Optimized tag moiety

Abstract

The present disclosure provides an improved peptide tag moiety, with affinity for a binding molecule. The peptide tag moiety is for example useful as a part of a tag construct, e.g., together with a cargo moiety. It exhibits unexpected benefits as part of a biopharmaceutical product complex, when compared to known tag moieties having a similar amino acid sequence. Also disclosed are binding molecules and bispecific conjugates with affinity for the peptide tag moiety, and complexes comprising the peptide tag moiety non-covalently bound to a such binding molecules or bispecific conjugates. In such complexes, the tag moiety may for example form part of a tag construct further comprising an antigen as cargo moiety, for antigen delivery to immune cells. Medical uses of the tag moiety, tag construct and complex of the disclosure are also provided.

Description

[0001] OPTIMIZED TAG MOIETY

[0002] Technical field

[0003] The present disclosure relates to an improved peptide tag moiety, with affinity for a binding molecule. The peptide tag moiety is for example useful as a part of a tag construct, e.g., together with a cargo moiety. It exhibits unexpected benefits as part of a biopharmaceutical product complex, when compared to known tag moieties having a similar amino acid sequence. Also disclosed are binding molecules and bispecific conjugates with affinity for the peptide tag moiety, and complexes comprising the peptide tag moiety non-covalently bound to a such binding molecules or bispecific conjugates. In such complexes, the tag moiety may for example form part of a tag construct further comprising an antigen as cargo moiety, for antigen delivery to immune cells. Medical uses of the tag moiety, tag construct and complex of the disclosure are also provided.

[0004] Background

[0005] Monoclonal antibodies (mAbs) which modulate immune responses are proving highly effective in cancer treatment, with increasing evidence that such responses can be harnessed to provide durable eradication of tumors. Various antibodies against different targets have been developed, e.g., targeting the immune checkpoints CTLA-4 and PD-1, which support the view that T cell immunity can provide an effective treatment for cancer. Promising clinical data have also been obtained with immunostimulatory mAbs that bind agonistically to the co-stimulatory receptor CD40 on antigen-presenting cells (APCs).

[0006] Effective stimulation (or priming) of T cells requires not only application of a stimulus to an APC, but also the presentation of antigen by the APC (in the context of an MHC) for recognition and binding by a T cell receptor (TOR). Thus, it is advantageous for the APC to cross-present antigen to T cells for the purpose of T cell stimulation, in other words to take up, process and present antigen (of extracellular origin) to the T cells. However, antigenic material may not always be present (for example if a tumor has been resected, or in the context of a vaccine against an infection), and CD40 agonists may have poor efficacy in driving effective T cell stimulation in such a situation. CD40 stimulation may also be insufficient for T cell activation (for example if there is a dose-limiting toxicity of the CD40 agonist as an infusion product). For these reasons, it is advantageous to deliver antigen to the APC at the same time as activating CD40 on the APC surface with an agonist. Bispecific conjugates for this purpose are described in W02020 / 104690 and WO2021 / 239968. The bispecific conjugates described therein comprise two binding proteins covalently coupled together. The first binding protein is specific for CD40; the second binding protein, rather than being directly specific for an antigen, is instead specific for a tag moiety. A tag construct is provided, in which the tag moiety is covalently coupled to an antigen, and forms a complex with the bispecific conjugate through the binding affinity of the second binding protein in the conjugate towards the tag moiety. Hence, by binding to the tag moiety, the bispecific conjugate is bound indirectly to the antigen, providing a flexible, modular approach by which the antigen in the tag construct can be varied, there being no chemical linkage between the antigen and the conjugate.

[0007] W02020 / 104690 provides a complex formed between the bispecific conjugate and the tag construct, which complex provides both a CD40 agonist (for activation of an APC) and an antigen (for presentation by the APC). This ensures that APC activation by the CD40 agonist leads to activation of T cells specific for the target antigen, and advantageously allows for flexibility in preparing conjugates and complexes for use in personalized medicine, as well as the use of this flexible platform for vaccine development for vaccination of individuals using the CD40- pathway to mount an effective anti-pathogen immune response.

[0008] The complex of W02020 / 104690 can also stimulate B cell responses against an antigen. The complex can form two interactions with a B cell: the anti-CD40 binding protein can bind CD40 on the surface of a B cell and the antigen in the tag construct can bind a specific B cell receptor. The combination of these two interactions activates B cells which recognize the antigen. Indeed, B cells activated in this manner using complexes as described in W02020 / 104690 may not require co-stimulation by helper T cells for full activation.

[0009] Rather than preparing conjugates comprising an antigen directly fused to a CD40 agonist, or an antigen-specific binder fused to the agonist, which would require the laborious synthesis and production of a separate conjugate for each patient (or at least each different tumor antigen) or for each pathogenic serotype, the bi-specific conjugate can be tailored for individual, personalized use by binding to different tag constructs containing different antigens but the same tag moiety, according to the need of a particular, individual, patient. The bispecific conjugate is by this strategy tailored to also adapt a personalized strategy to vaccinate against a pathogen with a high antigen drift ensuring a flexible vaccination strategy. In this way, only separate tag constructs need to be prepared, providing a benefit in the ease and costs of preparing patient / pathogen-specific therapeutic agents. It is further believed that the non-covalent binding of antigen to the CD40 agonist may be advantageous for the efficacy of the complex, as compared to a conjugate comprising antigen fused directly and covalently to the CD40 agonist, or as compared to providing the CD40 agonist and antigen separately.

[0010] WO2021 / 239968 discloses a further agonistic antibody (or related binding protein) against CD40, shown to bind CD40 with a high affinity and to display strong agonistic activity. This CD40 binding protein is particularly suited to use in the context of a therapeutic bispecific conjugate, which may similarly be used for cancer therapy or, alternatively, for treatment of or vaccination against an infection.

[0011] WO2011 / 115483 relates to conjugates comprising a tetanus toxoid peptide (“MTTE”) for induction of immune responses when administered to subjects having pre-existing antibodies to the tetanus toxoid. The PCT application defines that the peptides comprises at least 10 amino acids. A very large number of peptides are tested, and in section 2.5 on page 42, the best results are said to be achieved with a 22-mer peptide, while the shortest peptide bound by the test reagent comprises 10 amino acids. The goal of this disclosure is to optimize binding to polyclonal antibodies previously present in a host and directed against a universal B cell epitope.

[0012] W02020 / 104690 and WO2021 / 239968 suggest and test a large number of peptide tag moiety sequences as candidates for binding to the bispecific conjugate. Some of them are derived from tetanus toxin.

[0013] On page 19 lines 3-20 of W02020 / 104690, the MTTE peptide is presented as a candidate along with a number of other peptides derived from tetanus toxin. The MTTE peptide has 18 amino acids and is denoted “SEQ ID NO:6”, while other candidate tag peptides are presented as “SEQ ID NO:10-14”. No specific tag peptides consisting of fewer than 18 amino acids are disclosed, much less made and tested. There is a speculative discussion in this section about using shorter fragments of MTTE, but this discussion does not point out any individual part of the sequence as being useful as a fragment, and, importantly, there is no experimental data to the effect that any particular length or sequence of such a fragment may indeed be effectively used in the disclosed complex. Also, WO2021 / 239968 discusses the MTTE peptide, e.g., on page 24 lines 23-31. Here, the authors describe that the MTTE sequence was trimmed to a peptide consisting of the 12 N-terminal amino acids of the sequence, and the resulting sequence is presented as “SEQ ID NO: 16”. The following section, page 24 line 32 - page 25 line 4, teaches various sequence variants of the 12-mer sequence and longer sequences comprising it. Importantly, this disclosure is matched in WO2021 / 239968 by an experiment showing that the peptide could 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 Recognised by 14GIIICII and IBIIICI” (emphasis added) - this disclosure teaches the skilled person that the MTTE peptide cannot be further shortened. This message is made more clear by the disclosure of other peptide sequences presented as potentially useful in WO2021 / 239968 on page 25 lines 14-33, where the unrelated tetanus toxin sequences presented as “SEQ ID NO:11-15” are discussed (incidentally, these are the same sequences as those listed as “SEQ ID NQ:10-14” in WQ2020104690). In this section only, unlike for the shortened MTTE variants discussed on the previous pages, there are proposals to create shorter fragments, but only with respect to SEQ ID NO: 11-15. There is no disclosure of fragments of the MTTE peptide that are shorter than 12 amino acids. In other words, there is no disclosure of MTTE peptide fragments consisting of less than 12 amino acids.

[0014] Together, WQ2020 / 104690 and WO2021 / 239968 disclose a promising platform for the development of personalized biopharmaceuticals. However, the development and production of candidate products place high demands on all components or moieties comprised in the product, and there is therefore a general need to optimize and improve the platform.

[0015] Disclosure of the invention

[0016] It is an object of the disclosure to meet this need through the provision of a peptide tag moiety which improves on the previously tested peptide tags, and presents a smaller (shorter) epitope for binding by a bispecific conjugate than was previously believed possible, while maintaining efficacy.

[0017] It is another object of the disclosure to identify a narrow selection of peptide tag moiety sequences which are particularly useful as binding partners to a bispecific conjugate or binding molecule. It is a further object to enable the use of peptide tag moiety sequences which are unlikely to elicit an immune response to themselves, and / or for which it is unlikely that a subject has pre-existing antibodies that limit their use. In other words, it is an object to provide tag moieties that are less immunogenic in a host than previously known tag moieties.

[0018] Another object is to provide a peptide tag moiety which leaves the maximum amount of room for at least one cargo moiety when said peptide tag moiety and said at least one cargo moiety are comprised in a tag construct.

[0019] Yet another object is to provide a tag moiety sequence which simplifies as far as possible the recombinant production and / or chemical synthesis of the tag moiety, for example by being as short as possible.

[0020] A further object of the disclosure is to provide a tag moiety which is optimized for interaction with an engineered binding molecule, as opposed to relying on preexisting, endogenous binding entities in the subject.

[0021] It is another object of the disclosure to provide an alternative tag moiety which exhibits an increased compatibility with a binding molecule having affinity thereto, when used in the context of a complex as described for example in W02020 / 104690 and WO2021 / 239968.

[0022] These and other objects which are evident to the skilled person from the present disclosure are met by different aspects of the invention as claimed in the appended claims and as generally disclosed herein.

[0023] Thus, in a first aspect, there is provided a tag moiety, consisting of the amino acid sequence FIGITELXsXg (SEQ ID NO:1), wherein Xs is selected from K, L and H; and X9 is K or absent, said tag moiety comprising an epitope for binding by a binding molecule specific therefor.

[0024] In one embodiment, Xs is selected from K and L.

[0025] In one embodiment, Xs is selected from L and H.

[0026] In one embodiment, Xs is selected from K and H.

[0027] In one embodiment, Xs is K. In yet another embodiment, Xs is L. In still another embodiment, Xs is H.

[0028] In one embodiment, X9 is K. In another embodiment, X9 is absent.

[0029] Said alternative embodiments of the amino acids in positions Xs and X9 may be freely combined in any subgroup. For example, in one embodiment, the tag moiety consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2-7. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NO:2-4. In another embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NO:5-7.

[0030] The tag moiety of this aspect is useful as a minimal binding target, or epitope, for a binding molecule specific therefor. This property makes it useful e.g., in the context of a drug platform such as that described in WQ2020 / 104690 and WO2021 / 239968, insofar as this platform incorporates a binding molecule specific for the tag moiety in a bispecific conjugate molecule.

[0031] In this and other contexts, it may be of interest to incorporate the tag moiety in a tag construct, further comprising at least one cargo moiety. A cargo moiety may be any compound that is of interest to transport or carry, by way of the affinity between tag moiety and the specific binding molecule.

[0032] Thus, in a second aspect, the 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 the part of the tag construct that is designed to interact with the binding molecule specific therefor, whereas the cargo moiety is a part which is present in order to achieve some other function, for example elicit a specific immune response against a cargo representing an antigen. In one embodiment, the tag moiety and cargo moiety / moieties in the tag construct are covalently coupled. In the context of the tag construct as disclosed herein, the tag moiety and cargo moiety / moieties are non-overlapping. By way of example, illustrated by the case wherein the cargo moiety is a peptidic moiety, the amino acid sequence of the tag moiety is structurally and functionally distinct from the amino acid sequence of the cargo moiety. The skilled person understands that the cargo moiety is thus not part of the tag moiety. In one embodiment, a cargo moiety is a peptidic moiety. In another embodiment, a cargo moiety is a nucleic acid moiety.

[0033] In embodiments wherein the at least one cargo moiety is a nucleic acid moiety, the tag moiety and cargo moiety can be arranged in either order in a polypeptide-nucleic acid combination. In one such embodiment, the cargo moiety is an siRNA molecule.

