chlamydiae species such as chlamydia (caused by Chlamydia trachomatis) and pneumonia (caused by Chlamydia pneumoniae), leprosy (caused by Mycobacterium leprae and Mycobacterium lepromatosis), and tuberculosis, including disseminated tuberculosis (caused by Mycobacterium tuberculosis), can be treated according to the present disclosure. Intracellular fungal or protozoal infections, including leishmaniasis (caused by trypanosomes of the genus Leishmania) and toxoplasmosis (caused by the apicomplexan Toxoplasma gondii), can also be treated according to the present disclosure. Thus, antigens can be derived from any of the aforementioned pathogens.

[0081] In the case of a conjugate of a tag moiety with a binding molecule contained in a bispecific conjugate comprising an anti-CD40 antibody or fragment thereof, agonism of CD40 can activate the immune system to fight infectious diseases (a principle similar to its use in cancer therapy). In the case of the conjugates of the present disclosure, as explained above, this can be provided with an antigen derived from the target pathogen, thus inducing a specific immune response against the pathogen. The vaccine platform is advantageously adaptable, for example, for pandemic situations. This adaptability can be provided, in particular, by using tag constructs containing antigens that can be modified to accommodate viral diversity and antigenic drift. Viral antigens can be selected based on HLA prevalence in specific regions, as well as viral serotype determinants.

[0082] In one embodiment, a bispecific conjugate of the present disclosure may comprise an antagonistic anti-CD40 antibody or antigen-binding fragment thereof. In such an embodiment, the bispecific conjugate is useful for tolerizing the immune system to targets that drive autoimmune disease. In one embodiment of the conjugate of the present disclosure, it comprises a bispecific conjugate whose second moiety is a CD40 antagonist and a tag construct comprising an antigen known to be a driver of autoimmune disease, such as T cell-driven autoimmune disease. In this case, the purpose of the bispecific conjugate in complex with the antigen-containing tag construct is to reeducate the immune system to tolerate the disease-causing antigen, thereby preventing excessive destruction of healthy tissue. Examples of antigens useful for tag constructs according to such embodiments are selected from the group consisting of antigens associated with SLE, type 1 diabetes, rheumatoid arthritis, vasculitis, myositis, multiple sclerosis, psoriasis, and allergies.

[0083] The antigen (e.g., a cancer antigen, a pathogen-derived antigen, or an antigen associated with an immune-mediated disease) can be selected to be recognized by a particular subset of T cells in the subject to be treated, the T cells expressing a TCR known to recognize the selected antigen. In particular, the antigen can be selected based on its recognition by the T cells to be used in adoptive cell therapy in the subject to be treated.

[0084] For example, in adoptive cell therapy, T cells can be obtained from a subject, and T cells that recognize an antigen of interest can be isolated. The isolated T cells can then be expanded and / or otherwise treated to stimulate their effector functionality, and then reinfused into the subject to be treated. In this context, the antigen recognized by the reinfused T cells can be used in a tag construct. The complex of the present disclosure can then be administered to the subject so that the antigen activates the reinfused T cells.

[0085] Alternatively, T cells can be obtained from the subject or donor to be treated and genetically modified to express a TCR that recognizes a target antigen. The genetically modified T cells can then be expanded and / or otherwise treated to stimulate their effector functionality, and then infused (or re-infused) into the subject to be treated. In this context, the antigen recognized by the genetically modified T cells can be used in a tag construct. The complex of the present disclosure can then be administered to the subject so that the antigen activates the infused T cells. The method in which administration of the complex of the present disclosure is combined with adoptive cell therapy is particularly useful for the treatment of cancer, in which case the antigen used in the tag construct is a cancer antigen.

[0086] Thus, the present disclosure provides a method for treating or preventing cancer, the method comprising administering to a subject a tag moiety or construct of the present disclosure, a conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0087] In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising: (i) obtaining T cells from a subject; (ii) isolating T cells that recognize the target cancer antigen and, optionally, expanding the isolated T cells; (iii) re-infusing the subject with the isolated T cells; and (iv) administering to the subject a conjugate of the present disclosure, wherein the tag construct comprises the target cancer antigen. Equivalently, in step (iv), the subject may alternatively be administered a binding molecule comprised in the bispecific conjugate of the present disclosure and a tag construct comprising the target cancer antigen separately.

[0088] In another embodiment, the present disclosure provides a method of treating cancer in a subject, the method comprising: (i) obtaining T cells from a subject or donor; (ii) genetically modifying T cells to express a TCR that recognizes a target cancer antigen, and optionally expanding the T cells before or after genetic modification; (iii) infusing the genetically modified T cells into the subject; (iv) administering to the subject a conjugate of the present disclosure, wherein the tag construct comprises the target cancer antigen. Equivalently, in step (iv), the subject may alternatively be administered a binding molecule comprised in the bispecific conjugate of the present disclosure and a tag construct comprising the target cancer antigen separately.

[0089] The present disclosure also provides the use of a tag moiety or tag construct of the present disclosure, or a conjugate of the present disclosure, in the manufacture of a medicament for the treatment of cancer prevention.

[0090] Similarly, the present disclosure provides a method of treating or preventing an infectious disease, the method comprising administering to a subject a tag moiety or tag construct of the present disclosure, a conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0091] The present disclosure also provides the use of a tag moiety or tag construct of the present disclosure, or a conjugate of the present disclosure, in the manufacture of a medicament for the treatment of prophylaxis of an infectious disease.

[0092] The present disclosure also provides a method for treating or preventing an autoimmune disease, the method comprising administering to a subject a tag moiety or tag construct of the present disclosure, a conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0093] The present disclosure also provides the use of a tag moiety or tag construct of the present disclosure, or a conjugate of the present disclosure, in the manufacture of a medicament for the treatment of prophylaxis of an autoimmune disease.

[0094] Throughout the above embodiments, reference to the use of a conjugate of the present disclosure includes the combined use of a binding molecule of the present disclosure (e.g., comprised in a bispecific conjugate) and a tag moiety or tag construct, which are administered separately or sequentially.

[0095] In an alternative embodiment, the conjugates of the present disclosure can be used in gene therapy. In this embodiment, a gene therapy vector or delivery system encoding both the binding molecule of the present disclosure (e.g., contained in a bispecific conjugate) and the tag moiety or tag construct of the present disclosure can be administered to a subject. Upon uptake by the subject's cells, the binding molecule and tag are expressed and secreted, forming a complex in vivo.

[0096] As described above, the tag moiety or tag construct of the present disclosure, or the conjugate of the present disclosure, can be used as a monotherapy or in combination with other therapeutic agents. Thus, in the treatment of cancer, the other therapeutic agent can be an anti-cancer agent, e.g., a chemotherapeutic agent, many classes of which are known in the art, or an immunological agent, including, for example, interferon, immune checkpoint inhibitors (e.g., anti-PD-1, -PD-L1, or -CTLA4 antibodies), and other immune-enhancing agents (e.g., anti-OX40 agonist antibodies). Other therapeutic agents, such as antiproliferative or anti-inflammatory cytokines, as well as antiproliferative, immunomodulatory, or blood coagulation-influencing factors, or angiogenesis inhibitors, can be beneficial in the treatment of cancer or infectious diseases. For the treatment of infectious diseases, the other (or second) therapeutic agent can be an antimicrobial agent, e.g., an antibiotic, antifungal, or antiviral agent.

[0097] The tag moiety, tag construct, conjugate, or pharmaceutical composition comprising the tag moiety, tag construct, or conjugate (or a component of the conjugate) can be administered via one or more routes of administration using one or more of a variety of methods known in the art. Similarly, the binding molecule and tag construct can be administered separately by these same methods. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, e.g., directly at the site of a tumor, e.g., by injection or infusion.

[0098] As used herein, the phrase "parenteral administration" refers to a form of administration other than enteral and topical administration, usually by injection. Alternatively, parenteral routes such as topical, epidermal, or mucosal administration routes may be used. Local administration is preferred, including peritumoral, near-tumoral, intratumoral, intralesional, perilesional, intracavity injection, intravesical administration, and inhalation. However, the tag moiety, tag construct, complex, or composition may also be administered systemically.

[0099] In embodiments in which the binding molecule (e.g., comprised in a bispecific conjugate) and the tag moiety or tag construct are administered separately, i.e., they form a complex without first being premixed, they must be administered via the same route. Preferably, they are both administered topically, e.g., intradermally, to the same (or substantially the same) site so that the two components mix and thus combine to form a complex rapidly after administration. In these embodiments, the two components must be administered to a subject simultaneously or quickly one after the other to avoid a delay between the administration of the first component and the administration of the second component. This ensures that the second component is administered and allows complex formation before the first component has time to degrade or diffuse excessively away from the administration site.

[0100] Suitable dosages of the specific tag moieties or tag constructs of the present disclosure, or the conjugates of the present disclosure, can be determined by those skilled in the art. The actual dosage levels of the active ingredients in the pharmaceutical compositions and products of the present disclosure can be varied to obtain an effective amount of the active ingredient to achieve the desired therapeutic response for a particular subject, i.e., patient, without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular conjugate used, the route of administration, the time of administration, the rate of excretion of the conjugate, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0101] A suitable dose of a tag moiety, tag construct, or conjugate of the present disclosure can be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg of body weight of the patient being treated. For example, a suitable dosage can be about 0.1 μg / kg to about 10 mg / kg of body weight per day, or about 10 μg / kg to about 5 mg / kg of body weight per day.

[0102] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions into dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the necessary pharmaceutical carrier.

[0103] The tag moiety, tag construct, or conjugate (or a combination of a binding molecule and a tag construct) may be administered in a single dose or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different sites. Alternatively, the conjugate may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency may vary depending on the half-life of the administered species in the patient and the desired duration of treatment. The dosage and frequency of administration may also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage may be administered at relatively infrequent intervals over an extended period of time. In therapeutic applications, a relatively high dosage may be administered, for example, until the patient shows partial or complete improvement in disease symptoms. In an exemplary dosage regimen, the conjugate (or combination of the conjugate and tag construct) is administered to a subject once a week, once every two weeks, or once every three weeks, in a cycle repeated 2 to 10 times.

[0104] The combined administration of two or more agents can be achieved in several different ways. In one embodiment, the conjugate and the other agent can be administered together in a single composition. In another embodiment, the conjugate and the other agent can be administered in separate compositions as part of a combination therapy. For example, the conjugate can be administered before, after, or simultaneously with the other agent. The conjugates of the present disclosure can be administered in combination with or sequentially with tumor-targeting antibodies, targeted therapies, pathway inhibitors, or other immunomodulatory antibodies targeting, for example, PD-1, PD-L1, CD137, GITR, OX40, CTLA-4, CD27, HVEM, LTβR, and LAG3. Furthermore, the conjugates can also be combined with local radiation. Similarly, such additional therapies can be co-administered when the conjugate and tagged construct are administered separately to the subject.

[0105] The present disclosure also provides an in vitro or ex vivo method for activating T cells that express a TCR that recognizes an antigen, the method comprising: i) a tag construct of the present disclosure and a binding molecule described herein (e.g., comprised in a bispecific conjugate), wherein the tag construct comprises an antigen recognized by the TCR; or ii) contacting with a complex of the present disclosure, wherein the tag construct of the complex comprises an antigen recognized by the TCR.

[0106] Thus, the conjugates of the present disclosure can be used to activate APCs in vitro or ex vivo, as well as in vivo. Thus, the conjugates have both medical and non-medical uses, and all such uses are encompassed herein. For example, isolated or cultured APCs can be contacted with the conjugate, for example, in a laboratory setting, for example, for research, development, or testing purposes. This can be achieved by premixing the conjugate and tag construct to form a complex, and then applying the complex to the APC. Alternatively, the conjugate and tag construct can be applied separately to the APC so that the complex is formed within the APC culture.

[0107] The complex can be used to activate T cells expressing a TCR that recognizes the antigen present in the tag construct. Specifically, the TCR recognizes the antigen when presented by an APC (i.e., in the context of an MHC). Thus, an APC activated by the complex, or activated for activation by the complex, can be contacted with a T cell. Thus, for example, an APC can be cultured or incubated in the presence of the complex (or a component thereof), and then the APC can be contacted with a T cell, e.g., co-cultured, or further incubated in the presence of the T cell. Alternatively, the complex (or a component thereof), APC, and T cell can be incubated or co-cultured together. Thus, the antigen is delivered to the APC and presented to the T cell, resulting in their activation.

[0108] The invention is further illustrated by the following non-limiting figures and examples. [Brief explanation of the drawings]

[0109] [Figure 1A] 1 shows four sensorgrams obtained from SPR measurements of the interaction of tag constructs with the bispecific binding molecule SP027, as described in Example 2. The tag constructs were the following peptides: A) UU0179 (SEQ ID NO: 30), B) UU0185 (SEQ ID NO: 31), C) UU0186 (SEQ ID NO: 30), and D) UU0086 (SEQ ID NO: 25). The amino acid sequence of the scFv in the SP027 construct is represented by SEQ ID NO: 107. [Figure 1B] 1 shows four sensorgrams obtained from SPR measurements of the interaction of tag constructs with the bispecific binding molecule SP027, as described in Example 2. The tag constructs were the following peptides: A) UU0179 (SEQ ID NO: 30), B) UU0185 (SEQ ID NO: 31), C) UU0186 (SEQ ID NO: 30), and D) UU0086 (SEQ ID NO: 25). The amino acid sequence of the scFv in the SP027 construct is represented by SEQ ID NO: 107. [Figure 1C]1 shows four sensorgrams obtained from SPR measurements of the interaction of tag constructs with the bispecific binding molecule SP027, as described in Example 2. The tag constructs were the following peptides: A) UU0179 (SEQ ID NO: 30), B) UU0185 (SEQ ID NO: 31), C) UU0186 (SEQ ID NO: 30), and D) UU0086 (SEQ ID NO: 25). The amino acid sequence of the scFv in the SP027 construct is represented by SEQ ID NO: 107. [Figure 1D] 1 shows four sensorgrams obtained from SPR measurements of the interaction of tag constructs with the bispecific binding molecule SP027, as described in Example 2. The tag constructs were the following peptides: A) UU0179 (SEQ ID NO: 30), B) UU0185 (SEQ ID NO: 31), C) UU0186 (SEQ ID NO: 30), and D) UU0086 (SEQ ID NO: 25). The amino acid sequence of the scFv in the SP027 construct is represented by SEQ ID NO: 107. [Figure 2] Figure 1 shows the results of an in vitro screen of endogenous antibodies against a panel of tag constructs containing the indicated trimmed versions of the tag moiety compared to the original 18-mer peptide tag moiety UU0024 (SEQ ID NO: 84), as described in Example 3. The figure is divided into three sections, each showing the results for one of three serum donors. [Figure 3] Figure 1 shows a summary of the in vitro CD4+ T cell proliferation potential of tag constructs UU0060 (SEQ ID NO: 97), UU0129 (SEQ ID NO: 28), UU0130 (SEQ ID NO: 39), and UU0131 (SEQ ID NO: 35), each comprising a tag moiety disclosed herein together with a cargo moiety comprising the peptide OTII antigen sequence SEQ ID NO: 19, when used at the concentrations indicated in combination with the bispecific binding molecule SP019 described in Example 4. Y-axis: % proliferating OTII cells. X-axis: tag constructs tested, positive control (ConA), and negative control (mAb). [Figure 4]

[0023] Figure 1 shows the results of enzyme-linked immunospot (ELISpot) assays of T cell responses in the four indicated donors, A-D, to tag constructs containing the tag partial peptide UU0086 (SEQ ID NO: 2), UU0002 (SEQ ID NO: 24), UU0008 (SEQ ID NO: 100), UU0152 (SEQ ID NO: 98), or UU0165 (SEQ ID NO: 99) at 10 μM, as described in Example 5. T cell responses are presented as SFU per 3 x 10 cells on the y-axis of each plot (error bars are shown) 24 hours after stimulation with the tag construct tested. [Figure 5]

[0023] Figure 1 shows the results of an in vivo T cell proliferation assay in mice, as described in Example 6. The organs harvested and tagged constructs are indicated on the x-axis. The T cell immune response is illustrated by the percentage of proliferating Thy1.1 cells in each respective organ, shown on the y-axis. From left to right within each organ group, the tagged constructs used in the immunization procedure were UU0032 (SEQ ID NO: 102) (negative control), UU0030 (SEQ ID NO: 101), UU0120 (SEQ ID NO: 103), UU0142 (SEQ ID NO: 104), and UU0146 (SEQ ID NO: 105). [Figure 6]

[0023] Figure 1 shows the results of an in vivo T cell proliferation assay in draining lymph nodes harvested from mice, as described in Example 7. The constructs tested are indicated on the x-axis. The T cell immune response is illustrated by the percentage of proliferating CD8+Thy1.1+ T cells for each respective construct and is indicated on the y-axis. The tagged constructs used in the immunization procedure were UU0030 (SEQ ID NO: 101), UU0120 (SEQ ID NO: 103), UU0146 (SEQ ID NO: 105), UU0142 (SEQ ID NO: 104), and UU0032 (SEQ ID NO: 102) (negative control). [Figure 7]

[0023] Figure 1 shows the results of in vivo T cell expansion as described in Example 9. After administration of the indicated constructs, relevant T cell populations were quantified in the indicated organs by flow cytometry and given as CFSElow percentages of CD3+ and CD4+ cells. [Figure 8] 1 is a survival graph showing the survival probability in the three groups of mice indicated in the experiment described in Example 10. HD: high dose. LD: low dose. [Figure 9] Figure 9 shows the results of the in vivo T cell proliferation assay described in Example 8. Following administration of the indicated constructs, single cell suspensions were prepared from organs. CD4+ T cell proliferation, as defined by CFSElow, and CD4+ T cell activation, as defined by ICOS expression, were assessed, and the results are shown in Figures 9A and B, respectively. The y-axis indicates the cell marker used. Constructs used in the immunization procedure, from left to right on the x-axis, were "vehicle" (negative control), UU0060 (SEQ ID NO: 97), UU0126 (SEQ ID NO: 27), UU0127 (SEQ ID NO: 38), and UU0128 (SEQ ID NO: 34). [Figure 10] Figure 1 shows the difference in anti-tumor efficacy (illustrated as tumor growth volume, mm3) between mice immunized with the bispecific binding conjugate SP027 and either a tag construct (UU0170, SEQ ID NO: 147) comprising a 7-mura tag portion and an immunogenic epitope derived from the E7 antigen (SEQ ID NO: 140) as the cargo portion, or a tag construct (UU0169, SEQ ID NO: 141) comprising a 9-mura tag portion and an immunogenic epitope derived from the E7 antigen (SEQ ID NO: 140) as the cargo portion, all compared to mice receiving vehicle (negative control). [Example]

[0110] The following examples disclose the development of novel, improved, non-immunogenic tag moieties generated by utilizing advanced binding experiments and methodologies. The comprehensive amino acid sequences of the tag moieties described herein are listed in the Sequence Listing as SEQ ID NO: 1. The examples further describe the characterization of different variants of the tag moieties, as well as tag constructs and complexes containing them, and demonstrate their in vitro and in vivo functionality.

[0111] Example 1 Surface plasmon resonance analysis of trimmed tagged portions Previously presented data suggest that a tag construct containing an 18-amino acid residue (aa) long peptide tag portion (UU0024, SEQ ID NO: 84) can be shortened to a 12-aa long tag portion and retain its binding affinity to the IBIIICI murine scFv containing SEQ ID NO: 15 (see Example 17 on pp. 67-68 of WO 2021 / 239968). Below, the binding kinetics of a selection of further trimmed peptide tag portions were examined using surface plasmon resonance (SPR). The 18-aa long peptide tag portion was shortened to 11, 10, 9, 8, and 7-aa long peptides, and the binding kinetics to a humanized variant of IBIIICI, e.g., the one designated "IBIIICI CDR-grafted scFv SG" containing the amino acid sequence SEQ ID NO: 107, was investigated.