[0034] In embodiments wherein the at least one cargo moiety is peptidic, the tag moiety and cargo moiety can be arranged in either order in a polypeptide chain. As such, in one embodiment, the C terminus of the tag moiety is covalently bound to the N terminus of the at least one cargo moiety. In another embodiment, the N terminus of the tag moiety is covalently bound to the C terminus of the at least one cargo moiety. 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 cytotoxic agents, anti-inflammatory agents, kinase inhibitors, receptor kinase inhibitors, nonreceptor tyrosine kinase inhibitors, serine / threonine kinase inhibitors, epigenetic inhibitors, BCL2 inhibitors, hedgehog pathway inhibitors, proteasome inhibitors, PART inhibitors and indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

[0035] In an alternative embodiment, the at least one cargo moiety is an antigen. In a more specific embodiment, the at least one cargo moiety is a peptidic antigen. In such an embodiment, the peptidic antigen comprises a target antigen amino acid sequence, to which it may be of interest to generate an immune response. It is understood that said at least one cargo moiety comprised in the tag construct may be more than one cargo moiety, such as a plurality of cargo moieties. In a specific embodiment, said 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, such as at least five cargo moieties. It is understood that the number of cargo moieties comprised in the tag construct is not limited to a specific number of moieties. In embodiments wherein an at least one cargo moiety is a peptidic antigen, it suitably comprises a target antigen amino acid sequence. In such embodiments, the target antigen amino acid sequence may either constitute the entire at least one cargo moiety, or the at least one cargo moiety may comprise additional amino acids that are not as such part of the target antigen amino acid sequence but present for another purpose, e.g. as linker between the tag moiety and the target antigen sequence within the cargo moiety. Furthermore, it is understood that said target antigen amino acid sequence may in turn comprise amino acid sequences corresponding to a plurality of peptidic antigens. Said plurality of peptidic antigens comprised in the cargo moiety, or comprised in the target amino acid sequence, could be directly or indirectly fused to each other. Furthermore, as described above, said at least one cargo moiety may comprise additional amino acids that are not as such part of the target antigen amino acid sequence but present for another purpose, e.g. as linker between the tag moiety and the target antigen sequence within the cargo moiety or as a linker between multiple cargo moieties or between multiple peptidic antigen sequences.

[0036] In embodiments wherein the at least one cargo moiety is a peptidic antigen moiety, the target antigen amino acid sequence may for example be selected from the group consisting of a cancer antigen, a self-antigen and an antigen derived from a pathogen. It is understood that the tag construct comprising the at least one cargo moiety may comprise more than one cargo moiety. In one such embodiment, it is a self-antigen. 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 wherein the target antigen amino acid sequence is a cancer antigen, it may suitably be selected from the group consisting of a neoantigen, a tumor-associated antigen and an antigen derived from an onco-virus. In another embodiment said cancer antigen is a plurality of cancer antigens, such as a plurality of neoantigens, such as a plurality of tumor-associated antigens or such as a plurality of antigens derived from an onco-virus.

[0038] Specific but non-limiting examples of amino acid sequences which may constitute the target antigen amino acid sequence in a tag construct according to the disclosure and have been tested in the Examples to follow are those that are selected from the group consisting of SEQ ID NO:17-23, 25, 116 and 140. The skilled person is aware of many other antigen sequences for which it may be of interest to generate an immune response, e.g., a T cell immune response. In a specific embodiment, the target antigen amino acid sequence comprised in a tag construct according to the disclosure is selected from the group consisting of SEQ ID NO:21-23, 25 and 116. In one such embodiment, said sequence is SEQ ID NO:21. In another such embodiment, said sequence is SEQ ID NO:22. In another such embodiment, said sequence is SEQ ID NO:23. In another such embodiment, said sequence is SEQ ID NO:25. In another such embodiment, said sequence is SEQ ID NO:116.

[0039] When combining the non-limiting examples of alternative sequences for the at least one cargo moiety with the embodiments of the tag moiety sequences (SEQ ID NO:2-7), it is clear that in an embodiment of the tag construct according to the disclosure, the amino acid sequence of the tag construct is selected from the group consisting of SEQ ID NO: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 NO: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 NO:41-67, 90-91 , 118-123 and 144-146.

[0040] As described herein, the tag moiety of the disclosure consists of an amino acid sequence as described herein, and comprises an epitope for binding by a binding molecule specific therefor. By extension, the tag construct of the disclosure, comprising said tag moiety, comprises an epitope for binding by a binding molecule specific therefor. In one embodiment, the tag moiety, or the tag construct comprising the tag moiety, is capable of binding to the binding molecule such that the KD value of the interaction is at most 1 x 10'9M, for example at most 1 x 10'1° M, for example at most 1 x 10'11M.

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

[0042] In one embodiment, the binding molecule specific for the tag moiety comprises an immunoglobulin heavy chain variable region (VH) comprising three complementarity determining domains (CDRs), wherein: VHCDR1 has the sequence set forth in SEQ ID NO:68;

[0043] VHCDR2 has the sequence IGRIDPEXaXbDAEYVP (SEQ ID NO:69), wherein XaXbis selected from the group consisting of SG, GG, QG, DG, NA and NG; and

[0044] VHCDR3 has the sequence set forth in SEQ ID NQ:70; and an immunoglobulin light chain variable region (VL) comprising three complementarity determining domains (CDRs), wherein: VLCDR1 has the sequence set forth in SEQ ID NO:71;

[0045] VLCDR2 has the sequence set forth in SEQ ID NO:72; and

[0046] VLCDR3 has the sequence set forth in SEQ ID NO:73.

[0047] Put differently, the different alternative amino acid pairs for XaXbin VHCDR2 mean that the VHCDR2 amino acid sequence is selected from the group consisting of SEQ ID NO:124-129.

[0048] In an alternative embodiment, the binding molecule specific for the tag moiety comprises an immunoglobulin heavy chain variable region (VH) comprising three complementarity determining domains (CDRs), wherein:

[0049] VHCDR1 has the sequence set forth in SEQ ID NO: 134;

[0050] VHCDR2 has the sequence set forth in SEQ ID NO: 135;

[0051] VHCDR3 has the sequence set forth in SEQ ID NO: 136; and an immunoglobulin light chain variable region (VL) comprising three complementarity determining domains (CDRs), wherein: VLCDR1 has the sequence set forth in SEQ ID NO:137;

[0052] VLCDR2 has the sequence set forth in SEQ ID NO: 138; and

[0053] VLCDR3 has the sequence set forth in SEQ ID NO: 139.

[0054] In one embodiment, said binding molecule comprises an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: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 NO:14, 131 and 133 and amino acid sequences having at least 90 % identity thereto. In specific embodiments, one or more of these VH and VL sequence has at least 91 %, such as at least 92 %, such as at least 93 %, such as at least 94 %, such as at least 95 %, such as at least 96 %, such as at least 97 %, such as at least 98 %, such as at least 99 % identity to any one of the specifically listed sequences.

[0055] As described herein, binding molecules having amino acid sequences with 90 % or more identity to the listed sequences are also contemplated to fall within the ambit of the disclosure. Such variant sequences may be modified relative to the listed sequences by substitution, insertion and / or deletion of one or more amino acids.

[0056] An amino acid substitution relative to a listed sequence may be a conservative amino acid substitution. 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 thus a conservative substitution of an amino acid important for the structure or function of a polypeptide may be expected to affect polypeptide structure / function less than a nonconservative amino acid substitution at the same position. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine), non-polar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative amino acid substitution may be considered to be a substitution in which a particular amino acid residue is substituted for a different amino acid in the same family. However, an amino acid substitution may equally be a non-conservative substitution, in which one amino acid is substituted for another with a side-chain belonging to a different family.

[0057] As detailed above, according to the present disclosure variants of the listed sequences have at least 90 % sequence identity to the respective listed sequence. Sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programs that make pairwise or multiple alignments of sequences are useful, for instance EMBOSS Needle or EMBOSS stretcher (both Rice et al. (2000), Trends Genet., 16(6):276-277) may be used for pairwise sequence alignments, while Clustal Omega (Sievers et al. (2011), Mol. Syst. Biol. 7:539) or MUSCLE (Edgar (2004), Nucleic Acids Res. 32(5):1792-1797) may be used for multiple sequence alignments, though any other appropriate program may be used. Whether the alignment is pairwise or multiple, it must be performed globally (i.e., across the entirety of the reference sequence) rather than locally.

[0058] Sequence alignments and % identity calculations may be determined using for instance standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1. Alternatively, the 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.

[0059] As described above, the binding molecule may in some embodiments be an scFv molecule, in which the VH and VL domains are joined together in a single polypeptide chain in a manner familiar to a person of skill in the art, e.g., 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 NO:15-16 and 107-112. In one embodiment, said amino acid sequence is selected from the group consisting of SEQ ID NO:107-112. In a more specific embodiment, said amino acid sequence is selected from the group consisting of SEQ ID NQ:107 and 112. In an even more particular embodiment, said amino acid sequence is SEQ ID NQ:107.

[0060] As explained above, the tag construct is useful as a component in the biopharmaceutical platform described in WQ2020 / 104690 and WO2021 / 239968. As such, the disclosure also provides a complex, such as a non-covalent complex, formed between the tag construct (comprising the tag moiety) on the one hand, and the binding molecule specific for the tag moiety on the other. In other words, a third aspect of the disclosure provides a complex, such as a non-covalent complex, comprising a tag construct comprising a tag moiety according to the first aspect and a binding molecule specific for said tag moiety. With respect to embodiments of the complex, all the embodiments described above in connection with the tag moiety aspect, the tag construct aspect and the binding molecule specific for the tag moiety apply equally to the complex according to this aspect, and will for the sake of brevity not be repeated here.

[0061] In a complex according to this aspect, it may be beneficial for the binding molecule to, in turn, be comprised in, and be covalently linked to a second moiety which is an antibody or antigen binding fragment thereof, for example 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, said second moiety is an antibody, such as an antibody of the lgG2 subtype.

[0062] In a particular embodiment of said complex in which the binding molecule is covalently linked to a second moiety, said second moiety is an anti-CD40 antibody or an 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, ChiLob 7 / 4, SEA-CD40 and ABS-1150 / 1151 and an antibody comprising an antigen binding fragment derived from any one or more of said antibodies. In a more preferred embodiment, the anti-CD40 antibody or antigen binding fragment thereof comprises six CDR:s, wherein:

[0063] VLCDR1 has the sequence set forth in SEQ ID NO:74;

[0064] VLCDR2 has the sequence set forth in SEQ ID NO:75;

[0065] VLCDR3 has the sequence set forth in SEQ ID NO:76;

[0066] VHCDR1 has the sequence set forth in SEQ ID NO:77;

[0067] VHCDR2 has the sequence set forth in SEQ ID NO:78; and

[0068] VHCDR3 has the sequence set forth in SEQ ID NO:79.

[0069] 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 NQ: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 an embodiment in which said anti- CD40 antibody or antigen binding fragment thereof is a full-length antibody, it may for example comprise 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.

[0070] As detailed above with reference to the binding molecule which is specific for the tag moiety, one or more of these VH, VL, LC and HC sequences has at least 91 %, such as at least 92 %, such as at least 93 %, such as at least 94 %, such as at least 95 %, such as at least 96 %, such as at least 97 %, such as at least 98 %, such as at least 99 % identity to any one of the specifically listed sequences.

[0071] In a fourth aspect of the disclosure, there is provided a polynucleotide encoding a tag moiety or tag construct as described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector. Also encompassed by this disclosure is a method of producing a tag moiety or tag construct of the disclosure, comprising culturing said host cell under conditions allowing expression of said tag moiety or tag construct from its expression vector, and isolating the tag moiety or tag construct.

[0072] In an alternative aspect, the tag moiety or tag construct according to the disclosure is produced using de novo peptide synthesis, employing methods available to a person of ordinary skill in the art. As such, the disclosure provides a method of production of a tag moiety or tag construct according to the disclosure, comprising the stepwise addition of amino acid monomers to a growing polypeptide chain, until a final tag moiety or tag construct as described herein has been formed.

[0073] In a further aspect, the disclosure provides a pharmaceutical composition comprising (i) a tag moiety as described above; (ii) a tag construct as described above, or (iii) a complex as described above. In addition to the tag moiety, tag construct or complex, the pharmaceutical composition also comprises at least one pharmaceutically acceptable carrier or excipient.

[0074] Also provided by the present disclosure are kits and products 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 products, the binding molecule and tag moiety or tag construct may be separately provided in compositions containing a pharmaceutically acceptable carrier or excipient.

[0075] 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. 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, complex or constituent component thereof may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate or denature it.

[0076] Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions, kits and products include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride and the like.

[0077] The pharmaceutical composition, product or kit also may include a pharmaceutically acceptable antioxidant. They may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0078] 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.

[0079] Sterile injectable solutions can be prepared by incorporating the active agent (e.g., complex) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0080] Pharmaceutical compositions, products and kits may comprise additional active ingredients as well as the tag moiety, tag construct, complex or component thereof, for example they may comprise additional therapeutic or prophylactic agents. Thus, the complex may be used as a monotherapy or as part of a combination therapy, e.g., in the treatment of cancer. A kit or combination product as described herein may additionally contain instructions for use.

[0081] The tag moiety of the present disclosure, tag construct of the present disclosure, complex of the present disclosure, pharmaceutical composition of the present disclosure, kit of the present disclosure and combination product of the present disclosure may be used in therapy. The disclosure thus provides the tag moiety, tag construct, complex, pharmaceutical composition or kit of the disclosure for use in therapy. By therapy is meant the treatment of a subject. By “therapy” as used herein is meant the treatment of any medical condition. Such treatment may be prophylactic (i.e., preventative), curative (or treatment intended to be curative), or palliative (i.e., treatment designed merely to limit, relieve or improve the symptoms of a condition). In curative and palliative applications, complexes or compositions are administered to a subject already suffering from a disorder 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 decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a ’’therapeutically effective amount”. Effective amounts for a given purpose will depend on the disease or condition to be treated, its severity and the size / weight and general state of the subject.