[0112] Materials and Methods Affinity measurements of IBIIICI CDR-grafted scFv variants SG (SEQ ID NO: 107), GG (SEQ ID NO: 108), QG (SEQ ID NO: 109), DG (SEQ ID NO: 110), NA (SEQ ID NO: 111), and NG (SEQ ID NO: 112) for peptides UU0024, UU0084-UU0088, UU0090-UU0092, and p003 (Table 1) were performed by SPR using a Biacore T200 instrument (GE Healthcare) and single-cycle kinetics. Anti-FLAG M2 antibody (Sigma-Aldrich, #F3165) was immobilized on a Series S CM5 chip by primary amine coupling using NHS-EDC chemistry according to the manufacturer's instructions, allowing capture of each scFv via its FLAG tag. A 5-fold dilution series containing five different peptide concentrations (0.16 nM to 100 nM) was sequentially injected into the flow cell to allow binding to the captured scFv. After the dissociation step, surface regeneration was achieved under acidic conditions using 10 mM glycine-HCl, pH 2.1. Response unit sensorgrams for all peptides were obtained by subtracting the response curve of a reference surface with only anti-FLAG antibody immobilized thereon. Data were analyzed using BIAeval v.3.1 (GE Healthcare).

[0113] [Table 1]

[0114] result The kinetic parameters of the binding of IBIIICI CDR-grafted scFv variant SG (SEQ ID NO: 107) to different peptides are shown in Table 2. As described above in connection with Example 17 of WO2021 / 239968, the inventors previously showed that the binding of IBIIICI murine scFv was not affected by shortening the tag portion peptide length from 18 to 12 amino acids. The results here suggest that the tag portion can be further shortened. Indeed, the binding of IBIIICI CDR-grafted scFv variant SG was maintained even when the peptide was shortened to 8 aa. Peptide UU0091, containing an 8 aa tag portion FIGITELK (SEQ ID NO: 90) fused at the C-terminus with the ovalbumin epitope SIINFEKL, exhibited retained binding affinity for IBIIICI CDR-grafted scFv variant SG. However, by reducing the length of the tag portion to 7 aa (FIGITEL, SEQ ID NO: 89) or mutating position 8 of the peptide from K to H (FIGITELH, SEQ ID NO: 91), binding was retained, but the affinity was significantly reduced (more than 10-fold, as shown in a pairwise comparison of the results obtained with UU0090 (SEQ ID NO: 89) and UU0091 (SEQ ID NO: 90)).

[0115] Furthermore, the results suggest that the shortened tag moiety UU0091 can be modified at the C-terminus without loss of binding to the IBIIICI CDR-grafted scFv variant SG. In contrast, modification of the N-terminus causes a complete loss of binding to the scFv (UU0088, SEQ ID NO: 88). Similar binding properties were observed for all other IBIIICI CDR-grafted scFv variants tested (data not shown).

[0116] [Table 2]

[0117] Example 2 SPR analysis of binding of scFv-containing conjugates to tag constructs containing a 9aa tag moiety and a C-terminal antigen The binding kinetics of a panel of tag constructs comprising a 9 aa peptide tag moiety, with or without the addition of an antigenic cargo sequence at the C-terminus of the 9 aa tag moiety, to the IBIIICI CDR-grafted scFv variant SG in the context of a bispecific conjugate was examined using SPR.

[0118] Materials and Methods The IBIIICI CDR-grafted scFv variant SG (SEQ ID NO: 107) was placed into a conjugate in a context analogous to the constructs described in WO2020 / 104690 and WO2021 / 239968. Briefly, the scFv was designed to form part of a bispecific conjugate construct in which one copy of the peptide-binding scFv was conjugated to each heavy chain of another binding molecule, e.g., a full-length antibody against CD40. See Figure 6A of WO2020 / 104690 for a schematic diagram of the construct design. The anti-CD40 antibody used in this example is a variant of the antibody designated "A9" in WO2021 / 239968. The bispecific conjugate construct herein was designated SP027. SP027 comprises two copies of one polypeptide chain having SEQ ID NO: 113 constituting the heavy chain of the A9 anti-CD40 antibody linked to scFv variant SG, and two copies of one polypeptide chain having SEQ ID NO: 114 constituting the light chain of the anti-CD40 antibody.

[0119] The tag constructs tested contained a 9-aa tag subsequence with or without an attached cargo sequence. Affinity measurements of the binding of tag constructs UU0086 (SEQ ID NO: 2), UU0179 (SEQ ID NO: 30), UU0185 (SEQ ID NO: 31), and UU0186 (SEQ ID NO: 32) to the bispecific construct SP027 were performed by SPR using a Cytiva BiaCore 8K instrument (Cytiva) and a multi-cycle kinetic approach. The bispecific constructs were immobilized on a Series S CM5 chip by primary amine coupling using NHS-EDC chemistry (Cytiva, #BR100633). A two-fold dilution series comprising six concentrations (15.6 nM to 1000 nM) of the tested tag constructs UU0086, UU0179, UU0185, and UU0186 was sequentially injected through the flow cell and allowed to bind to the captured antibody. After the dissociation step, surface regeneration was achieved under acidic conditions using 10 mM HCl, pH 2.1. Response unit sensorgrams for all peptides were obtained by subtracting the response curve of a reference surface with immobilized SP027. Data were analyzed using BIAevaluate software (Cytiva) using a multi-cycle kinetic model for fitting 1:1 binding events.

[0120] result The resulting kinetic properties of the tag constructs tested are shown in Figure 1 (A: UU0179, B: UU0185, C: UU0186, and D: UU0086). Overall, the binding properties of the tag construct containing only the 9aa peptide tag portion (UU0086) were very similar to those of the tag construct containing the 9aa peptide tag portion together with a cargo portion, which is herein a peptide sequence. The similar binding properties shown in the four panels of Figure 1 suggest that the addition of a cargo portion at the C-terminus of the 9aa tag portion does not affect the binding affinity of the tag portion with the IBIIICI CDR-grafted scFv variant SG.

[0121] Example 3 Investigating the presence of endogenous antibodies against different tag moieties in donor plasma The original 18-mer tag portion is of non-human origin and contains a known B-cell epitope. Therefore, recipients of the tag construct according to the present disclosure may have pre-existing endogenous antibodies against the tag portion, limiting its usefulness due to the risk of antibody binding competition. Therefore, the amount of endogenous antibodies in donor plasma against tag constructs containing trimmed versions of the original tag portion was investigated and compared with the amount of endogenous antibodies against tag constructs containing the original 18-mer peptide sequence UU0024 (SEQ ID NO: 84).

[0122] Materials and Methods Sera from three healthy human donors were used to screen for endogenous binding to the tag moiety in ELISA experiments. On day 0, exemplary tag constructs containing 9-mer, 10-mer, and 11-mer tag moieties (see Table 3) were diluted to 1 μM in PBS. 100 μl of each diluted tag construct was added to a pre-coated streptavidin plate and incubated overnight at 4°C. On day 1, the plate was washed three times with PBS containing 0.05% Tween-20. This was repeated after each incubation step, followed by the addition of plasma samples, secondary antibodies, and TMB (3,3',5,5'-tetramethylbenzidine). Plate wells were blocked with PBS containing 10% BSA and 0.05% Tween-20 for 2 hours at room temperature before the addition of donor human plasma. Before addition to the wells, donor human plasma was serially diluted 1:1 in PBS containing 1% BSA and 0.05% Tween-20, starting at 1:200 and ending at 1:1600. After dilution, 100 μl of donor human plasma was added to each well and incubated for 1 hour at room temperature. The final step was the addition of a 1:4000 dilution of total IgG secondary antibody-HRP (Dako, #P0214) in PBS containing 1% BSA and 0.05% Tween-20 to each well, followed by incubation for 1 hour at room temperature. The plate was developed with TMB, and the reaction was stopped by the addition of 1 M H2SO4. The absorbance at 450 nm was measured.

[0123] [Table 3]

[0124] result Endogenous antibodies bound to each tag construct were measured at 450 nm for the tag constructs listed in Table 3. The results are shown in Figure 2 and indicate that sera from all three donors contained pre-existing endogenous antibodies that bound to the 18-aa peptide tag portion (UU0024) containing a known B cell epitope. The lowest endogenous antibody reactivity was observed with constructs containing a 9-aa-long peptide tag portion (exemplified by UU0135, UU0154, and UU0155). Point mutations in the 9-aa constructs (UU0154, mutation K8L; UU0155, mutation K8H) had different effects on endogenous antibody reactivity. The K8L mutation in the 9-aa peptide tag (UU0154) clearly increased endogenous reactivity in two of the donors compared with the original 9-aa-long peptide tag portion (UU0135). In contrast, the K8H mutation in the 9aa peptide tag (UU0155) did not induce endogenous antibody binding compared to the original 9aa-long peptide tag portion (UU0135). Compared to the unmutated 9aa-long peptide tag portion (UU0135), higher endogenous antibody reactivity was observed in all donors with constructs containing 10aa and 11aa-long tag portions (UU0136, UU0148, UU0137, and UU0149). The K8L mutation in the 10aa and 11aa-long tag portions, in particular, resulted in endogenous reactivity in all three donors. Donor serum samples showed no endogenous binding to the negative control (data not shown).

[0125] Example 4 T cell proliferation potential of tagged constructs measured in a T cell proliferation assay The ability of tagged constructs to stimulate CD4+ T cell proliferation was tested. The tagged constructs tested were UU0060 (SEQ ID NO: 97), UU0129 (SEQ ID NO: 28), UU0130 (SEQ ID NO: 39), and UU0131 (SEQ ID NO: 35), each containing a tag moiety as disclosed herein with a cargo moiety containing the peptide OTII antigen sequence SEQ ID NO: 19. The tagged constructs were used in complex with a bispecific construct designated SP019 and evaluated using CD4+ OTII cells, which express a T cell receptor specific for the MHC class II-presented chicken ovalbumin peptide 323-339.

[0126] Materials and Methods Bone marrow cells isolated from hCD40 transgenic mice (provided by the University of Southampton and owned by Dartmouth College) were differentiated into immature myeloid dendritic cells (imBMDCs) in 8-day culture with GM-CSF (20 ng / ml, Peprotech, 315-03). Cells were harvested on day 8, and cell maturation was verified by flow cytometry for the expression of CD11b (antibody M1 / 70, Biolegend), CD11c (antibody N418, Biolegend), human CD40 (antibody 5C3, Biolegend), CD86 (antibody GL-1, Biolegend), and MHC-II (antibody M5 / 114.15.2, Biolegend). imBMDCs were cultured at 5 × 10 cells. 5 The cells were diluted to a concentration of 1000 cells / ml and plated at 50 μl / well in a 96-well tissue culture-treated plate in the presence of GM-CSF (60 ng / ml, Peprotech).

[0127] The bispecific binding construct SP019 used in this experiment is similar to the SP027 construct used in Example 2 and comprises two copies of one polypeptide chain with SEQ ID NO: 113 constituting the heavy chain of the A9 anti-CD40 antibody linked to the scFv variant SG, and two copies of one polypeptide chain with SEQ ID NO: 115 constituting the light chain of the A9 anti-CD40 antibody.

[0128] SP019 (10 nM) was mixed separately with each of the peptides UU0060, UU0129, UU0130, and UU0131 (at two concentrations of 100 and 20 nM, respectively) and preincubated for 30 min at room temperature. Different pairwise mixes of bispecific binding constructs and peptides were added to imBMDCs and incubated for 2 h at 37°C with 5% CO2. Unbound constructs and free peptides were removed by washing the cells twice at 300 g for 5 min.

[0129] CD4+ OT-II cells were isolated from the spleen and inguinal lymph nodes of OTII transgenic mice. The organs were made into single-cell suspensions, and red blood cells were lysed with RBC lysis buffer (Invitrogen, 00-4333-57). CD4+ cells were isolated using the Dynabeads™ Untouched™ Mouse CD4 Cell Kit (Invitrogen, 11415D). Isolated CD4+ OT-II cells were stained with CFSE (2 μM, Invitrogen, C34570) and co-cultured with 25,000 BMDCs per 50,000 CD4+ OT-II cells at 37°C with 5% CO2 for 96 hours. After co-culture, cells were harvested and stained with anti-CD3, anti-CD4, and ICOS, and proliferation was assessed by flow cytometry.

[0130] result The results are shown in Figure 3. Peptides UU0060, UU0129, UU0130, and UU0131 all induced greater than 90% OTII T cell proliferation as determined by CFSE dilution. Two proliferation controls, ConA (positive control) and mAb (negative control IgG2 antibody), were included and analyzed in the proliferation assay. mAb alone did not induce any cell proliferation. The data demonstrate that tag constructs containing 9-Mur tag moieties (UU0129, UU0130, and UU0131) promote the same level of CD4+ T cell proliferation as tag constructs containing the 18-Mur tag moiety sequence UU0060 and the positive proliferation control ConA.

[0131] Example 5 Analysis of peptide-specific immune responses by ELISpot The peptide-specific immune response to the tag moiety alone was compared to that of the tag moiety in the tag construct context (tag moiety together with the antigen moiety) using an enzyme-linked immunospot (ELISpot) assay.

[0132] Materials and Methods Peripheral blood mononuclear cells (PBMCs) from four healthy donors were tested for reactivity to peptides UU0086 (SEQ ID NO: 2), UU0002 (SEQ ID NO: 24), UU0008 (SEQ ID NO: 100), UU0152 (SEQ ID NO: 98), and UU0165 (SEQ ID NO: 99). PBMCs were isolated from freshly drawn buffy coats (<8 hours after collection) via density gradient centrifugation using Ficoll-Paque™ Premium (Cytiva, 17-5442-03) and SepMate™ tubes (STEMCELL Technologies, 85450). Blood was diluted 1:1 with 1x PBS and added on top of the density gradient medium. After centrifugation (1200g, 10 min), the PBMC layer was poured into a new tube and washed twice with 1x PBS. The obtained PBMCs were stored frozen at -150°C in 90% FBS / 10% DMSO until further use.

[0133] Cryopreserved PBMCs were thawed in RPMI1640 GlutaMAX™ medium (Gibco, 61870036) supplemented with 10% FBS, 1% penicillin-streptomycin, 1% HEPES, and 50 U / ml Pierce™ nuclease (Thermo Scientific, 88701), washed, and resuspended in the same medium (minus the nuclease) to a concentration of 2 × 10 cells. 6 The cells were incubated at a concentration of 1000 cells / ml for 2 hours at 37°C in 5% CO2. During cell resting, a pre-coated ELISpot plate (anti-IFNγ, Mabtech, 3420-4APT) was washed and blocked. After resting, the cells were harvested and 3 x 10 cells were added. 5The cells were seeded onto ELISpot plates at a concentration of 10 ...

[0134] result The results are shown in Figure 4. In all four donors tested, 24-hour stimulation with peptides UU0002 (SEQ ID NO: 24), UU0008 (SEQ ID NO: 100), and UU0152 (SEQ ID NO: 98) induced IFNγ secretion, indicating that all four donors responded to NLV peptides derived from CMV. UU0086 (SEQ ID NO: 2), i.e., the tag portion alone, did not induce spot formation in any of the donors tested. Two of the four donors responded to peptide UU0165 (SEQ ID NO: 99), a mutant peptide derived from the oncogenic KRAS protein. These data demonstrate that tag constructs comprising the disclosed 9-mer tag portion linked to an antigenic peptide induce peptide-specific immune responses. Antigen-peptide-specific immune responses directed against the T cell antigenic portion of a synthetic peptide extension comprising a tag portion and a T cell epitope vary due to individual intrinsic factors. The data also demonstrate that the tag moiety alone (UU0086) does not induce a measurable T cell response.

[0135] Example 6 In vivo T cell proliferation Utilizing an in vivo T cell proliferation experimental setup, the immune response of mice to a tagged construct in the context of a complex with an antibody-scFv conjugate delivered together with a tagged construct comprising a tag moiety and an antigen moiety was analyzed compared to the immune response of mice to a tagged construct lacking the tag moiety sequence (i.e., using an antibody-scFv conjugate delivered together with only the antigen moiety without any tag moiety sequence).

[0136] Materials and Methods This experiment was conducted to evaluate the immunogenic potential of tag constructs containing cargo moieties containing the antigenic sequence exemplified by human melanoma gp100 (SEQ ID NO: 20) along with tag moieties of a range of lengths. The tag constructs used were UU0030 (SEQ ID NO: 101), UU0146 (SEQ ID NO: 105), UU0142 (SEQ ID NO: 142), and UU0120 (SEQ ID NO: 103), containing tag moiety lengths of 18, 11, 10, and 9 aa, respectively. In the tag constructs, the tag moiety sequence was C-terminally linked to the N-terminus of the antigen sequence and delivered to mice in a complex context, i.e., the tag construct was delivered together with the bispecific antibody construct SP019 (see Example 4). A negative control was utilized in the form of UU0032 (SEQ ID NO: 102), which lacks the tag moiety sequence and contains only the cargo moiety gp100. UU0032 was also delivered together with the bispecific construct SP019.

[0137] Twenty mice (B-hCD40, Biocytogen) were divided into five groups (four test groups and one control group), each consisting of four mice. On day -1 (pre-immunization), cells were isolated from the spleen and inguinal lymph nodes of pmel-1 transgenic mice (pmel-1). CD8+ T cells in pmel-1 transgenic mice express a T cell receptor specific for the melanoma antigen gp100 (SEQ ID NO: 20). Erythrocytes were lysed with RBC lysis buffer (Invitrogen, #00-4333-57), and single-cell suspensions were prepared from harvested organs. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and 11 x 10 cells were collected in 100 μl of 1x sterile PBS.6 CFSE-labeled pmel-1 cells were transferred into B-hCD40 mice by intravenous injection. On day 0, one day after adoptive transfer of pmel-1 cells, 20 B-hCD40 mice were immunized by subcutaneous (sc) needle injection into the heel joint according to the settings presented in Table 4. Each mouse received a 20 μl injection containing 15 pmol of the bispecific construct SP019 and 37.5 pmol of the tag construct.

[0138] [Table 4]

[0139] To assess the outcome of in vivo T cell proliferation, organs (spleen, draining and non-draining popliteal and inguinal lymph nodes) were harvested and collected on day 4 post-immunization. Single cell suspensions were prepared from the harvested organs and 1 × 10 cells were collected per organ. 6 Cells were stained for Thy1.1, CD3+, and CD8+, and antigen-specific T cell proliferation was assessed by flow cytometry using the congenic marker Thy1.1 as a tracker.

[0140] result The results are shown in Figure 5, where the harvested organs are displayed on the x-axis and the immune response is illustrated by the percentage (%) of proliferating Thy1.1 cells on the y-axis. All tested tag constructs, including tag subsequences exemplified by UU0030 (18-mer), UU0120 (9-mer), UU0142 (10-mer), and UU0146 (11-mer), induced higher Thy1.1 CD8+ T cell proliferation than tag constructs without any tag subsequences (cargo moiety only), exemplified by UU0032. This was observed in all harvested organs, and the data indicate that the targeted delivery system used induces a potent immune response compared to cargo moiety administration alone. Interestingly, the construct containing the shortest tag subsequence (9 aa in length, UU0120) elicited a significantly higher % T cell response in a length-dependent manner and was the only tag moiety that generated proliferating cells in the spleen at the doses evaluated in the repeated dosing schedule. UU0120 induced 35%, 35%, and 20% Thy1.1 CD8+ T cell proliferation in the inguinal drainage, popliteal drainage, and spleen, respectively.