[0082] Prophylactic treatment may include the prevention of a condition, or a delay in the development or onset of a condition. For example, the complex may be used to prevent an infection, or to reduce the extent to which an infection may develop, or to prevent, delay or reduce the extent of a cancer developing, or recurring, or for example to prevent or reduce the extent of metastasis.

[0083] A subject, as defined herein, refers to any mammal, e.g., a farm 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.

[0084] The combination product of the disclosure comprises a binding molecule (e.g., comprised 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 to say, when the combination product disclosed herein is used according to the disclosure, i.e. , in therapy, the binding molecule and tag moiety or tag construct are administered simultaneously or sequentially to the subject. Similarly, when the kit of the disclosure is used in therapy, the binding molecule and tag moiety or tag construct are administered simultaneously or sequentially to the subject. By “simultaneous” administration, as used herein, means that the two components are administered to the subject at the same time, or at least substantially the same time, by the same administrative route and at substantially the same location. By “sequential” administration, as used herein, is meant that the two components are administered to the subject at different times. In particular, administration of the first component is completed before administration of the second component commences.

[0085] Due to the nature of the present disclosure, sequential administration of the binding molecule and tag moiety or tag construct requires both to be administered by the same route and at substantially the same location. Furthermore, although the administration of the binding molecule and tag moiety or tag construct may be temporally spaced, the interval between the administrations should be such as to allow a complex to be formed, when both components have been administered. Thus, for example, both components may be administered within 1 hour of each other, or more particularly within 40, 30, 20, 15, 10, 8, 7, 6, 5, 4, 3, 2 or 1 minute, or less than a minute, of each other.

[0086] Mention of the complex of the disclosure (or a pharmaceutical composition comprising such a complex) being administered to a subject for therapeutic purposes is to be understood as referring to a pre-mixed composition (e.g. solution) comprising both components of the complex (i.e. a binding molecule and tag moiety or tag construct as described above), which components may exist in a dynamic equilibrium comprising the complex and its two individual components.

[0087] In particular, the tag moiety, tag construct, binding molecule (e.g., comprised in a bispecific conjugate), complex, composition, kit or combined product of the disclosure may be used in the treatment or prevention of cancer. By cancer is meant any malignant or pre-malignant neoplastic condition. The cancer may thus be any cancer, of any organ, tissue or cell type. Cancers which present as solid tumors, and which do not exhibit solid tumors are included. Accordingly, haemopoietic cancers are included.

[0088] 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 cancer, bone cancer, brain cancer, esophageal cancer, eye cancer, gastric cancer, oral cancer, penile cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer. Mast cell tumors and hemangiosarcoma may also be treated according to the present disclosure. The cancer may be newly diagnosed and naive to treatment or it may be relapsed or refractory, or relapsed and refractory, primary or metastatic.

[0089] When present in a bispecific conjugate together with a binding molecule of the disclosure, a CD40-specific second moiety activates the immune system by agonizing or stimulating CD40 on APCs, particularly on dendritic cells. In particular, this may lead to T cell activation. The subsequent immune response exerts an anticancer effect on neighboring or accessible tumor cells, without regard to CD40 expression by the tumor. A bispecific conjugate of this embodiment, or complex containing it, may therefore be effective against both CD40-positive and CD40- negative cancers. Such a bispecific conjugate can also exert its effect as an adjuvant within a vaccination regimen against a pathogen. If a vaccine platform (e.g., an attenuated virus or a DNA / RNA-based vaccine) does not stimulate a sufficient immune response by itself, CD40 activation may be required to stimulate effective anti-pathogen immune responses resulting in neutralizing antibodies or T cell responses.

[0090] In addition to the agonistic immune activating effect provided by the CD40- specific second moiety in embodiments wherein it is present, the complex of the disclosure also provides an antigen, which may be processed and presented to T cells which are activated, and thus T cells may be primed to target cancer cells which express the antigen or to a virally infected cell. This may be of particular benefit in circumstances where cancer antigen presence is low or reduced, for example where a tumor has been surgically removed, in cases where an anti-CD40 therapeutic cannot be delivered intra-tumorally, or when antigen presentation is not ideally performed at the tumor site due to suppressive factors secreted by the tumor. The complex according to this embodiment provides a means for providing cancer antigen in the vicinity of the agonistic activation signal, e.g., at a non-tumor site to ensure T cell priming and activation that can later travel to the tumor and exert their function.

[0091] In some embodiments, the cancer antigen delivered by the complex may be selected based on the subject and the particular cancer, thus allowing personalized medicine. For example, the cancer of the subject may be subjected to genetic profiling, allowing a suitable antigen to be selected. A bank or library of antigens, or tag constructs comprising antigens, may be provided from which a suitable tag construct may be prepared or selected depending on the cancer type of the subject.

[0092] The tag moiety, tag construct, binding molecule (e.g., comprised in a bispecific conjugate), complex, composition, kit or combined product of the disclosure may also be useful in the treatment or prevention of an infection. The present disclosure may in particular be used in therapy for (e.g., vaccination against) viral infections, in particular infections caused by RNA viruses. Infections caused by RNA viruses which may be treated of prevented (by vaccination) according to the present disclosure include infections caused by coronaviruses (such as SARS-CoV 1 (the causative agent of SARS), SARS CoV 2 (the causative agent of COVID 19) and MERS-CoV), influenza viruses, ebola virus, hepatitis C virus (HCV), hepatitis E virus (HEV), rabies virus, poliovirus, Ross River virus and measles virus.

[0093] The disclosure may also be used in treatment for or vaccination against infections caused by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes viruses (e.g., HHV 6), parvovirus B19 and human papillomavirus (HPV), though any viral infection can, in principle, be treated or prevented according to the present disclosure.

[0094] Intracellular bacterial infections may also be treated according to the present disclosure, e.g. Brucellosis (caused by bacterial species of the genus Brucella), Q fever (caused by Coxiella burnetii), diseases caused by species of Chlamydiae, 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). Intracellular fungal or protozoal infections may also be treated by the current disclosure, including leishmaniasis (caused by trypanosomes of the genus Leishmania) and toxoplasmosis (caused by the apicomplexan Toxoplasma gondii). The antigen may thus be derived from any of the aforementioned pathogens.

[0095] In the case of a complex of the tag moiety with a binding molecule comprised in a bispecific conjugate comprising an anti-CD40 antibody or fragment thereof, agonism of CD40 may activate the immune system to fight the infection (on a similar principle to use in therapy for cancer). In the case of the complex of the disclosure, as described above this may be provided with an antigen derived from the target pathogen, thus inducing a specific immune response against the pathogen. The vaccine platform is beneficially adaptable for e.g., a pandemic situation. This adaptability can in particular be provided by using a tag construct comprising an antigen that can be modified for viral diversity and antigen drift. Viral antigens can be selected based on HLA prevalence in a certain region along with viral serotype determinants.

[0096] The bispecific conjugate of the disclosure may, in one embodiment, comprise an antagonistic anti-CD40 antibody or antigen binding fragment thereof. In such an embodiment, the bispecific conjugate is useful to tolerize the immune system against a target that drives an autoimmune disease. In one embodiment of the complex of the disclosure, it comprises a bispecific conjugate in which the second moiety is an antagonist of CD40 and a tag construct comprising an antigen known to be the driver for an autoimmune disease, such as an autoimmune disease caused by T cells. The purpose of the bispecific conjugate in complex with the tag construct comprising antigen in this case is to re-educate the immune system to tolerize against the antigen causing disease, and it may thereby prevent the excessive destruction of healthy tissue. Examples of antigens useful in tag constructs according to such an embodiment are selected from the group consisting of antigens associated with SLE, type I diabetes, rheumatoid arthritis, vasculitis, myositis, multiple sclerosis, psoriasis and allergy.

[0097] The antigen (e.g., cancer antigen, pathogen-derived antigen or antigen associated with an immune-mediated disease) may 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 chosen antigen. In particular, the antigen may be selected on the basis that it is recognized by T cells used in adoptive cell therapy in the subject to be treated.

[0098] For instance, in adoptive cell therapy, T cells may be obtained from the subject, and T cells which recognize an antigen of interest isolated. The isolated T cells may then be expanded and / or otherwise treated to stimulate their effector functionality, and then re-infused into the subject to be treated. In this context, the antigen recognized by the re-infused T cells may be used in the tag construct. A complex of the disclosure may then be administered to the subject, so that the antigen activates the re-infused T cells.

[0099] Alternatively, T cells may be obtained from the subject to be treated or a donor, and genetically modified to express a TCR which recognizes a target antigen. The genetically modified T cells may 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 may be used in the tag construct. A complex of the disclosure may then be administered to the subject, so that the antigen activates the infused T cells. Methods in which administration of a complex of the disclosure is combined with adoptive cell therapy are particularly useful in the treatment of cancer, in which case the antigen used in the tag construct is a cancer antigen.

[0100] The disclosure thus provides a method of treating or preventing cancer, comprising administering to a subject a tag moiety or construct of the disclosure, a complex of the disclosure or a pharmaceutical composition of the disclosure.

[0101] In a particular embodiment, the disclosure provides a method of treating cancer in a subject, the method comprising:

[0102] (i) obtaining T cells from the subject;

[0103] (ii) isolating T cells which recognize a target cancer antigen, and optionally expanding the isolated T cells;

[0104] (iii) re-infusing the isolated T cells into the subject; and

[0105] (iv) administering to the subject a complex of the present disclosure, wherein the tag construct comprises the target cancer antigen. Equivalently, in step (iv) the subject could alternatively be separately administered a binding molecule comprised in a bispecific conjugate of the disclosure and a tag construct comprising the target cancer antigen.

[0106] In another embodiment, the disclosure provides a method of treating cancer in a subject, the method comprising:

[0107] (i) obtaining T cells from the subject or a donor;

[0108] (ii) genetically modifying the T cells to express a TCR which 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; and

[0109] (iv) administering to the subject a complex of the present disclosure, wherein the tag construct comprises the target cancer antigen. Equivalently, in step (iv) the subject could alternatively be separately administered a binding molecule comprised in a bispecific conjugate of the disclosure and a tag construct comprising the target cancer antigen.

[0110] The disclosure similarly provides the use of a tag moiety or tag construct of the disclosure or a complex of the disclosure in the manufacture of a medicament for the treatment of prevention of cancer.

[0111] Similarly, the disclosure provides a method of treating or preventing an infection, comprising administering to a subject a tag moiety or tag construct of the disclosure, a complex of the disclosure or a pharmaceutical composition of the disclosure.

[0112] The disclosure also provides the use of a tag moiety or tag construct of the disclosure or a complex of the disclosure in the manufacture of a medicament for the treatment of prevention of an infection.

[0113] Also, the disclosure provides a method of treating or preventing an autoimmune disease, comprising administering to a subject a tag moiety or construct of the disclosure, a complex of the disclosure or a pharmaceutical composition of the disclosure.

[0114] The disclosure also provides the use of a tag moiety or construct of the disclosure or a complex of the disclosure in the manufacture of a medicament for the treatment of prevention of an autoimmune disease.

[0115] Throughout the above embodiments, reference to the use of a complex of the disclosure includes the combined use of a binding molecule (e.g., comprised in a bispecific conjugate) of the disclosure and a tag moiety or tag construct, which are separately or sequentially administered.

[0116] In an alternative embodiment, the complex of the disclosure may be used in gene therapy. In this embodiment, a gene therapy vector or delivery system encoding both a binding molecule (e.g., comprised in a bispecific conjugate) of the disclosure and a tag moiety or tag construct of the disclosure may be administered to the subject. Upon take up by cells of the subject, the binding molecule and tag are expressed and secreted, and form a complex in vivo.

[0117] As noted above, the tag moiety or tag construct of the disclosure or complex of the disclosure may be used as a monotherapy, or in conjunction with other therapeutic agents. Thus, in the treatment of cancer the other therapeutic agent may be an anti-cancer agent, such as a chemotherapeutic agent, numerous classes of which are known in the art, or an immunological agent, including for example, interferons, immune checkpoint inhibitors (e.g. anti-PD-1 , -PD-L1 or -CTLA4 antibodies) and other immune-enhancing agents (e.g. anti-OX40 agonistic antibodies). Other therapeutic agents may be beneficial in the treatment of cancer or an infection, e.g., anti-proliferative or anti-inflammatory cytokines, and antiproliferative, immunomodulatory or factors influencing blood clotting, or inhibitors of angiogenesis. For treatment of an infection, the other (or second) therapeutic agent may be an anti-microbial agent, e.g., an antibiotic, anti-fungal or anti-viral agent.

[0118] The tag moiety, tag construct, complex, or pharmaceutical composition comprising the tag moiety, tag construct or complex (or the components of the complex) may 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 may be individually administered by these same methods. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion, e.g., directly to the site of a tumor.

[0119] The phrase ’’parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection. Alternatively, a non-parenteral route may be used, such as a topical, epidermal or mucosal route of administration. Local administration is preferred, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intra cavity infusion, intravesicle administration, and inhalation. However, the tag moiety, tag construct, complex or composition may also be administered systemically.

[0120] In embodiments where the binding molecule (e.g., comprised in a bispecific conjugate) and tag moiety or tag construct are administered individually, i.e., they are not first pre-mixed to form the complex, they must be administered via the same route. Preferably, they are both administered locally, e.g., intradermally, at the same (or substantially the same) site, such that the two components mix, and thus combine to form the complex, rapidly after administration. In these embodiments, the two components must be administered to the subject either simultaneously or rapidly one after the other, avoiding delay between administration of the first component and administration of the second component. This ensures the second component is administered, and complex formation is enabled, before the first component degrades or becomes excessively disseminated from the administration site.