[0141] Example 7 In vivo T cell proliferation This experiment was conducted to evaluate the immunogenic potential of cargo moieties containing antigenic peptides, exemplified by human melanoma gp100, delivered with 18-mer or 9-mer tag moieties attached to the C-terminus of the cargo moiety. The immune responses induced by these combinations were compared to those induced by gp100 peptides delivered with CpG ODN1826 as a benchmark adjuvant.

[0142] Materials and Methods Thirty mice, strain B-hCD40 (Biocytogen), were divided into test and control groups with 3-5 mice per group. On day -1, one day before the start of immunization, cells were isolated from the spleen and inguinal lymph nodes of pmel-1 transgenic mice (pmel-1). CD8+ T cells from pmel-1 transgenic mice express T cell receptors specific for MHC class I epitopes derived from the known immunogenic melanoma gp100. Red blood cells were lysed with RBC lysis buffer (Invitrogen, 00-4333-57), and single-cell suspensions were prepared from the spleen and inguinal lymph nodes. 1 x 10 cells were collected in 100 μl of 1x sterile PBS. 7 pmel-1 cells were transferred into B-hCD40 mice by intravenous injection. Mice were immunized twice with 5 days between immunizations according to the scheme presented in Table 5, with the first immunization starting on day 0, one day after pmel-1 cell adoptive transfer. The bispecific antibody SP019 and peptides UU0032 (SEQ ID NO: 102), UU0030 (SEQ ID NO: 101), and UU0120 (SEQ ID NO: 103) were each mixed separately with SP019 before injection. Treatments were administered by subcutaneous needle injection into the right heel joint in a total volume of 20 μl. Groups of mice were immunized according to the following scheme:

[0143] [Table 5]

[0144] For the second immunization, 150 pmol of the bispecific antibody SP019 was used in the antibody-peptide mixture according to the same configuration.

[0145] Draining popliteal and inguinal lymph nodes were harvested 2 days after the second immunization to assess T cell proliferation. Single cell suspensions were prepared from the organs and diluted to 1 x 10 6 The cells were stained for Thy1.1, CD3+, and CD8+, and T cell proliferation was assessed by flow cytometry.

[0146] result The results are shown in Figure 6. Peptide UU0032 (gp100 without a tag portion) did not induce any immune response when delivered alone or together with SP019. Peptide UU0030 (gp100 with an 18-Mur tag portion) did not induce any immune response when delivered together with SP019. In contrast, peptide UU0120 (gp100 with a 9-Mur tag portion) induced 10% Thy1.1+CD8+ T cell proliferation in the draining lymph nodes. This level of immune induction is similar to the immune response detected with UU0032 delivered at a 10x higher dose together with CpG ODN1826. The data demonstrate that gp100 peptides conjugated to the 9-Murtag moiety and delivered with the bispecific antibody construct SP019 induce significantly higher and more potent immune responses compared to the immune responses of gp100 peptides conjugated to the 18-Murtag moiety and delivered with the bispecific antibody construct SP019.

[0147] Example 8 In vivo T cell proliferation The immune response induced by tag constructs in combination with the bispecific binding conjugate SP027 (see Example 2) was evaluated. All tag constructs used in this experiment contain a synthetic long peptide corresponding to a known immunogenic epitope from ovalbumin (SEQ ID NO: 19). The amino acid sequence and SEQ ID NO for each respective tag construct, as well as the immunogenic epitope from ovalbumin, are detailed in Table 6. Experiments were performed to evaluate the immunogenic potential of tag constructs comprising an ovalbumin-derived antigenic peptide and a tag moiety of different length or sequence (either an 18-Mur tag moiety or a 9-Mur tag moiety) attached to the N-terminus of the ovalbumin-derived peptide in complex with SP027.

[0148] Materials and Methods Adult htgCD40 mice (10-11 weeks old) from Biocytogen were divided into test and control groups (n = 3-5). Cells were isolated from the spleen and inguinal lymph nodes of OT-II transgenic mice on day -1, one day before the start of immunization. These mice were CD4 + They express mouse alpha and beta chain T cell receptors paired with co-receptors and are specific for known immunogenic helper epitopes derived from the ovalbumin protein. Single cell suspensions were prepared by passing the spleen and lymph nodes through a 70 μM strainer, and red blood cells (RBCs) in the spleen suspension were lysed with RBC lysis buffer (eBioscience™ Catalog No. 00-4300-54). The remaining cells were stained with CFSE (Thermo Fisher Scientific, C34554) and then diluted with PBS. 1 × 10 cells were collected in 100 μl of sterile PBS. 7 CFSE-stained cells were injected intravenously into htgCD40 mice. On day 0, htgCD40 mice were immunized with vehicle or 150 pmol of SP027 mixed with 450 pmol of each tagged construct: UU0060, UU0126, UU0127, or UU0128. Treatment administration was by subcutaneous injection into the right heel joint in a total volume of 50 μl.

[0149] [Table 6]

[0150] To assess T cell proliferation, draining lymph nodes (popliteal and inguinal) were collected and pooled on day 2 post-immunization. Single cell suspensions were prepared from the organs, and the cells were purified to CD3 + , CD4 + T cell activation was assessed by flow cytometry by staining with , and ICOS (Biolegend) markers.

[0151] result The results are presented in Figure 9 and show CD4 +Upregulation of the activation marker ICOS on T cells results in CFSE low The tag constructs containing the Murtag moiety (UU0126, UU0127, and UU0128) and an immunogenic helper epitope derived from ovalbumin as the cargo moiety, together with the bispecific binding conjugate SP027, showed higher CD4 T cell proliferation compared to vehicle, defined as activation. + It induced an average increase of 3-6 fold in T cell activation compared to a tagged construct containing an 18-mer tag moiety (UU0060) and an immunogenic epitope from ovalbumin as a cargo moiety, delivered together with the bispecific binding conjugate SP027.

[0152] Thus, this example shows that an ovalbumin antigen peptide conjugated to a 9-mer tag moiety contained in a tag construct according to the present invention and delivered with the bispecific antibody construct SP027 induced a higher immune response than an ovalbumin peptide conjugated to a comparative prior art 18-mer tag moiety and delivered with the same bispecific conjugate SP027.

[0153] Example 9 In vivo T cell proliferation A study essentially as described in Example 8 was carried out using test peptide UU0126 (SEQ ID NO: 27) and control peptide UU0138 (SEQ ID NO: 19) in combination with the bispecific conjugate SP027 (see Example 2).

[0154] Materials and Methods Adult htgCD40 mice (n = 7, data pooled from two experiments; 8-14 weeks; provided by the University of Southampton and owned by Dartmouth College) were used for in vivo T cell proliferation assays. CFSE-labeled (10 × 10 6OT-II-derived cells (1000 cells) were injected intravenously via the tail vein. The next day, mice were injected subcutaneously on the right side of the ankle with the SP027 bispecific conjugate (15 pmol) along with either the immunogenic epitope from ovalbumin UU0138 alone (37.5 pmol) or the 9-MurTag moiety (37.5 pmol) linked to the ovalbumin epitope cargo moiety in construct UU0126. The draining popliteal fossa, draining inguinal lymph nodes, non-draining inguinal lymph nodes, and spleens were harvested 2–3 days after the last injection. Organs were passed through a 70 μm cell strainer, and RBCs in the spleen were lysed using RBC lysis buffer before surface marker staining and analysis by flow cytometry.

[0155] result The results are presented in Figure 7 and show that the MHC class II-driven antigen presentation pathway was supported by antibody-mediated peptide delivery to antigen-presenting cells. + The ability to induce T cell activation was assessed using the OT-II model comparing peptide delivery with or without the 9-mer tag moiety defined by SEQ ID NO: 2. The affinity interaction between the tag moiety contained in the tag construct and the bispecific conjugate was shown to mediate the activation of CD4 T cells in the draining lymph nodes. + This proved crucial for T cell proliferation, with a 2- to 7-fold increase shown for UU0126 (a tag construct containing a 9-mer tag moiety and an ovalbumin epitope cargo moiety) when compared with a peptide (ovalbumin epitope) without a tag moiety (UU0138).

[0156] Example 10 Tumor treatment effectiveness We investigated the antitumor response induced by repeated vaccination with a peptide in combination with the bispecific conjugate SP027 in the TC-1 tumor model. By local vaccination at a non-tumor site, a tagged construct containing a tumor-associated antigen (derived from a virus or neoantigen) was delivered to dendritic cells via binding to CD40 of the bispecific conjugate, which then internalized and released the antigenic peptide. The antigenic peptide was processed and presented to T cells. Upon repeated injections, the antigen-specific T cells proliferated, migrated, and targeted tumor cells. The antitumor effect of the complex of the bispecific conjugate and tagged construct was compared with that of the peptide alone and / or the bispecific conjugate alone, similarly injected at a non-tumor site.

[0157] Materials and Methods Mice of strain B-hCD40 (Biocytogen) (9-12 weeks old) were divided into three treatment groups (6-7 mice per group): Group 1: a high dose of 30 μg of antigenic peptide UU0171 (SEQ ID NO: 140, a synthetic long peptide derived from immunogenic E7, HPV16 E7 [HPV16 E7 44-62]); Group 2: bispecific binding conjugate SP027 alone, 50 μg for the first injection on day 5 and 30 μg for injections on days 10 and 15; Group 3: bispecific binding conjugate SP027 complexed with tag construct UU0169 (SEQ ID NO: 141), comprising a 9 aa tag moiety linked to an HPV peptide and administered at a low dose of 3 μg in the conjugate mixture per dose, 50 μg for the first injection on day 5 and 30 μg for injections on days 10 and 15. Approximately 5×10 5 Tumor cells were injected sc into the right flank on day 0. Mice received treatment injections sc into the left ankle on days 5, 10, and 15; no therapy was administered at the site of tumor growth. Tumor growth and survival were monitored three times weekly, with tumor size reaching a maximum of 1000 mm as the experimental endpoint. 3 Mice were sacrificed when they reached a humane endpoint (e.g., health status in terms of wounds, weight, and appearance) or when they reached a humane endpoint. Mice that reached a humane endpoint while not reaching the experimental endpoint were excluded.