[0121] A suitable dosage of a specific tag moiety or tag construct of the disclosure or complex of the disclosure may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions and products of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, i.e. , patient, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular complex employed, the route of administration, the time of administration, the rate of excretion of the complex, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0122] A suitable dose of a tag moiety, tag construct or complex of the disclosure may be, for example, in the range of from about 0.1 pg / kg to about 100 mg / kg body weight of the patient to be treated. For example, a suitable dosage may be from about 0.1 pg / kg to about 10 mg / kg body weight per day or from about 10 pg / kg to about 5 mg / kg body weight per day.

[0123] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0124] The tag moiety, tag construct or complex (or combination of binding molecule and tag construct) may be administered in a single dose or in multiple doses. The multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, complexes can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the administered species in the patient and the duration of treatment that is desired. The dosage and frequency of administration can 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 a long period of time. In therapeutic applications, a relatively high dosage may be administered, for example until the patient shows partial or complete amelioration of symptoms of disease. In an exemplary dosage regime, the complex (or combination of the conjugate and tag construct) is administered to the subject once a week, once a fortnight or once every three weeks, in a cycle repeated from 2 to 10 times.

[0125] Combined administration of two or more agents may be achieved in a number of different ways. In one embodiment, the complex and the other agent may be administered together in a single composition. In another embodiment, the complex and the other agent may be administered in separate compositions as part of a combined therapy. For example, the complex may be administered before, after or concurrently with the other agent. The complex of the disclosure may be administered in combination with or sequentially to tumor targeting antibodies, target therapy, pathway inhibitors or other immunomodulatory antibodies targeting e.g., PD- 1 , PD-L1, CD137, GITR, 0X40, CTLA-4, CD27, HVEM, LT R, and LAG3. Further the complex may also be combined with local radiation. Similarly, such additional therapies may be co-administered when the conjugate and tag construct are individually administered to the subject.

[0126] The disclosure also provides an in vitro or ex vivo method of activating a T cell expressing a TCR which recognizes an antigen, said method comprising contacting an antigen-presenting cell with: i) a tag construct of the disclosure and a binding molecule (e.g., comprised in a bispecific conjugate) as described herein, wherein said tag construct comprises the antigen recognized by said TCR; or ii) a complex of the disclosure, wherein a tag construct of said complex comprises the antigen recognized by said TCR.

[0127] Thus, the complex of the disclosure may be used to activate APCs in vitro or ex vivo, as well as in vivo. The complex thus have both medical and non-medical uses, and all such uses are encompassed herein. For example, isolated or cultured APCs may be contacted with the complex, e.g., in a laboratory setting, for example for research, development or testing purposes. This may be achieved by pre-mixing the conjugate and tag construct, to form the complex, and then applying the complex to the APCs. Alternatively, the conjugate and tag construct can be individually applied to the APCs, such that the complex forms within the APC culture.

[0128] The complex may be used to activate a T cell which expresses a TCR which 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 for activation by the complex, may be contacted with the T cell. Thus, for example, APCs may be cultured or incubated in the presence of the complex (or constituent parts thereof), following which the APCs may be contacted with the T cells, e.g., co-cultured, or further incubated in the presence of the T cells. Alternatively, the complex (or constituent parts thereof), APCs and T cells may be incubated or co-cultured together. Thus, the antigen is delivered to the APCs and presented to the T cells, resulting in their activation.

[0129] The invention is further illustrated by the following non-limiting figures and examples:

[0130] Brief description of the figures

[0131] Figure 1 shows four sensorgrams obtained from SPR measurements of the interaction of tag constructs with bispecific binding molecule SP027 as described in Example 2. The tag constructs were the following peptides: A) ULI0179 (SEQ ID NO:30); B) UU0185 (SEQ ID NO:31); C) UU0186 (SEQ ID NQ: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.

[0132] Figure 2 shows the results of an in vitro screen for endogenous antibodies towards a panel of tag constructs comprising the indicated trimmed versions of the tag moiety compared to the original 18-mer peptide tag moiety ULI0024 (SEQ ID NO:84), as described in Example 3. The figure is divided into three sections, each section showing the results for each of three serum donors.

[0133] Figure 3 shows a summary of the in vitro CD4+ T cell proliferation capability of tag constructs UU0060 (SEQ ID NO:97), UU0129 (SEQ ID NO:28), UU0130 (SEQ ID NO:39) and ULI0131 (SEQ ID NO:35) each comprising a tag moiety as disclosed herein together with a cargo moiety comprising the peptidic OTII antigen sequence SEQ ID NO:19, when used at the indicated concentrations in combination with bispecific binding molecule SP019 as described in Example 4. Y-axis: % proliferating OTII cells. X-axis: tested tag constructs, positive control (ConA) and negative control (mAb).

[0134] Figure 4 shows the results of an enzyme-linked immunospot (ELISpot) assay of the T cell response in the indicated four donors A-D towards tag constructs comprising tag moiety peptide ULI0086 (SEQ ID NO:2), ULI0002 (SEQ ID NO:24), UU0008 (SEQ ID NQ:100), UU0152 (SEQ ID NO:98) or UU0165 (SEQ ID NO:99) at 10 pM as described in Example 5. T cell response is presented on the y-axis of each plot as SFU per 3 x 105cells after 24 h of stimulation with the tested tag constructs (error bars indicated).

[0135] Figure 5 shows the results of an in vivo T cell proliferation assay in mice, as described in Example 6. Harvested organs and tag constructs are displayed on the x-axis. The T cell immune response, illustrated by the percentage of proliferating Thy1.1 cells (in %) in each respective organ, is displayed on the y-axis. From left to right within each organ group, the tag constructs used in the immunization procedure were UU0032 (SEQ ID NQ:102) (negative control), UU0030 (SEQ ID NQ:101), UU0120 (SEQ ID NQ:103), UU0142 (SEQ ID NQ:104) and UU0146 (SEQ ID NO: 105).

[0136] Figure 6 shows the results of an in vivo T cell proliferation assay in harvested draining lymph nodes from mice, as described in Example 7. Tested constructs are displayed on the x-axis. The T cell immune response, illustrated by the percentage of proliferating CD8+ Thy1.1+ T cells (in %) for each respective tested construct, is displayed on the y-axis. The tag constructs used in the immunization procedure were UU0030 (SEQ ID NQ:101), UU0120 (SEQ ID NQ:103), UU0146 (SEQ ID NQ:105), UU0142 (SEQ ID NQ:104) and UU0032 (SEQ ID NQ:102) (negative control).

[0137] Figure 7 shows the results of the in vivo T cell proliferation described in Example 9. After administration of the indicated constructs, the relevant T cell population was quantified in the indicated organs by flow cytometry and given as percentage CFSE|OWof the CD3+and CD4+cells.

[0138] Figure 8 is a survival graph showing the probability of survival in the indicated three groups of mice in the experiment described in Example 10. HD: high dose. LD: low dose.

[0139] Figure 9 shows the results of the in vivo T cell proliferation described in Example 8. After administration of the indicated constructs, a single cell suspension was prepared from organs. CD4+T cell proliferation, as defined by CFSE|OW, and CD4+T cell activation, as defined by ICOS expression, was assessed and results are shown in Figure 9A and B, respectively. On the Y axis the cell markers used is displayed. On the X axis, from left to right, the constructs used in the immunization procedure were “Vehicle” (negative control), ULI0060 (SEQ ID NO:97), ULI0126 (SEQ ID NO:27), UU0127 (SEQ ID NO:38), and UU0128 (SEQ ID NO:34).

[0140] Figure 10 shows the difference in anti-tumor efficacy (illustrated as tumor growth volume, in mm3) between mice immunized with bispecific binding conjugate SP027 and either a tag construct (ULI0170, SEQ ID NO:147) comprising a 7-mer tag moiety and an immunogenic epitope derived from the E7 antigen (SEQ ID NO: 140) as cargo moiety, a tag construct (ULI0169, SEQ ID NO:141) comprising or a 9-mer tag moiety and an immunogenic epitope derived from the E7 antigen (SEQ ID NO: 140) as cargo moiety, all in comparison to mice receiving vehicle (negative control).

[0141] The following examples disclose the development of an improved and novel non-immunogenic tag moiety, generated by utilizing advanced binding experiments and methodology. The generic amino acid sequence of the tag moiety described herein is listed in the sequence listing with SEQ ID NO:1. The examples further describe the characterization of different variants of the tag moiety, as well as tag constructs and complexes comprising them, and demonstrate their in vitro and in vivo functionality.

[0142] Example 1 Surface plasmon resonance analysis of trimmed tag moieties Previously presented data suggest that a tag construct comprising an 18 amino acid residues (aa) long peptide tag moiety (ULI0024; SEQ ID NO:84) can be shortened to a 12 aa long tag moiety and retain its binding affinity towards the IBIIICI mouse scFv comprising SEQ ID NO: 15 (see Example 17 on p 67-68 of WQ2021 / 239968). Below, the binding kinetics of a selection of further trimmed peptide tag moieties were studied using surface plasmon resonance (SPR). The 18 aa long peptide tag moiety was shortened to 11 , 10, 9, 8 and 7 aa long peptides, and the binding kinetics towards humanized variants of IBIIICI, e.g., one denoted “IBIIICI CDR graft scFv SG” and comprising the amino acid sequence SEQ ID NQ:107, were investigated. instrument (GE Healthcare) and single cycle kinetics. The anti-FLAG M2 antibody (Sigma-Aldrich, #F3165) was immobilized onto a Series S CM5 chip by primary amine coupling using NHS-EDC chemistry according to the manufacturer’s instructions, allowing capture of the respective scFv through their FLAG tags. A 5- fold dilution series with five concentrations (0.16 nM to 100 nM) of the different peptides was sequentially injected over the flow cells, allowing binding to the captured scFv. Following a dissociation phase, regeneration of the surface was accomplished under acidic conditions using 10 mM glycine-HCI at pH 2.1. By subtracting the response curve of a reference surface having only the anti-FLAG antibody immobilized thereto, response unit sensorgrams for all peptides were obtained. Data was analyzed using BIAeval v.3.1 (GE Healthcare).

[0143] Table 1 : Peptides used in Examples 1 and 2. The tag moiety part is in bold.

[0144] Results

[0145] The kinetic parameters of binding of IBIIICI CDR graft scFv variant SG (SEQ ID NO: 107) to the different peptides are displayed in Table 2. As stated above with reference to Example 17 of WO2021 / 239968, we previously showed that the binding of the IBIIICI mouse scFv was unaffected by shortening of the tag moiety peptide length from 18 to 12 amino acids. The results here suggest that the tag moiety can be shortened even further. In fact, the binding of the IBIIICI CDR graft scFv variant SG was maintained also when shortening the peptide to 8 aa. The peptide ULI0091 contains the 8 aa tag moiety FIGITELK (SEQ ID NO:90) fused C-terminally to the ovalbumin epitope SIINFEKL, and exhibited retained binding affinity to IBIIICI CDR graft scFv variant SG. However, by reducing the length of the tag moiety to 7 aa (FIGITEL; SEQ ID NO:89), or by mutating position 8 of the peptide from K to H (FIGITELH; SEQ ID NO:91), binding was retained but affinity decreased considerably (over 10-fold, as seen when pairwise comparing the results obtained with UU0090 (SEQ ID NO:89) and UU0091 (SEQ ID NQ:90)).

[0146] Furthermore, the results suggest that the shortened tag moiety ULI0091 can be C-terminally modified without loss of binding to the IBIIICI CDR graft scFv variant SG. In contrast, modification of the N-terminal causes a complete loss of binding to the scFv (ULI0088, SEQ ID NO:88). Similar binding properties were observed with all other tested IBIIICI CDR graft scFv variants (data not shown).

[0147] Table 2: SPR kinetic data for binding of tested peptides to scFv variant SG

[0148] Example 2

[0149] SPR analysis of binding of conjugate comprising scFv to tag constructs comprising a 9 aa tag moiety and C-terminal antigens

[0150] The binding kinetics of a panel of tag constructs comprising a 9 aa peptide tag moiety, with or without the addition of antigenic cargo seguences C-terminally of the 9 aa tag moiety, towards the IBIIICI CDR graft scFv variant SG in the context of a bispecific conjugate were studied using SPR.

[0151] Materials and methods The IBIIICI CDR graft scFv variant SG (SEQ ID NO:107) was placed in the conjugate context in analogy with the constructs described in W02020 / 104690 and WO2021 / 239968. Briefly, the scFv was designed to form part of a bispecific conjugate construct wherein one copy of the peptide binding scFv was conjugated to another binding molecule, e.g., to each heavy chain of a full-length antibody against CD40. See Figure 6A of W02020 / 104690 for a schematic illustration of the construct design. The anti-CD40 antibody used in this Example is a variant of the antibody denoted “A9” in WO2021 / 239968. The bispecific conjugate construct herein was denoted SP027. SP027 comprises two copies of one polypeptide chain having SEQ ID NO: 113, which constitutes the heavy chain of the A9 anti-CD40 antibody linked to the scFv variant SG, and two copies of one polypeptide chain having SEQ ID NO: 114, which constitutes the light chain of the anti-CD40 antibody.