[0158] result The results are shown in Figure 8. The TC-1 peptide derived from the oncogenic HPV protein E7, coupled to a 9-muR tag moiety (UU0169) and delivered in a complex with the bispecific conjugate construct SP027, induced a strong immune response and significantly reduced tumor volume compared to control groups receiving either the bispecific conjugate SP027 alone or a high dose of the antigenic peptide without the tag moiety (UU0171), even though the therapy was injected at a low dose at a non-tumor site.

[0159] Example 11 Tumor treatment effectiveness Antitumor efficacy was investigated by immunizing mice with the bispecific binding conjugate SP027 in combination with either tag construct UU0170 (SEQ ID NO: 147) (containing a 7-Mur tag portion, SEQ ID NO: 148) or tag construct UU0169 (SEQ ID NO: 141) (containing a 9-Mur tag portion, SEQ ID NO: 2). Both tag constructs contained cargo moieties corresponding to immunogenic epitopes (SEQ ID NO: 140) derived from the E7 antigen. The tag construct containing the 9-Mur tag portion (UU0169) bound to the bispecific binding conjugate SP027, and targeted peptide delivery was promoted to antigen-presenting cells. The tag construct containing the 7-Mur tag portion (UU0170) had minimal or no binding to the bispecific binding conjugate SP027; therefore, this configuration was considered to represent uncoupled delivery of the tag construct and the bispecific binding conjugate.

[0160] Materials and Methods Sixteen mice (B-hCD40) were divided into three groups of 4–6 mice per group. The murine lung tumor human papillomavirus-16 (HPV-16) E6 / E7 TC-1 cell line was used as a tumor model. TC-1 cells were cultured at a density of 3 × 10 cells in RPMI Glutamax (Gibco, #11360070) supplemented with 10% FBS (Thermofisher, Catalog #10500064), 1% penicillin-streptomycin (Gibco, Catalog #11548876), 1 mM sodium pyruvate (Gibco, Catalog #11360070), and 10 mM HEPES (Gibco, Catalog #11560496). 4 pieces / cm 2 The cells were cultured using a seeding density of 0.5 × 10 in 100 μl of PBS. The cells were split every 2–3 days by using trypsin (Gibco, catalog #11580626). On day 0, 5 days before the start of treatment, 0.5 × 10 cells were split in 100 μl of PBS. 5 TC-1 cells were injected subcutaneously (sc) into the shaved right flank of mice. Mice were treated twice, on days 5 and 10. The bispecific binding conjugate SP027 and tag construct were mixed prior to injection. Treatment was administered by sc needle injection into the left heel joint in a total volume of 50 μl. Groups of mice were treated according to the following configuration: Group 1: 25 mM histidine buffer pH 6; Group 2: 250 pmol of antibody SP027 mixed with 750 pmol of UU0170; Group 3: 250 pmol of antibody SP027 mixed with 750 pmol of UU0169.

[0161] Tumor growth was assessed by determining whether the tumor volume increased or decreased over 1000 mm according to the ellipsoid formula (4 ÷ 3 × π × (width ÷ 2 × length ÷ 2 × height ÷ 2)). 3 The blood pressure was monitored every 2-3 days by measurement with an electronic caliper until the blood pressure reached 0.05°C.

[0162] [Table 7]

[0163] result The results are shown in Figure 10. Tumor inoculation was successful in all animals included in the study. Differences in tumor efficacy (anti-tumor efficacy measured as tumor growth volume) were observed from day 12 onwards.

[0164] SP027 mixed with tagged construct UU0169, which contains a 9-MurTag moiety and an E7 peptide as the cargo moiety, prevented tumor growth and extended mouse survival compared to a control group that received SP027 with tagged construct UU0170, which contains a 7-MurTag moiety and an E7 peptide as the cargo moiety. Mice that received SP027 with a tagged construct containing a 7-MurTag moiety (Group 2, representing non-binding bispecific binding conjugate and tagged construct delivery) had tumor growth similar to mice that received vehicle (Group 1, control group).

[0165] This data demonstrates that targeted delivery of the bispecific binding molecule SP027 (Group 3) with a tag construct comprising a 9-mer tag moiety and a peptide cargo moiety according to the present invention induced superior cellular responses resulting in improved anti-tumor efficacy compared to non-linked tag constructs and bispecific binding conjugates.

[0166] Unofficial sequence listing SEQ ID NO: 1 FIGITELXK SEQ ID NO: 2 FIGITELKK SEQ ID NO: 3 FIGITELLK SEQ ID NO:4 FIGITELHK SEQ ID NO:5 FIGITELK SEQ ID NO:6 FIGITELL SEQ ID NO:7 FIGITELH SEQ ID NO:8 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO:9 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEGGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 10 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 11 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEDGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 12 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 13 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSS SEQ ID NO: 14 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 15 EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSSGGGG SGGGGSGGGGSGGGGSELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK SEQ ID NO: 16 QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPSDNFSNLNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLTVSSGGGGSGGGGS GGGGSGGGGSDIVMTQATPSVLVTPGEAVSISCRASRSLLHSNGITYLYWFLQRPGQSPQVLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEFPYTFGGGTKLEIK SEQ ID NO: 17 NLVPMVATV SEQ ID NO: 18 SIINFEKL SEQ ID NO: 19 ISQAVHAAHAEINEAGR SEQ ID NO: 20 KVPRNQDWL SEQ ID NO: 21 YKLVVVGAVGVGKSALT SEQ ID NO: 22 YKLVVVGARGVGKSALT SEQ ID NO: 23 YKLVVVGADGVGKSALT SEQ ID NO: 24 FIGITELKKAGILARNLVPMVATVQGQNLKY SEQ ID NO: 25 YKLVVVGACGVGKSALT SEQ ID NO: 26 FIGITELKKAAYLEQLESIINFEKLAAAAAK SEQ ID NO: 27 FIGITELKKISQAVHAAHAEINEAGR SEQ ID NO: 28 FIGITELKKAAYISQAVHAAHAEINEAGR SEQ ID NO: 29 FIGITELKK-OH-PEG3-Biotin SEQ ID NO: 30 FIGITELKKYKLVVVGAVGVGKSALT SEQ ID NO: 31 FIGITELKKYKLVVVGARGVGKSALT SEQ ID NO: 32 FIGITELKKYKLVVVGADGVGKSALT SEQ ID NO: 33 FIGITELHKAGILARNLVPMVATVQGQNLKY-FITC SEQ ID NO: 34 FIGITELHKISQAVHAAHAEINEAGR SEQ ID NO: 35 FIGITELHKAAYISQAVHAAHAEINEAGR SEQ ID NO: 36 FIGITELHK-OH-PEG3-Biotin SEQ ID NO: 37 FIGITELLKAGILARNLVPMVATVQGQNLKY-FITC SEQ ID NO: 38 FIGITELLKISQAVHAAHAEINEAGR SEQ ID NO: 39 FIGITELLKAAYISQAVHAAHAEINEAGR SEQ ID NO: 40 FIGITELLK-OH-PEG3-Biotin SEQ ID NO: 41 FIGITELKAGILARNLVPMVATVQGQNLKY SEQ ID NO: 42 FIGITELLAGILARNLVPMVATVQGQNLKY SEQ ID NO: 43 FIGITELHAGILARNLVPMVATVQGQNLKY SEQ ID NO: 44 FIGITELKAAYLEQLESIINFEKLAAAAAK SEQ ID NO: 45 FIGITELLAAYLEQLESIINFEKLAAAAAK SEQ ID NO: 46 FIGITELHAAYLEQLESIINFEKLAAAAAK SEQ ID NO: 47 FIGITELKISQAVHAAHAEINEAGR SEQ ID NO: 48 FIGITELLISQAVHAAHAEINEAGR SEQ ID NO: 49 FIGITELHISQAVHAAHAEINEAGR SEQ ID NO:50 FIGITELKAAYISQAVHAAHAEINEAGR SEQ ID NO:51 FIGITELLAAYISQAVHAAHAEINEAGR SEQ ID NO:52 FIGITELHAAYISQAVHAAHAEINEAGR SEQ ID NO:53 FIGITELK-OH-PEG3-Biotin SEQ ID NO:54 FIGITELL-OH-PEG3-Biotin SEQ ID NO: 55 FIGITELH-OH-PEG3-Biotin SEQ ID NO:56 FIGITELKYKLVVVGAVGVGKSALT SEQ ID NO:57 FIGITELLYKLVVVGAVGVGKSALT SEQ ID NO:58 FIGITELHYKLVVVGAVGVGKSALT SEQ ID NO:59 FIGITELKYKLVVVGARGVGKSALT SEQ ID NO: 60 FIGITELLYKLVVVGARGVGKSALT SEQ ID NO: 61 FIGITELHYKLVVVGARGVGKSALT SEQ ID NO: 62 FIGITELKYKLVVVGADGVGKSALT SEQ ID NO: 63 FIGITELLYKLVVVGADGVGKSALT SEQ ID NO: 64 FIGITELHYKLVVVGADGVGKSALT SEQ ID NO: 65 FIGITELKAGILARNLVPMVATVQGQNLKY-FITC SEQ ID NO: 66 FIGITELLAGILARNLVPMVATVQGQNLKY-FITC SEQ ID NO: 67 FIGITELHAGILARNLVPMVATVQGQNLKY-FITC SEQ ID NO: 68 FNIKDFNI SEQ ID NO: 69 IGRIDPEX a X b DAEYVP SEQ ID NO: 70 TTGSYDLDVE SEQ ID NO:71 HASQNINVWLS SEQ ID NO:72 KASTLHT SEQ ID NO: 73 QQGQSYPLT SEQ ID NO:74 QSISSY SEQ ID NO: 75 AAS SEQ ID NO:76 QQGYPYPFT SEQ ID NO:77 GFTFSSYA SEQ ID NO:78 ISGYSGST SEQ ID NO:79 ARYYSYYGYYYFDY SEQ ID NO: 80 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK SEQ ID NO: 81 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSS SEQ ID NO:82 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 83 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:84 FIGITELKKLESKINKVFK-Biotin SEQ ID NO: 85 FIGITELKKLE SEQ ID NO:86 FIGITELKKL SEQ ID NO:87 FIGITELK SEQ ID NO: 88 ANSKFIGITELK SEQ ID NO:89 FIGITELLEQLESIINFEKLAAAAAK SEQ ID NO: 90 FIGITELKLEQLESIINFEKLAAAAAK SEQ ID NO: 91 FIGITELHLEQLESIINFEKLAAAAAK SEQ ID NO:92 Biotin-IDIKNDLYEKTLNDYKAIANKLSQV SEQ ID NO: 93 FIGITELKKL-OH-PEG3-Biotin SEQ ID NO:94 FIGITELKKLE-OH-PEG3-Biotin SEQ ID NO: 95 FIGITELLKL-OH-PEG3-Biotin SEQ ID NO:96 FIGITELLKLE-OH-PEG3-Biotin SEQ ID NO:97 FIGITELKKLESKINKVFISQAVHAAHAEINEAGR SEQ ID NO: 98 FIGITELLKAGILARNLVPMVATVQGQNLKY SEQ ID NO: 99 FIGITELKKYKLVVVGACGVGKSALT SEQ ID NO: 100 AGILARNLVPMVATVQGQNLKY SEQ ID NO: 101 FIGITELKKLESKINKVFAVGALKVPRNQDWLGVPRQL SEQ ID NO: 102 AVGALKVPRNQDWLGVPRQL SEQ ID NO: 103 FIGITELKKAVGALKVPRNQDWLGVPRQL SEQ ID NO: 104 FIGITELKKLAVGALKVPRNQDWLGVPRQL SEQ ID NO: 105 FIGITELKKLEAVGALKVPRNQDWLGVPRQL SEQ ID NO: 106 FIGITELLKAVGALKVPRNQDWLGVPRQL SEQ ID NO: 107 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 108 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEGGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 109 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 110 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEDGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 111 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 112 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPENGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 113 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPESGDAEYVPKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK SEQ ID NO: 114 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 115 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 116 YKLVVVGAAGVGKSALT SEQ ID NO: 117 FIGITELKKYKLVVVGAAGVGKSALT SEQ ID NO: 118 FIGITELKYKLVVVGACGVGKSALT SEQ ID NO: 119 FIGITELLYKLVVVGACGVGKSALT SEQ ID NO: 120 FIGITELHYKLVVVGACGVGKSALT SEQ ID NO: 121 FIGITELKYKLVVVGAAGVGKSALT SEQ ID NO: 122 FIGITELLYKLVVVGAAGVGKSALT SEQ ID NO: 123 FIGITELHYKLVVVGAAGVGKSALT SEQ ID NO: 124 IGRIDPESGDAEYVP SEQ ID NO: 125 IGRIDPEGGDAEYVP SEQ ID NO: 126 IGRIDPEQGDAEYVP SEQ ID NO: 127 IGRIDPEDGDAEYVP SEQ ID NO: 128 IGRIDPENADAEYVP SEQ ID NO: 129 IGRIDPENGDAEYVP SEQ ID NO: 130 EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSS SEQ ID NO: 131 ELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK SEQ ID NO: 132 QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPSDNFSNLNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLTVSS SEQ ID NO: 133 SDIVMTQATPSVLVTPGEAVSISCRASRSLLHSNGITYLYWFLQRPGQSPQVLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEFPYTFGGGTKLEIK SEQ ID NO: 134 GYTFTDYW SEQ ID NO: 135 IDPSDNFS SEQ ID NO: 136 AVEDY SEQ ID NO: 137 RSLLHSNGITY SEQ ID NO: 138 RMS SEQ ID NO: 139 MQHLEFPYT SEQ ID NO: 140 QAEPDRAHYNIVTFCCKCD SEQ ID NO: 141 FIGITELKKQAEPDRAHYNIVTFCCKCD SEQ ID NO: 142 FIGITELLKQAEPDRAHYNIVTFCCKCD SEQ ID NO: 143 FIGITELHKQAEPDRAHYNIVTFCCKCD SEQ ID NO: 144 FIGITELKQAEPDRAHYNIVTFCCKCD SEQ ID NO: 145 FIGITELLQAEPDRAHYNIVTFCCKCD SEQ ID NO: 146 FIGITELHQAEPDRAHYNIVTFCCKCD SEQ ID NO: 147 GITELKKQAEPDRAHYNIVTFCCKCD SEQ ID NO: 148 GITELKK