[0152] The tag constructs tested comprised a 9 aa tag moiety sequence with or without a coupled cargo sequence. Affinity measurements of binding of tag constructs UU0086 (SEQ ID NO:2), UU0179 (SEQ ID NQ:30), UU0185 (SEQ ID NO:31) and ULI0186 (SEQ ID NO:32) to the bispecific construct SP027 was performed by SPR using a Cytiva BiaCore 8K instrument (Cytiva) and a multi-cycle kinetics approach. The bispecific construct was immobilized onto a Series S CM5 chip by primary amine coupling using NHS-EDC chemistry (Cytiva, #BR100633). A 2-fold dilution series comprised of six concentrations (15.6 nM to 1000 nM) of the tested tag constructs ULI0086, ULI0179, ULI0185 and ULI0186 were sequentially injected over the flow cells, allowing binding to the captured antibody. Following a dissociation phase, regeneration of the surface was accomplished under acidic conditions using 10 mM HCI at pH 2.1. By subtracting the response curve of a reference surface having SP027 immobilized thereto, response unit sensorgrams for all peptides were obtained. Data was analyzed using BIAevaluate software (Cytiva) using multi-cycle kinetics models for fitting of a 1:1 binding event.

[0153] Results

[0154] The resulting kinetic properties of the tested tag constructs are displayed in Figure 1 (A: UU0179; B: UU0185; C: UU0186, and D: UU0086). Overall, the binding properties of the tag construct comprising a 9 aa peptide tag moiety only (ULI0086) were very similar to those of tag constructs comprising the 9 aa peptide tag moiety together with a cargo moiety, herein a peptide sequence. The similar binding properties shown in the four panels of Figure 1 suggests that the addition of cargo moieties C-terminally of the 9-mer tag moiety does not affect the tag moiety’s binding affinity to IBIIICI CDR graft scFv variant SG.

[0155] Example 3 Investigation of the presence in donor plasma of endogenous antibodies to different tag moieties

[0156] The original 18-mer tag moiety is of non-human origin and contains a known B-cell epitope. It is therefore possible that recipients of tag constructs according to the disclosure have pre-existing, endogenous antibodies against the tag moiety, limiting its usefulness due to the risk of antibody binding competition. The amount in donor plasma of endogenous antibodies against tag constructs comprising trimmed versions of the original tag moiety was therefore investigated and compared to the amount of endogenous antibodies against a tag construct comprising the original 18- mer peptide seguence ULI0024 (SEQ ID NO:84).

[0157] Materials and methods

[0158] Serum from three healthy human donors was used to screen for endogenous binding to the tag moiety in an ELISA experiment. On day 0, exemplified tag constructs (see Table 3) comprising 9-mer, 10-mer and 11-mer tag moieties were diluted to 1 pM in PBS. 100 pl of each diluted tag construct was added to pre-coated streptavidin plates and incubated at 4 °C overnight. On day 1 , the plates were washed 3 times with PBS containing 0.05 % Tween-20. This was repeated after each incubation step prior to addition of plasma samples, secondary antibody and TMB (3,3’,5,5’-tetramethylbenzidine). The plate wells were blocked with PBS containing 10 % BSA and 0.05 % Tween-20 for 2 h at RT prior to addition of donor human plasma. Before addition to the wells, the donor human plasma was serially diluted 1 :1 in PBS containing 1 % BSA and 0.05 % Tween-20 starting at a 1 :200 dilution down to 1 : 1600. After dilution, 100 pl of the donor human plasma was added to each well and incubated for 1 h at RT. The final step was the addition of 1:4000 diluted total IgG secondary antibody-HRP (Dako, #P0214) in PBS containing 1 % BSA and 0.05 % Tween-20 to each well, followed by 1 h of incubation at RT. Development of the plate was done with TMB and the reaction was stopped by addition of 1 M H2SO4. The absorbance at 450 nm was measured. Table 3: Tag constructs used in the study of binding to endogenous antibodies. The tag moiety part is in bold.

[0159] Results

[0160] Endogenous antibodies bound to the respective tag construct were measured at 450 nm for the tag constructs listed in Table 3. The results are shown in Figure 2, and show that the serum of all three donors contained pre-existing endogenous antibodies binding to the 18 aa peptide tag moiety (ULI0024), containing a known 13- cell epitope. The lowest endogenous antibody reactivity was seen for constructs comprising the 9 aa long peptide tag moiety (exemplified by ULI0135, ULI0154 and ULI0155). Point mutations in the 9 aa constructs (ULI0154, mutation K8L; ULI0155, mutation K8H) had varying effects on endogenous antibody reactivity. The K8L mutation in a 9 aa peptide tag (ULI0154) clearly increased endogenous reactivity in two of the donors when compared to the original 9 aa long peptide tag moiety (ULI0135). In contrast, the K8H mutation in a 9 aa peptide tag (ULI0155) did not induce endogenous antibody binding when compared to the original 9 aa long peptide tag moiety (ULI0135). A higher endogenous antibody reactivity was seen in all donors for constructs comprising 10 aa and 11 aa long tag moieties (ULI0136, ULI0148, ULI0137, ULI0149) compared to the unmutated, 9 aa long peptide tag moiety (ULI0135). The K8L mutation in the 10 aa and 11 aa long tag moieties, in particular, led to endogenous reactivity in all three donors. No donor serum sample showed endogenous binding to the negative control (data not shown).

[0161] Example 4

[0162] T cell proliferation capacity of tag constructs measured in a T cell proliferation assay CD4+ T cell proliferation capacity of tag constructs was tested. The tag constructs studied were 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 as disclosed herein together with a cargo moiety comprising the peptidic OTII antigen sequence SEQ ID NO:19. Tag constructs were used in complex with a bispecific construct denoted SP019 and evaluated using CD4+ OTII cells. The CD4+ OTII cells express a T cell receptor specific for chicken ovalbumin peptide position 323-339 presented on MHC class II.

[0163] Materials and methods

[0164] Bone marrow cells isolated from hCD40 transgenic mice (provided by the University of Southampton and owned by Dartmouth College) were differentiated into immature bone marrow dendritic cells (imBMDCs) during an 8-day culture with GM- CSF (20 ng / ml, Peprotech, 315-03). On day 8, the cells were harvested, and cell maturation was verified by checking the expression of CD11b (using antibody M1 / 70, Biolegend), CD11c (using antibody N418, Biolegend), human CD40 (using antibody 5C3, Biolegend), CD86 (using antibody GL-1 , Biolegend) and MHC-II (using antibody M5 / 114.15.2, Biolegend) by flow cytometry. The imBMDCs were diluted to a concentration of 5 x 105cells / ml and plated at 50 pl / well in a 96-well tissue culture treated plate in the presence of GM-CSF (60 ng / ml, Peprotech).

[0165] The bispecific binding construct SP019 used in this experiment is analogous to the SP027 construct used in Example 2, and comprises two copies of one polypeptide chain having SEQ ID NO: 113, which constitutes the heavy chain of the A9 anti-CD40 antibody linked to the scFv variant SG, and two copies of one polypeptide chain having SEQ ID NO: 115, which constitutes the light chain of the A9 anti-CD40 antibody.

[0166] SP019 (10 nM) was separately mixed with each one of the peptides UU0060, UU0129, UU0130 and UU0131 (each at the two concentrations 100 and 20 nM) and pre-incubated for 30 min at room temperature. The different pairwise mixes of bispecific binding construct with peptide were added to the imBMDCs and incubated for 2 h at 37 °C with 5 % CO2. Unbound binding construct and free peptides were removed by washing the cells twice at 300 g for 5 min.

[0167] CD4+ OT-II cells were isolated from spleens and inguinal lymph nodes of OTII transgenic mice. The organs were made into a single cell suspension and the red blood cells lysed with RBC lysis buffer (Invitrogen, 00-4333-57). The CD4+ cells were isolated using the Dynabeads™ Untouched™ Mouse CD4 Cells Kit (Invitrogen, 11415D). Isolated CD4+ OTII cells were stained with CFSE (2 pM, Invitrogen, C34570) and then co-cultured with 25,000 BMDCs per 50,000 CD4+ OTII cells for 96 h at 37 °C with 5 % CO2. After co-culture, the cells were collected and stained with anti-CD3, anti-CD4 and ICOS to assess proliferation by flow cytometry.

[0168] Results

[0169] The results are shown in Figure 3. All of the peptides ULI0060, ULI0129, ULI0130 and ULI0131 induced >90 % OTII T cell proliferation as determined by CFSE dilution. Two proliferation controls, ConA (positive control) and mAb (negative control lgG2 antibody), were included in the proliferation assay and analyzed. mAb alone did not induce any cell proliferation. The data demonstrate that tag constructs comprising a 9-mer tag moiety (ULI0129, ULI0130 and ULI0131) promote the same level of CD4+ T cell proliferation as the tag construct comprising the 18-mer tag moiety sequence ULI0060 and as the positive proliferation control ConA.

[0170] Example 5

[0171] Analysis of peptide-specific immune responses by ELISpot

[0172] An enzyme-linked immunospot (ELISpot) assay was utilized to analyze and compare the peptide-specific immune response to a tag moiety alone to those of a tag moiety in the tag construct context (tag moiety with antigen moiety).

[0173] Materials and methods

[0174] Peripheral blood mononuclear cells (PBMCs) from four healthy donors were tested for reactivity against the peptides ULI0086 (SEQ ID NO:2), ULI0002 (SEQ ID NO:24), UU0008 (SEQ ID NQ:100), UU0152 (SEQ ID NO:98) and UU0165 (SEQ ID NO:99). PMBCs were isolated from freshly drawn buffy coats (<8 hours postdonation) 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 media. After centrifugation (1200 g, 10 min), the PBMC layer was poured into a new tube and washed twice with 1x PBS. The resulting PBMCs were cryopreserved in 90% FBS / 10% DMSO at -150 °C until further use.

[0175] 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, resuspended in the same medium (excluding nuclease) and were left to rest at a concentration of 2x106cells / ml for 2 h at 37 C, 5% CO2. During cell rest, pre-coated ELISpot plates (anti-IFNy, Mabtech, 3420-4APT) were washed and blocked. After resting, cells were harvested and seeded in the ELISpot plates at a concentration of 3x105cells / well. Subsequently, each peptide was added in triplicate wells (10 pM final concentration) followed by each donor’s cells, and the plates were incubated at 37 C, 5% CO2 for 24 h. Plates were washed and an alkaline-phosphatase- conjugated detection antibody (1:200, 7-B6-1-ALP, Mabtech) was added to the plates for 2 h. Plates were washed and the BCIP / NBT substrate (Mabtech, 3420- 4APT) was added for 10 min to develop the plates. The plates were washed and left to dry overnight and were then analyzed using a Mabtech IRIS™ reader coupled with Mabtech Apex™ software for spot enumeration.

[0176] Results

[0177] The results are shown in Figure 4. 24 h stimulation with peptides ULI0002 (SEQ ID NO:24), UU0008 (SEQ ID NO:100) and UU0152 (SEQ ID NO:98) induced IFNy secretion in all four donors tested, indicating that all four donors respond to the CMV derived NLV peptide. ULI0086 (SEQ ID NO:2), i.e. , the tag moiety alone, did not induce any spot formation in any of the tested donors. Two of four donors responded to peptide ULI0165 (SEQ ID NO:99), a mutated peptide derived from the oncogenic KRAS protein. These data demonstrate that tag constructs comprising the 9-mer tag moiety of the disclosure coupled to antigen peptides induce peptidespecific immune responses. The antigen peptide-specific immune responses directed against the T cell antigenic part of the synthetic peptide stretches that include tag moiety and T cell epitopes vary due to individual inherent factors. The data also demonstrate that the tag moiety alone (ULI0086) does not induce any measurable T cell responses.

[0178] Example 6 T cell proliferation in vivo

[0179] An in vivo T cell proliferation experimental set-up was utilized to analyze the immune responses of mice to tag constructs, in the context of a complex with an antibody-scFv conjugate delivered together with a tag construct comprising a tag moiety and an antigen moiety, compared to the immune responses of mice to tag constructs lacking a tag moiety sequence (i.e. using an antibody-scFv conjugate delivered together with only the antigen moiety without any tag moiety sequence).

[0180] Materials and methods

[0181] This experiment was performed to evaluate the immunogenic potential of tag constructs comprising cargo moieties including an antigenic sequence, exemplified by human melanoma gp1OO (SEQ ID NO:20), together with tag moieties ranging in length. The tag constructs used were ULI0030 (SEQ ID NO:101), ULI0146 (SEQ ID NQ:105), UU0142 (SEQ ID NO:142) and UU0120 (SEQ ID NQ:103), comprising tag moiety lengths of 18, 11 , 10 and 9 aa, respectively. In the tag constructs, the tag moiety sequences were C-terminally attached to the N-terminus of the antigen sequence and delivered to mice in the complex context, i.e., the tag construct was delivered together with the bispecific antibody construct SP019 (see Example 4). A negative control in the form of ULI0032 (SEQ ID NO: 102) was utilized which lacked a tag moiety sequence and only included the cargo moiety gp100. Also, ULI0032 was delivered together with the bispecific construct SP019.

[0182] Twenty mice (B-hCD40, Biocytogen) were divided into five groups (4 test groups and 1 control group), each group consisting of 4 mice. On day -1 (preimmunization), cells were isolated from spleens and inguinal lymph nodes from pmel- 1 transgenic mice (pmel-1). CD8+ T cells in pmel-1 transgenic mice express T cell receptors specific for the melanoma antigen gp100 (SEQ ID NQ:20). Red blood cells were lysed with RBC lysis buffer (Invitrogen, #00-4333-57) and single cell suspensions were prepared from the harvested organs. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and 11x106CFSE labeled pmel-1 cells in 100 pl 1x sterile PBS were transferred to B-hCD40 mice through intravenous injection. On day 0, one day after pmel-1 cell adoptive transfer, the 20 B-hCD40 mice were immunized via needle injection subcutaneously (s.c.) in the hock according to the set-up presented in Table 4. Each mouse received a 20 pl injection comprising 15 pmol of the bispecific construct SP019 and 37.5 pmol of tag construct.