[0167] Itemized List of Embodiments 1. A tag portion having an amino acid sequence FIGITELX8X9 (SEQ ID NO: 1) wherein X8 is selected from K, L, and H; X9 is K or absent; A tag moiety that contains an epitope for binding by a specific binding molecule. 2. The tag moiety according to item 1, wherein X8 is selected from K and L. 3. The tag moiety according to item 1, wherein X8 is selected from L and H. 4. The tag moiety according to item 1, wherein X8 is selected from K and H. 5. The tag moiety of any one of items 2 and 4, wherein X8 is K. 6. The tag moiety according to any one of items 2 to 3, wherein X8 is L. 7. The tag moiety according to any one of items 3 to 4, wherein X8 is H. 8. The tag moiety of any of the preceding items, wherein X9 is K. 9. The tag moiety according to any one of items 1 to 7, wherein X9 is absent. 10. The tag portion according to any one of the preceding items, which consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7. 11. A tag construct comprising: - a tag portion according to any of the preceding items; - at least one cargo moiety. 12. The tag construct according to item 11, wherein the at least one cargo moiety is an antigen moiety. 13. The tag construct according to item 12, wherein the at least one antigen moiety is a peptide antigen moiety comprising a target antigen amino acid sequence. 14. The tag construct according to any one of items 11 to 13, wherein the C-terminus of the tag moiety is covalently linked to the N-terminus of the at least one cargo moiety. 15. The tag construct according to any one of items 11 to 13, wherein the N-terminus of the tag moiety is covalently linked to the C-terminus of the at least one cargo moiety. 16. The tag construct according to any one of items 13 to 15, wherein the target antigen amino acid sequence is selected from the group consisting of a cancer antigen, an autoantigen, and an antigen derived from a pathogen. 17. The tag construct according to item 16, wherein the target antigen sequence is a cancer antigen. 18. The tag construct according to item 17, wherein the cancer antigen is selected from the group consisting of neoantigens, tumor-associated antigens, and antigens derived from oncoviruses. 19. The tag construct according to any one of items 11 to 18, wherein the target antigen sequence is selected from the group consisting of SEQ ID NOs: 17 to 23, 25, 116, and 140. 20. The tag construct according to any one of items 11 to 19, wherein the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NOs: 24, 26 to 67, 88, 90 to 91, 98 to 99, 103, 106, 117 to 123, and 141 to 146. 21. The tag construct according to item 20, wherein the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NOs: 24, 26 to 40, 88, 98 to 99, 103, 106, 117, and 141 to 143. 22. The tag construct according to item 20, wherein the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NOs: 41 to 67, 90 to 91, 118 to 123, and 144 to 146. 23. The tag construct according to item 11, wherein the at least one cargo moiety is a nucleic acid moiety, such as an siRNA molecule. 24.K for Interaction D The value is up to 1×10 -9 M, e.g., up to 1 x 10 -10 M, e.g., up to 1 x 10 -11 M is a nucleotide sequence of the tag moiety according to any one of items 1 to 10 or a tag construct according to any one of items 11 to 23, which is capable of binding to the binding molecule such that M is a nucleotide sequence of the tag moiety according to any one of items 1 to 10 or a tag construct according to any one of items 11 to 23 25. A tag moiety or construct according to any of the preceding items, wherein the binding molecule specific for the epitope in the tag moiety is an antibody construct. 26. The tag moiety or construct according to item 25, wherein the antibody construct is an antibody or an antigen-binding fragment thereof. 27. The tag moiety or construct according to item 26, wherein the binding molecule is an antibody fragment that is an scFv. 28. The binding molecule is an immunoglobulin heavy chain variable region (VH) comprising three complementarity-determining domains (CDRs), VHCDR1 has the sequence set forth in SEQ ID NO: 68, VHCDR2 has the sequence IGRIDPEX a X b DAEYVP (SEQ ID NO: 69) a X b is selected from the group consisting of SG, GG, QG, DG, NA, and NG; VH, wherein VHCDR3 has the sequence set forth in SEQ ID NO: 70; an immunoglobulin light chain variable region (VL) comprising three complementarity-determining domains (CDRs), VLCDR1 has the sequence set forth in SEQ ID NO: 71; VLCDR2 has the sequence set forth in SEQ ID NO: 72; 28. The tag moiety or construct according to any one of items 25 to 27, comprising a VL, and a VLCDR3 having the sequence set forth in SEQ ID NO: 73. 29. The binding molecule is an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 13, 130, and 132, and amino acid sequences having at least 90% identity thereto; - an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 131, and 133, and amino acid sequences having at least 90% identity thereto. 30. The tag moiety or construct according to item 29, wherein the binding molecule comprises a complete scFv sequence selected from the group consisting of SEQ ID NOs: 15-16 and 107-112, for example selected from the group consisting of SEQ ID NOs: 107-112, for example selected from the group consisting of SEQ ID NOs: 107 and 112, for example SEQ ID NO: 107. 31. The binding molecule is an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 130 and amino acid sequences having at least 90% identity thereto; - an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 131 and amino acid sequences having at least 90% identity thereto. 32. The tag moiety or construct according to item 31, wherein the binding molecule comprises the complete scFv sequence SEQ ID NO: 15. 33. The binding molecule is an immunoglobulin heavy chain variable region (VH) comprising three complementarity-determining domains (CDRs), VHCDR1 has the sequence set forth in SEQ ID NO: 134, VHCDR2 has the sequence set forth in SEQ ID NO: 135, VH, wherein VHCDR3 has the sequence set forth in SEQ ID NO: 136; an immunoglobulin light chain variable region (VL) comprising three complementarity-determining domains (CDRs), VLCDR1 has the sequence set forth in SEQ ID NO: 137; VLCDR2 has the sequence set forth in SEQ ID NO: 138; 28. The tag moiety or construct according to any one of items 25 to 27, comprising a VL, and a VLCDR3 having the sequence set forth in SEQ ID NO: 139. 34. The binding molecule is an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 132 and amino acid sequences having at least 90% identity thereto; - an immunoglobulin light chain variable region (VL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 133 and amino acid sequences having at least 90% identity thereto. 35. A complex, - a tag construct comprising a tag moiety according to any one of items 1 to 10, - a binding molecule specific for the tag moiety. 36. The complex according to item 35, wherein the tag construct is a tag construct according to any one of items 11 to 23. 37. The conjugate according to any one of items 35 to 36, wherein the binding molecule is as defined in any one of items 25 to 34. 38. The conjugate of any one of items 35 to 37, wherein the binding molecule is covalently linked to a second moiety that is an antibody or an antigen-binding fragment thereof, such as a fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fc fragment, an Fv fragment, a single-chain (scFv) fragment, an (scFv)2 fragment, and a domain antibody. 39. The conjugate of item 38, wherein the second moiety is an antibody. 40. The conjugate according to item 39, wherein the second portion is of the IgG2 subtype. 41. The conjugate according to any one of items 38 to 40, wherein the second moiety is an anti-CD40 antibody or an antigen-binding fragment thereof. 42. The conjugate of item 41, 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 antibodies comprising an antigen-binding fragment derived from any one or more of said antibodies. 43. The anti-CD40 antibody or antigen-binding fragment thereof comprises six complementarity-determining domains (CDRs); VLCDR1 has the sequence set forth in SEQ ID NO: 74, VLCDR2 has the sequence set forth in SEQ ID NO: 75; VLCDR3 has the sequence set forth in SEQ ID NO: 76; VHCDR1 has the sequence set forth in SEQ ID NO: 77, VHCDR2 has the sequence set forth in SEQ ID NO: 78, 42. The conjugate of item 41, wherein VHCDR3 has the sequence set forth in SEQ ID NO: 79. 44. the light chain variable domain of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 80 and an amino acid sequence having at least 90% sequence identity thereto; 44. The conjugate of item 43, wherein the heavy chain variable domain of the anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO: 81 and an amino acid sequence having at least 90% sequence identity thereto. 45. The anti-CD40 antibody is a light chain comprising an amino acid sequence selected from SEQ ID NO: 82 and an amino acid sequence having at least 90% sequence identity thereto; 45. The conjugate of item 44, comprising a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 83 and amino acid sequences having at least 90% sequence identity thereto. 46. ​​A polynucleotide encoding the tag moiety or tag construct according to any one of items 1 to 34. 47. An expression vector comprising the polynucleotide according to item 46. 48. A host cell comprising the expression vector according to item 47. 49. A method for producing a tag moiety or tag construct according to any one of items 1 to 34, the method comprising: - culturing a host cell according to item 48 under conditions allowing expression of the tag moiety or tag construct from the expression vector; - isolating said tag moiety or tag construct. 50. A composition comprising a tag moiety or tag construct according to any one of items 1 to 34 and at least one pharmaceutically acceptable excipient or carrier. 51. A composition comprising a conjugate according to any one of items 35 to 45 and at least one pharmaceutically acceptable excipient or carrier. 52. A tag moiety or tag construct according to any one of items 1 to 34, a conjugate according to any one of items 35 to 45, or a composition according to any one of items 50 to 51 for use in therapy. 53. The tag moiety or tag construct according to any one of items 1 to 34, the conjugate according to any one of items 35 to 45, or the composition according to any one of items 50 to 51 for use as an in vitro diagnostic agent, an in vivo diagnostic agent, an in vitro prognostic agent and / or an in vitro prognostic agent. 54. A tag moiety, tag construct, conjugate, or composition for use according to item 52 in the treatment or prevention of cancer, an autoimmune disease, or an infectious disease. 55. A tag moiety, tag construct, complex or composition for use according to item 54 in the treatment or prevention of cancer. 56. A method for treating or preventing cancer, an autoimmune disease, or an infectious disease, comprising administering to a subject in need thereof an effective amount of the tag moiety or tag construct according to any one of items 1 to 34, the conjugate according to any one of items 35 to 45, or the composition according to any one of items 50 to 51. 57. The method according to item 56, for the treatment or prevention of cancer. 58. Use of a tag moiety or tag construct according to any one of items 1 to 34, a conjugate according to any one of items 35 to 45, or a composition according to any one of items 50 to 51 in the manufacture of a medicament. 59. The use according to item 58, wherein the medicinal product is intended for the treatment or prevention of cancer, an autoimmune disease, or an infectious disease. 60. The use according to item 59, wherein the medicinal product is intended for the treatment or prevention of cancer. 61. A tag moiety, tag construct, complex or composition for use according to item 53 as a diagnostic agent in the in vitro or in vivo diagnosis of a disease, disorder and / or infection. 62. The tag moiety, tag construct, complex, or composition for use according to item 53 as a prognostic agent in the in vitro or in vivo prognosis of a disease, disorder, and / or infection. 63. A diagnostic or prognostic method for determining the presence of a disease, disorder, or infection in a subject, comprising: a. contacting a subject or a sample isolated from a subject with a tag moiety or tag construct according to any one of items 1 to 34, a complex according to any one of items 35 to 45, or a composition according to any one of items 50 to 51; b. obtaining a value corresponding to the amount of tag moiety, tag construct, complex, or composition bound to said subject or said sample. 64. The diagnostic or prognostic method according to item 63, wherein the contacting in step a) is a method for in vivo diagnosis or in vivo prognosis, e.g., medical imaging, comprising contacting the subject with the tag moiety, tag construct, complex, or composition. 65. The diagnostic or prognostic method according to item 63, which is an in vitro method, and wherein the contacting in step a) comprises contacting a sample previously isolated from the subject with the tag moiety, tag construct, complex, or composition.

Claims

1. The tag portion is an amino acid sequence FIGITELX ８ X ９ (SEQ ID NO: 1) wherein X ８ is selected from K, L, and H; X ９ is K or absent, A tag portion comprising an epitope for binding by a specific binding molecule.

2. The tag portion according to claim 1, which consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7.

3. A tag construct comprising: a tag portion according to any one of the preceding claims; - at least one cargo moiety.

4. The tag construct of claim 3 , wherein the at least one cargo moiety is an antigen moiety.

5. 5. The tag construct of claim 4, wherein the at least one antigen moiety is a peptide antigen moiety comprising a target antigen amino acid sequence.

6. 6. The tag construct of claim 5, wherein the target antigen amino acid sequence is selected from the group consisting of a cancer antigen, an autoantigen, and an antigen derived from a pathogen.

7. 7. The tag construct of claim 6, wherein the target antigen amino acid sequence is a cancer antigen, for example, a cancer antigen selected from the group consisting of neoantigens, tumor-associated antigens, and antigens derived from oncoviruses.

8. 4. The tag construct of claim 3, wherein the at least one cargo moiety is a nucleic acid moiety, such as an siRNA molecule.

9. K of Interaction Ｄ The value is up to 1 x 10 －９ M, for example, up to 1 × 10 －１０ M, for example, up to 1 × 10 －１１ 10. A tag moiety or tag construct according to any one of the preceding claims, capable of binding to said binding molecule such that M is

10. 10. A tag moiety or construct according to any one of the preceding claims, wherein the binding molecule specific for the epitope in the tag moiety is an antibody construct, such as an antibody or an antigen-binding fragment thereof, or such as an scFv.

11. A complex, a tag construct comprising a tag moiety according to any one of claims 1 to 2 and 9 to 10; - a binding molecule specific for said tag moiety.

12. The complex according to claim 11, wherein the tag construct is a tag construct according to any one of claims 3 to 10.

13. 13. The conjugate of claim 11 or 12, wherein the binding molecule is as defined in claim 10.

14. The binding molecule is an antibody or an antigen-binding fragment thereof, such as a Fab fragment, a Fab' fragment, or a F(ab') ２ fragment, Fc fragment, Fv fragment, single chain (scFv) fragment, (scFv) ２ and a fragment selected from the group consisting of a domain antibody, e.g., an anti-CD40 antibody or an antigen-binding fragment thereof.

15. A polynucleotide encoding the tag moiety or tag construct of any one of claims 1 to 10.

16. 15. A composition comprising any of the tag moieties or tag constructs of any one of claims 1 to 10, or the conjugates of any one of claims 11 to 14, and at least one pharmaceutically acceptable excipient or carrier.

17. A tag moiety or tag construct according to any one of claims 1 to 10, a complex according to any one of claims 11 to 14, or a composition according to claim 16, for use in therapy, in vivo diagnosis, or in vivo prognosis.

18. 18. A tag moiety, tag construct, conjugate or composition for use according to claim 17 in the treatment or prevention of cancer, an autoimmune disease or an infectious disease.

19. 20. A tag moiety, tag construct, complex or composition for use according to claim 18 in the treatment or prevention of cancer.

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