[0183] Table 4: Overview of immunization set-up

[0184] To evaluate the results 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 1x106cells per organ were stained with Thy1.1 , CD3+ and CD8+ to assess the proliferation of antigen-specific T cells by flow cytometry using the congenic marker Thy1 .1 as a tracker.

[0185] Results

[0186] The results are displayed in Figure 5, with the harvested organs displayed on the x-axis and the immune response, illustrated by the percentage of proliferating Thy1.1 cells (in %), on the y-axis. All tested tag constructs comprising a tag moiety sequence, exemplified by ULI0030 (18-mer), ULI0120 (9-mer), ULI0142 (10-mer) and ULI0146 (11-mer), induced higher a Thy1.1 CD8+ T cell proliferation than a tag construct not comprising any tag moiety sequence, exemplified by ULI0032 (cargo moiety only). This is observed in all harvested organs, and the data indicate that the targeted delivery system used induces potent immune responses compared to cargo moiety administration alone. Interestingly, the construct comprising the shortest tag moiety sequence (9 aa long, ULI0120) 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 assessed dose with the repeating dosing schedule. ULI0120 induced 35 %, 35 % and 20 % Thy1.1 CD8+ T cell proliferation in inguinal draining, popliteal draining and spleen, respectively.

[0187] Example 7 T cell proliferation in vivo

[0188] This experiment was performed to evaluate the immunogenic potential of cargo moieties comprising antigenic peptides, as exemplified by human melanoma gp100, delivered with an 18-mer or 9-mer tag moiety attached to the C-terminus of the cargo moiety sequence. The immune responses induced by these combinations were compared to the response induced by the gp100 peptide delivered with CpG ODN 1826 as a benchmark adjuvant. Materials and methods

[0189] Thirty mice, strain B-hCD40 (Biocytogen) were divided into test and control groups of 3 to 5 mice per group. On Day -1 , one day before start of immunizations, cells were isolated from spleens and inguinal lymph nodes from pmel-1 transgenic

[0190] 5 mice (pmel-1). CD8+ T cells from pmel-1 transgenic mice express a T cell receptor specific to a known immunogenic melanoma gp1OO derived MHC class I epitope. Red blood cells were lysed with RBC lysis buffer (Invitrogen, 00-4333-57) and single cell suspensions were prepared from the spleens and inguinal lymph nodes. 1 x 107pmel-1 cells in 100 pl 1X sterile PBS were transferred to the B-hCD40 mice through

[0191] 10 intravenous injection. The mice were immunized twice according to the set-up presented in Table 5, with 5 days between immunizations, 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 ULI0120 (SEQ ID NO:103) were each mixed separately with SP019 prior to

[0192] 15 injections. T reatments were administered by subcutaneous needle injection into the right foot hock in a total volume of 20 pl. The groups of mice were immunized according to the following format:

[0193] Table 5: Overview of immunization set-up in Example 7

[0194] In the second immunization, 150 pmol bispecific antibody SP019 was used in the antibody peptide mixes following the same format.

[0195] Draining popliteal and inguinal lymph nodes were collected 2 days after the

[0196] 5 second immunization to evaluate T cell proliferation. Single cell suspensions were prepared from organs and 1x106cells were stained with Thy1.1 , CD3+ and CD8+ to assess T cell expansion by flow cytometry.

[0197] Results

[0198] 10 The results are shown in Figure 6. Peptide ULI0032 (gp100 without a tag moiety) did not induce any immune responses when delivered alone or together with SP019. Peptide ULI0030 (gp100 with 18-mer tag moiety) did not induce any immune response when delivered with SP019. In contrast, peptide ULI0120 (gp100 with 9- mer tag moiety) did induce 10 % Thy 1.1+ CD8+ T cell expansion in draining lymph

[0199] 15 nodes. This level of immune induction is similar to the immune response detected with ULI0032 delivered at a 10x higher dose together with CpG ODN 1826. The data demonstrate that the gp100 peptide coupled to the 9-mer tag moiety and delivered with the bispecific antibody construct SP019 induces a potent immune response that is significantly higher compared to the immune response of the gp100 peptide

[0200] 20 coupled to the 18-mer tag moiety and delivered with the bispecific antibody construct SP019.

[0201] Example 8

[0202] 25 T cell proliferation in vivo

[0203] The immune responses induced by tag constructs in combination with bispecific binding conjugate SP027 (see Example 2) was evaluated. All tag constructs used in this experiment comprise a synthetic long peptide corresponding to a known immunogenic epitope from ovalbumin (SEQ ID NO: 19). Amino acid

[0204] 30 sequence and SEQ ID NO for each respective tag construct, as well as for the immunogenic epitope from ovalbumin, are detailed in Table 6. The experiment was performed to evaluate the immunogenic potential of the tag constructs comprising an ovalbumin-derived antigen peptide and a tag moiety of differing length or sequence (either an 18-mer tag moiety or 9-mer tag moieties) attached to the N-terminus of the ovalbumin-derived peptide when in complex with SP027.

[0205] Materials and methods

[0206] Adult htgCD40 mice, htgCD40 from Biocytogen (10-11 weeks old), were divided into test and control groups (n=3-5). Day -1 , one day before start of immunizations, cells were isolated from spleens and inguinal lymph nodes from OT-II transgenic mice. These mice express the mouse alpha-chain and beta-chain T cell receptor that pairs with the CD4+co-receptor and is specific for a known immunogenic helper epitope derived from the ovalbumin protein. A single cell suspension was prepared by passing the spleens and lymph nodes through a 70 pM strainer and red blood cells (RBCs) in the spleen suspensions were lysed with RBC lysis buffer (eBioscience™ cat. No.00-4300-54). Remaining cells were CFSE stained (Thermo Fisher Scientific, C34554) before being diluted in PBS. 1x107CFSE stained cells in 100 pl of sterile PBS was injected intravenously in the htgCD40 mice. At day 0, the htgCD40 mice were immunized with vehicle or 150 pmol SP027 mixed with 450 pmol of respective tag constructs, ULI0060, ULI0126, ULI0127 or ULI0128. The treatment administration was performed by subcutaneous injection of the total volume of 50 pl into the right hock.

[0207] Table 6: Immunization set-up (tag moiety is indicated in bold)

[0208] To evaluate T cell proliferation, at day 2 post-immunization, the draining lymph nodes (popliteal and inguinal) was collected and pooled. A single cell suspension was prepared from organs and the cells were stained with CD3+, CD4+and ICOS (Biolegend) marker to assess T cell activation by flow cytometry.

[0209] Results

[0210] The results are presented in Figure 9 and show proliferating CD4 T cells defined as CFSEi0Wand activation by upregulation of the activation marker ICOS on the CD4+T cells in the draining lymph nodes 48 h after vaccination. Tag constructs, comprising a 9-mer tag moiety (ULI0126, ULI0127 and ULI0128) and an immunogenic helper epitope derived from the ovalbumin protein as the cargo moiety, together with the bispecific binding conjugate SP027, induced higher CD4+T cell activation compared to vehicle and 3-6-fold mean increase compared to a tag construct, comprising an 18-mer tag moiety (ULI0060) and an immunogenic epitope from the ovalbumin antigen as the cargo moiety, delivered together with the bispecific binding conjugate SP027.

[0211] Thus, the present example shows that the ovalbumin antigen peptide coupled to 9-mer tag moieties comprised in a tag construct according to the present invention and delivered with the bispecific antibody construct SP027 induced a higher immune response than the immune response of the ovalbumin peptide coupled to the comparative prior art 18-mer tag moiety and delivered with the same bispecific conjugate SP027.

[0212] Example 9 T cell proliferation in vivo

[0213] The study essentially described in Example 8 was carried out with 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).

[0214] Materials and methods

[0215] Adult htgCD40 mice (n = 7, pooled data from 2 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-labelled (10 x 106cells) OT-II derived cells were injected intravenously via the tail vein. Next day, the mice were injected subcutaneously on the right side of the hock with SP027 bispecific conjugate (15 pmol) together with either the immunogenic epitope from ovalbumin UU0138 alone (37.5 pmol)) or the 9-mer tag moiety linked to the ovalbumin epitope cargo moiety in the construct UU0126 (37.5 pmol). The draining popliteal, draining inguinal, non-draining inguinal lymph nodes and spleens were collected 2-3 days after the last injection. The organs were passed through a 70 pm cell strainer, and the RBCs in the spleens were lysed by using RBC lysis buffer prior to surface marker staining and analysis by flow cytometry. Results

[0216] The results are presented in Figure 7 and show that the MHC class II derived antigen-presentation pathway was supported by antibody-mediated peptide delivery to antigen-presenting cells. The ability to induce CD4+T cell activation was assessed with the OT-II model comparing peptide delivery with and without the 9-mer tag moiety defined by SEQ ID NO:2. The affinity interaction between the tag moiety comprised in the tag construct and the bispecific conjugate was proven to be decisive for CD4+T cell proliferation in the draining lymph nodes, with a 2-7-fold increase seen for ULI0126 (tag construct comprising a 9-mer tag moiety and an ovalbumin epitope cargo moiety) when compared to the peptide (ovalbumin epitope) without the tag moiety (ULI0138).

[0217] Example 10 Efficacy of tumor treatment

[0218] The anti-tumor responses induced by repeated vaccination with peptides in combination with the bispecific binding conjugate SP027 in the TC-1 tumor model were investigated. By local vaccination at a non-tumor site, tag constructs comprising a tumor-associated antigen (of viral or neoantigen origin) were delivered to dendritic cells by the bispecific conjugate’s binding to CD40 and internalized to release the antigenic peptides. The antigen peptides were processed and presented to T cells. With repeated injections, antigen-specific T cells expand, migrate and target tumor cells. The anti-tumor effect of the complex of bispecific conjugate and tag construct was compared to the peptide alone and / or to the bispecific conjugate alone, also injected at a non-tumor site.

[0219] Materials and methods

[0220] Mice, strain B-hCD40 (Biocytogen) (9-12 weeks) were divided into the following three treatment groups (6-7 mice per group): Group 1 : a high dose of 30 pg antigenic peptide ULI0171 (SEQ ID NO:140; immunogenic E7 derived synthetic long peptide HPV16 E7 [HPV16 E7 44-62]); Group 2: bispecific binding conjugate SP027 alone, at 50 pg in first injection on day 5 and at 30 pg in injections on day 10 and 15; Group 3: bispecific binding conjugate SP027 at 50 pg in first injection on day 5 and at 30 pg in injections on day 10 and 15 in complex with tag construct ULI0169 (SEQ ID NO:141) comprising a 9 aa tag moiety linked to the HPV peptide and given at a low dose of 3 pg in the complex mixture at every dosing. Appr 5x105tumor cells were injected s.c. in the right flank at day 0. Injection of treatment to the mice was performed s.c. in the left hock on day 5, day 10 and day 15, i.e., therapy was not given at the site of tumor growth. Tumor growth and survival were monitored 3 times a week, and the mice are sacrificed when the tumor size reached a maximum of 1000 mm3as the experimental endpoint, or if reaching the humane endpoint (e.g., health status with regard to wounds, weight and appearance. Mice reaching the humane endpoint while not reaching the experimental endpoint were censored.

[0221] Results

[0222] The results are shown in Figure 8. The TC-1 peptide from the oncogenic HPV protein E7 coupled to a 9-mer tag moiety (ULI0169) and delivered in complex with the bispecific conjugate construct SP027 induced a potent immune response and reduced the tumor volume significantly compared to the control groups that were given either the bispecific conjugate SP027 alone or a high dose of the antigenic peptide without tag moiety (ULI0171), even though the therapy was injected at a nontumor site at a low dose.

[0223] Example 11 Efficacy of tumor treatment

[0224] Anti-tumor efficacy was investigated by immunizing mice with the bispecific binding conjugate SP027 in combination either the tag construct ULI0170 (SEQ ID NO:147) (comprising a 7-mer tag moiety, SEQ ID NO:148) or the tag construct ULI0169 (SEQ ID NO:141) (comprising a 9-mer tag moiety, SEQ ID NO:2). Both tag constructs comprised respectively a cargo moiety corresponding to an immunogenic epitope derived from the E7 antigen (SEQ ID NO: 140). The tag construct comprising the 9- mer tag moiety (ULI0169) binds to the bispecific binding conjugate SP027, and targeted peptide delivery is facilitated to antigen presenting cells. The tag construct comprising the 7-mer tag moiety (ULI0170) has minimal or no binding to the bispecific binding conjugate SP027, and therefore that set-up was considered to represent unlinked delivery of tag construct and bispecific binding conjugate.

[0225] 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 the tumor model. The TC-1 cell line was cultured in RPMI Glutamax (Gibco, cat#11360070) supplemented with 10% FBS (Thermofisher, cat#10500064) 1% Penicillin-Streptomycin (Gibco, cat#11548876, 1 mM Sodium pyruvate (Gibco, 11360070) and 10 mM HEPES (Gibco, cat#11560496) with a seeding density of 3x104cells / cm2. The cells were split every two-three days with the use of trypsin (Gibco, cat#11580626). On day 0, five days prior to the start of treatment, 0.5x105TC-1 cells in 100 pl PBS were injected subcutaneously (s.c.) to the shaved right flank of the mice. The mice were treated two times on day 5 and day 10. Bispecific binding conjugate SP027 and tag constructs were mixed prior to injection.

[0226] Treatments were administered by needle injection s.c. to left foot hock in a total volume of 50 pl. The groups of mice were treated according to the following format: Group-1 : 25 mM histidine buffer pH 6; Group-2: 250 pmol antibody SP027 mixed with 750 pmol ULI0170; Group-3: 250 pmol antibody SP027 mixed with 750 pmol UU0169.

[0227] Tumor growth was monitored every 2-3 days by measurement with an electronic calliper until the tumor volume reached to 1000 mm3according to ellipsoid formula (4-^3xTTx(width-^2x|ength-^2xheigh 2).

[0228] Table 7: Overview of immunization set-up in Example 11 Results

[0229] The results are shown in Figure 10. Tumor inoculation was successful in all animals included in the study. The difference in tumor effect (anti-tumor efficacy measured as tumor growth volume) was observed from day 12 and onwards.

[0230] SP027 mixed with the tag construct ULI0169, which comprises the 9-mer tag moiety and the E7 peptide as cargo moiety, prevented tumor growth and prolonged mouse survival compared to the control group receiving SP027 with the tag construct ULI0170, comprising the 7-mer tag moiety and the E7 peptide as cargo moiety. The mice receiving SP027 with the tag construct comprising the 7-mer tag moiety (group 2, representative of unlinked bispecific binding conjugate and tag construct delivery) had similar tumor growth as the mice receiving vehicle (group 1 , control group).

[0231] This data demonstrates that targeted delivery of bispecific binding molecule SP027 together with a tag construct (group 3), comprising a 9-mer tag moiety according to the present invention and a peptide cargo moiety, induced a superior cell response leading to improved anti-tumor efficacy compared to unlinked tag construct and bispecific binding conjugate.

[0232] Informal

[0233] SEQ ID NO:1

[0234] FIGITELXK

[0235] SEQ ID NO:2 FIGITELKK

[0236] SEQ ID NO:3 FIGITELLK

[0237] SEQ ID NO:4 FIGITELHK

[0238] SEQ ID NO:5

[0239] FIGITELK

[0240] SEQ ID NO:6

[0241] FIGITELL

[0242] SEQ ID NO:7

[0243] FIGITELH

[0244] SEQ ID NO:8

[0245] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPES

[0246] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0247] GTLVTVSS SEQ ID NO:9

[0248] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEG

[0249] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ GTLVTVSS

[0250] SEQ ID NO:1Q

[0251] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQ GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ GTLVTVSS

[0252] SEQ ID NO:11

[0253] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPED

[0254] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ GTLVTVSS

[0255] SEQ ID NO:12

[0256] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEN ADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ GTLVTVSS

[0257] SEQ ID NO:13

[0258] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEN GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ GTLVTVSS

[0259] SEQ ID NO:14

[0260] DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHT

[0261] GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0262] SEQ ID NO:15

[0263] EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENG

[0264] DAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQG

[0265] TTLTVSSGGGGSGGGGSGGGGSGGGGSELQMTQSPSSLSASLGDTVTITCHASQ NINVWLSWYQQRPGNIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIAT YYCQQGQSYPLTFGAGTKLELK

[0266] SEQ ID NO:16

[0267] QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPS

[0268] DNFSNLNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLT

[0269] VSSGGGGSGGGGSGGGGSGGGGSDIVMTQATPSVLVTPGEAVSISCRASRSLLH SNGITYLYWFLQRPGQSPQVLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAED VGVYYCMQHLEFPYTFGGGTKLEIK

[0270] SEQ ID NO:17

[0271] NLVPMVATV

[0272] SEQ ID NO:18

[0273] SIINFEKL

[0274] SEQ ID NO:19

[0275] ISQAVHAAHAEINEAGR SEQ ID NQ:20

[0276] KVPRNQDWL

[0277] SEQ ID NO:21

[0278] YKLVVVGAVGVGKSALT

[0279] SEQ ID NO:22

[0280] YKLWVGARGVGKSALT

[0281] SEQ ID NO:23

[0282] YKLVVVGADGVGKSALT

[0283] SEQ ID NO:24

[0284] FIGITELKKAGILARNLVPMVATVQGQNLKY

[0285] SEQ ID NO:25

[0286] YKLVVVGACGVGKSALT

[0287] SEQ ID NO:26

[0288] FIGITELKKAAYLEQLESIINFEKLAAAAAK

[0289] SEQ ID NO:27

[0290] FIGITELKKISQAVHAAHAEINEAGR

[0291] SEQ ID NO:28

[0292] FIGITELKKAAYISQAVHAAHAEINEAGR

[0293] SEQ ID NO:29

[0294] FIGITELKK-OH-PEG3-Biotin

[0295] SEQ ID NQ:30

[0296] FIGITELKKYKLVWGAVGVGKSALT

[0297] SEQ ID NO:31

[0298] FIGITELKKYKLVVVGARGVGKSALT

[0299] SEQ ID NO:32

[0300] FIGITELKKYKLVVVGADGVGKSALT

[0301] SEQ ID NO:33

[0302] FIGITELHKAGILARNLVPMVATVQGQNLKY-FITC

[0303] SEQ ID NO:34

[0304] FIGITELHKISQAVHAAHAEINEAGR

[0305] SEQ ID NO:35

[0306] FIGITELHKAAYISQAVHAAHAEINEAGR

[0307] SEQ ID NO:36

[0308] FIGITELHK-OH-PEG3-Biotin SEQ ID NO:37

[0309] FIGITELLKAGILARNLVPMVATVQGQNLKY-FITC

[0310] SEQ ID NO:38

[0311] FIGITELLKISQAVHAAHAEINEAGR

[0312] SEQ ID NO:39

[0313] FIGITELLKAAYISQAVHAAHAEINEAGR

[0314] SEQ ID NQ:40

[0315] FIGITELLK-OH-PEG3-Biotin

[0316] SEQ ID NO:41

[0317] FIGITELKAGILARNLVPMVATVQGQNLKY

[0318] SEQ ID NO:42

[0319] FIGITELLAGILARNLVPMVATVQGQNLKY

[0320] SEQ ID NO:43

[0321] FIGITELHAGILARNLVPMVATVQGQNLKY

[0322] SEQ ID NO:44

[0323] FIGITELKAAYLEQLESIINFEKLAAAAAK

[0324] SEQ ID NO:45

[0325] FIGITELLAAYLEQLESIINFEKLAAAAAK

[0326] SEQ ID NO:46

[0327] FIGITELHAAYLEQLESIINFEKLAAAAAK

[0328] SEQ ID NO:47

[0329] FIGITELKISQAVHAAHAEINEAGR

[0330] SEQ ID NO:48

[0331] FIGITELLISQAVHAAHAEINEAGR

[0332] SEQ ID NO:49

[0333] FIGITELHISQAVHAAHAEINEAGR

[0334] SEQ ID NQ:50

[0335] FIGITELKAAYISQAVHAAHAEINEAGR

[0336] SEQ ID NO:51

[0337] FIGITELLAAYISQAVHAAHAEINEAGR

[0338] SEQ ID NO:52

[0339] FIGITELHAAYISQAVHAAHAEINEAGR

[0340] SEQ ID NO:53

[0341] FIGITELK-OH-PEG3-Biotin

[0342] SEQ ID NO:54 FIGITELL-OH-PEG3-Biotin

[0343] SEQ ID NO:55

[0344] FIGITELH-OH-PEG3-Biotin

[0345] SEQ ID NO:56

[0346] FIGITELKYKLWVGAVGVGKSALT

[0347] SEQ ID NO:57

[0348] FIGITELLYKLWVGAVGVGKSALT

[0349] SEQ ID NO:58

[0350] FIGITELHYKLVWGAVGVGKSALT

[0351] SEQ ID NO:59

[0352] FIGITELKYKLWVGARGVGKSALT

[0353] SEQ ID NQ:60

[0354] FIGITELLYKLVVVGARGVGKSALT

[0355] SEQ ID NO:61

[0356] FIGITELHYKLVVVGARGVGKSALT

[0357] SEQ ID NO:62

[0358] FIGITELKYKLWVGADGVGKSALT

[0359] SEQ ID NO:63

[0360] FIGITELLYKLVVVGADGVGKSALT

[0361] SEQ ID NO:64

[0362] FIGITELHYKLVVVGADGVGKSALT

[0363] SEQ ID NO:65

[0364] FIGITELKAGILARNLVPMVATVQGQNLKY-FITC

[0365] SEQ ID NO:66

[0366] FIGITELLAGILARNLVPMVATVQGQNLKY-FITC

[0367] SEQ ID NO:67

[0368] FIGITELHAGILARNLVPMVATVQGQNLKY-FITC

[0369] SEQ ID NO:68

[0370] FNIKDFNI

[0371] SEQ ID NO:69

[0372] IGRIDPEXaXbDAEYVP

[0373] SEQ ID NQ:70

[0374] TTGSYDLDVE

[0375] SEQ ID NO:71

[0376] HASQNINVWLS SEQ ID NO:72

[0377] KASTLHT

[0378] SEQ ID NO:73

[0379] QQGQSYPLT

[0380] SEQ ID NO:74

[0381] QSISSY

[0382] SEQ ID NO:75

[0383] AAS

[0384] SEQ ID NO:76

[0385] QQGYPYPFT

[0386] SEQ ID NO:77

[0387] GFTFSSYA

[0388] SEQ ID NO:78

[0389] ISGYSGST

[0390] SEQ ID NO:79

[0391] ARYYSYYGYYYFDY

[0392] SEQ ID NQ:80

[0393] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG

[0394] VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK

[0395] SEQ ID NO:81

[0396] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGY

[0397] SGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDY WGQGTLVTVSS

[0398] SEQ ID NO:82

[0399] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG

[0400] VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPS

[0401] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

[0402] DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0403] SEQ ID NO:83

[0404] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGY

[0405] SGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDY

[0406] WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG

[0407] ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

[0408] KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN

[0409] WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPA

[0410] PIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ

[0411] PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

[0412] SEQ ID NO:84 FIGITELKKLESKINKVFK-Biotin

[0413] SEQ ID NO:85

[0414] FIGITELKKLE

[0415] SEQ ID NO:86

[0416] FIGITELKKL

[0417] SEQ ID NO:87

[0418] FIGITELK

[0419] SEQ ID NO:88

[0420] ANSKFIGITELK

[0421] SEQ ID NO:89

[0422] FIGITELLEQLESIINFEKLAAAAAK

[0423] SEQ ID NQ:90

[0424] FIGITELKLEQLESIINFEKLAAAAAK

[0425] SEQ ID NO:91

[0426] FIGITELHLEQLESIINFEKLAAAAAK

[0427] SEQ ID NO:92

[0428] Biotin-IDIKNDLYEKTLNDYKAIANKLSQV

[0429] SEQ ID NO:93

[0430] FIGITELKKL-OH-PEG3-Biotin

[0431] SEQ ID NO:94

[0432] FIGITELKKLE-OH-PEG3-Biotin

[0433] SEQ ID NO:95

[0434] FIGITELLKL-OH-PEG3-Biotin

[0435] SEQ ID NO:96

[0436] FIGITELLKLE-OH-PEG3-Biotin

[0437] SEQ ID NO:97

[0438] FIGITELKKLESKINKVFISQAVHAAHAEINEAGR

[0439] SEQ ID NO:98

[0440] FIGITELLKAGILARNLVPMVATVQGQNLKY

[0441] SEQ ID NO:99

[0442] FIGITELKKYKLVVVGACGVGKSALT

[0443] SEQ ID NQ:100

[0444] AGILARNLVPMVATVQGQNLKY

[0445] SEQ ID NO:1Q1

[0446] FIGITELKKLESKINKVFAVGALKVPRNQDWLGVPRQL SEQ ID NQ:102

[0447] AVGALKVPRNQDWLGVPRQL

[0448] SEQ ID NQ:103

[0449] FIGITELKKAVGALKVPRNQDWLGVPRQL

[0450] SEQ ID NQ:104

[0451] FIGITELKKLAVGALKVPRNQDWLGVPRQL

[0452] SEQ ID NQ:105

[0453] FIGITELKKLEAVGALKVPRNQDWLGVPRQL

[0454] SEQ ID NQ:106

[0455] FIGITELLKAVGALKVPRNQDWLGVPRQL

[0456] SEQ ID NQ:107

[0457] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPES

[0458] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0459] GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHAS

[0460] QNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGQSYPLTFGQGTKLEI K

[0461] SEQ ID NQ:108

[0462] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEG

[0463] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0464] GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHAS

[0465] QNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGQSYPLTFGQGTKLEI K

[0466] SEQ ID NQ:109

[0467] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEQ

[0468] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0469] GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHAS

[0470] QNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGQSYPLTFGQGTKLEI K

[0471] SEQ ID NO:11Q

[0472] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPED

[0473] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0474] GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHAS

[0475] QNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGQSYPLTFGQGTKLEI K

[0476] SEQ ID NO:111

[0477] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEN

[0478] ADAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0479] GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHAS

[0480] QNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGQSYPLTFGQGTKLEI K

[0481] SEQ ID NO:112 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVRQAPGQGLEWIGRIDPEN

[0482] GDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTTGSYDLDVEYWGQ

[0483] GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHAS

[0484] QNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGQSYPLTFGQGTKLEI K

[0485] SEQ ID NO:113

[0486] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGY

[0487] SGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDY

[0488] WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG

[0489] ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

[0490] KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFN

[0491] WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPA

[0492] PIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ

[0493] PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

[0494] SLSPGKGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDFNIHWVR

[0495] QAPGQGLEWIGRIDPESGDAEYVPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV

[0496] YYCTTGSYDLDVEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSP

[0497] SSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASTLHTGVPSRFSG

[0498] SGSGTDFTLTISSLQPEDFATYYCQQGQSYPLTFGQGTKLEIK

[0499] SEQ ID NO:114

[0500] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASFLQSG

[0501] VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPS

[0502] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

[0503] DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0504] SEQ ID NO:115

[0505] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG

[0506] VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPS

[0507] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

[0508] DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0509] SEQ ID NO:116

[0510] YKLVVVGAAGVGKSALT

[0511] SEQ ID NO:117

[0512] FIGITELKKYKLVWGAAGVGKSALT

[0513] SEQ ID NO:118

[0514] FIGITELKYKLWVGACGVGKSALT

[0515] SEQ ID NO:119

[0516] FIGITELLYKLVVVGACGVGKSALT

[0517] SEQ ID NQ:120

[0518] FIGITELHYKLVVVGACGVGKSALT

[0519] SEQ ID NO:121

[0520] FIGITELKYKLWVGAAGVGKSALT

[0521] SEQ ID NO:122 FIGITELLYKLWVGAAGVGKSALT

[0522] SEQ ID NO:123

[0523] FIGITELHYKLVWGAAGVGKSALT

[0524] SEQ ID NO:124

[0525] IGRIDPESGDAEYVP

[0526] SEQ ID NO:125

[0527] IGRIDPEGGDAEYVP

[0528] SEQ ID NO:126

[0529] IGRIDPEQGDAEYVP

[0530] SEQ ID NO:127

[0531] IGRIDPEDGDAEYVP

[0532] SEQ ID NO:128

[0533] IGRIDPENADAEYVP

[0534] SEQ ID NO:129

[0535] IGRIDPENGDAEYVP

[0536] SEQ ID NQ:130

[0537] EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENG DAEYVPKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQG TTLTVSS

[0538] SEQ ID NO:131

[0539] ELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPGNIPKLLIYKASTLHTG

[0540] VPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK

[0541] SEQ ID NO:132

[0542] QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPS DNFSNLNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLT VSS

[0543] SEQ ID NO:133

[0544] SDIVMTQATPSVLVTPGEAVSISCRASRSLLHSNGITYLYWFLQRPGQSPQVLIYRM

[0545] SNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEFPYTFGGGTKLEIK

[0546] SEQ ID NO:134

[0547] GYTFTDYW

[0548] SEQ ID NO:135

[0549] IDPSDNFS

[0550] SEQ ID NO:136

[0551] AVEDY

[0552] SEQ ID NO:137

[0553] RSLLHSNGITY SEQ ID NO:138

[0554] RMS

[0555] SEQ ID NO:139

[0556] MQHLEFPYT

[0557] SEQ ID NQ:140

[0558] QAEPDRAHYNIVTFCCKCD

[0559] SEQ ID NO:141

[0560] FIGITELKKQAEPDRAHYNIVTFCCKCD

[0561] SEQ ID NO:142

[0562] FIGITELLKQAEPDRAHYNIVTFCCKCD

[0563] SEQ ID NO:143

[0564] FIGITELHKQAEPDRAHYNIVTFCCKCD

[0565] SEQ ID NO:144

[0566] FIGITELKQAEPDRAHYNIVTFCCKCD

[0567] SEQ ID NO:145

[0568] FIGITELLQAEPDRAHYNIVTFCCKCD

[0569] SEQ ID NO:146

[0570] FIGITELHQAEPDRAHYNIVTFCCKCD

[0571] SEQ ID NO:147

[0572] GITELKKQAEPDRAHYNIVTFCCKCD

[0573] SEQ ID NO:148

[0574] GITELKK

[0575] ITEMIZED LISTING OF EMBODIMENTS

[0576] 1. A tag moiety, consisting of the amino acid sequence

[0577] FIGITELXsX9(SEQ ID NO:1) wherein

[0578] Xs is selected from K, L, and H; and X9 is K or absent; said tag moiety comprising an epitope for binding by a binding molecule specific therefor.

[0579] 2. Tag moiety according to item 1, wherein Xs is selected from K and L.

[0580] 3. Tag moiety according to item 1, wherein Xs is selected from L and H.

[0581] 4. Tag moiety according to item 1, wherein Xs is selected from K and H.

[0582] 5. Tag moiety according to any one of items 2 and 4, wherein Xs is K.

[0583] 6. Tag moiety according to any one of items 2-3, wherein Xs is L.

[0584] 7. Tag moiety according to any one of items 3-4, wherein Xs is H.

[0585] 8. Tag moiety according to any preceding item, wherein X9 is K.

[0586] 9. Tag moiety according to any one of items 1-7, wherein X9 is absent.

[0587] 10. Tag moiety according to any preceding item, consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2-7.

[0588] 11. A tag construct comprising a tag moiety according to any preceding item; and at least one cargo moiety.

[0589] 12. Tag construct according to item 11 , wherein said at least one cargo moiety is an antigen moiety.

[0590] 13. Tag construct according to item 12, wherein said at least one antigen moiety is a peptidic antigen moiety comprising a target antigen amino acid sequence.

[0591] 14. Tag construct according to any one of items 11-13, wherein the C terminus of said tag moiety is covalently bound to the N terminus of said at least one cargo moiety.

[0592] 15. Tag construct according to any one of items 11-13, wherein the N terminus of said tag moiety is covalently bound to the C terminus of said at least one cargo moiety.

[0593] 16. Tag construct according to any one of items 13-15, wherein said target antigen amino acid sequence is selected from the group consisting of a cancer antigen, a self-antigen and an antigen derived from a pathogen. 17. Tag construct according to item 16, wherein said target antigen sequence is a cancer antigen.

[0594] 18. Tag construct according to item 17, wherein said cancer antigen is selected from the group consisting of a neoantigen, a tumor-associated antigen and an antigen derived from an oncovirus.

[0595] 19. Tag construct according to any one of items 11-18, wherein said target antigen sequence is selected from the group consisting of SEQ ID NO: 17-23, 25, 116 and 140.

[0596] 20. Tag construct according to any one of items 11-19, wherein the amino acid sequence of said tag construct is selected from the group consisting of SEQ ID NO:24, 26-67, 88, 90-91, 98-99, 103, 106, 117-123 and 141-146.

[0597] 21. Tag construct according to item 20, wherein the amino acid sequence of said tag construct is selected from the group consisting of SEQ ID NO:24, 26-40, 88, 98- 99, 103, 106, 117 and 141-143.

[0598] 22. Tag construct according to item 20, wherein the amino acid sequence of said tag construct is selected from the group consisting of SEQ ID NO:41-67, 90-91 , US- 123 and 144-146.

[0599] 23. Tag construct according to item 11, wherein said at least one cargo moiety is a nucleic acid moiety, such and siRNA molecule.

[0600] 24. Tag moiety according to any one of items 1-10 or a tag construct according to any one of items 11-23, which is capable of binding to said binding molecule such that the KD value of the interaction is at most 1 x 10'9M, for example at most 1 x 10'1° M, for example at most 1 x 10'11M.

[0601] 25. Tag moiety or construct according to any preceding item, wherein said binding molecule specific for said epitope in the tag moiety is an antibody construct.

[0602] 26. Tag moiety or construct according to item 25, wherein said antibody construct is an antibody or antigen binding fragment thereof.

[0603] 27. Tag moiety or construct according to item 26, wherein said binding molecule is an antibody fragment which is an scFv.

[0604] 28. Tag moiety or construct according to any one of items 25-27, wherein said binding molecule comprises: an immunoglobulin heavy chain variable region (VH) comprising three complementarity determining domains (CDRs), wherein:

[0605] VHCDR1 has the sequence set forth in SEQ ID NO:68; VHCDR2 has the sequence IGRIDPEXaXbDAEYVP (SEQ ID NO:69), wherein XaXbis selected from the group consisting of SG, GG, QG, DG, NA and NG and;

[0606] VHCDR3 has the sequence set forth in SEQ ID NO:70; and an immunoglobulin light chain variable region (VL) comprising three complementarity determining domains (CDRs), wherein:

[0607] VLCDR1 has the sequence set forth in SEQ ID NO:71 ;

[0608] VLCDR2 has the sequence set forth in SEQ ID NO:72; and

[0609] VLCDR3 has the sequence set forth in SEQ ID NO:73.

[0610] 29. Tag moiety or construct according to item 28, wherein said binding molecule comprises: an immunoglobulin heavy chain variable region (VH) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: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 NO: 14, 131 and 133 and amino acid sequences having at least 90 % identity thereto.

[0611] 30. Tag moiety or construct according to item 29, wherein said binding molecule comprises a complete scFv sequence selected from the group consisting of SEQ ID NO: 15-16 and 107-112, for example selected from the group consisting of SEQ ID NO: 107-112, for example selected from the group consisting of SEQ ID NQ:107 and 112, for example SEQ ID NQ:107.

[0612] 31. Tag moiety or construct according to item 29, wherein said binding molecule comprises: 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; and 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.

[0613] 32. Tag moiety or construct according to item 31 , wherein said binding molecule comprises the complete scFv sequence SEQ ID NO:15.

[0614] 33. Tag moiety or construct according to any one of items 25-27, wherein said binding molecule comprises: an immunoglobulin heavy chain variable region (VH) comprising three complementarity determining domains (CDRs), wherein:

[0615] VHCDR1 has the sequence set forth in SEQ ID NO: 134;

[0616] VHCDR2 has the sequence set forth in SEQ ID NO: 135;

[0617] VHCDR3 has the sequence set forth in SEQ ID NO: 136; and an immunoglobulin light chain variable region (VL) comprising three complementarity determining domains (CDRs), wherein: VLCDR1 has the sequence set forth in SEQ ID NO:137;

[0618] VLCDR2 has the sequence set forth in SEQ ID NO: 138; and VLCDR3 has the sequence set forth in SEQ ID NO: 139. Tag moiety or construct according to item 33, wherein said binding molecule comprises: 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; and 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. A complex comprising: a tag construct comprising a tag moiety according to any one of items 1-10; a binding molecule specific for said tag moiety. Complex according to item 35, wherein said tag construct is a tag construct according to any one of items 11-23. Complex according to any one of items 35-36, wherein said binding molecule is as defined in any one of items 25-34. Complex according to any one of items 35-37, in which said binding molecule is covalently linked to a second moiety which is an antibody or antigen binding fragment thereof, for example 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. Complex according to item 38, wherein said second moiety is an antibody. Complex according to item 39, wherein said second moiety is of the lgG2 subtype. Complex according to any one of items 38-40, wherein said second moiety is an anti-CD40 antibody or antigen binding fragment thereof. 42. Complex according to item 41 , wherein said anti-CD40 antibody is selected from the group consisting of CP-870,893, APX005M, ADC-1013, ChiLob 7 / 4, SEA- CD40 and ABS-1150 / 1151 and an antibody comprising an antigen binding fragment derived from any one or more of said antibodies.

[0619] 43. Complex according to item 41 , wherein said anti-CD40 antibody or antigen binding fragment thereof comprises six complementarity determining domains (CDRs), wherein:

[0620] VLCDR1 has the sequence set forth in SEQ ID NO:74;

[0621] VLCDR2 has the sequence set forth in SEQ ID NO:75;

[0622] VLCDR3 has the sequence set forth in SEQ ID NO:76;

[0623] VHCDR1 has the sequence set forth in SEQ ID NO:77;

[0624] VHCDR2 has the sequence set forth in SEQ ID NO:78; and VHCDR3 has the sequence set forth in SEQ ID NO:79.

[0625] 44. Complex according to item 43, wherein the light chain variable domain of said anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NQ:80 and amino acid sequences having at least 90 % sequence identity thereto; and the heavy chain variable domain of said anti-CD40 antibody comprises an amino acid sequence selected from SEQ ID NO:81 and amino acid sequences having at least 90 % sequence identity thereto.

[0626] 45. Complex according to item 44, wherein said anti-CD40 antibody comprises a light chain 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 comprising an amino acid sequence selected from SEQ ID NO:83 and amino acid sequences having at least 90 % sequence identity thereto.

[0627] 46. A polynucleotide encoding a tag moiety or tag construct according to any one of items 1-34.

[0628] 47. An expression vector comprising a polynucleotide according to item 46.

[0629] 48. A host cell comprising an expression vector according to item 47.

[0630] 49. A method of producing a tag moiety or tag construct according to any one of items 1-34, the method comprising: culturing a host cell according to item 48 under conditions allowing expression of said tag moiety or tag construct from said expression vector; and isolating said tag moiety or tag construct. 50. A composition comprising a tag moiety or tag construct according to any one of items 1-34 and at least one pharmaceutically acceptable excipient or carrier.

[0631] 51. A composition comprising a complex according to any one of items 35-45 and at least one pharmaceutically acceptable excipient or carrier.

[0632] 52. A tag moiety or tag construct according to any one of items 1-34, a complex according to any one of items 35-45, or a composition according to any one of items 50-51 , for use in therapy.

[0633] 53. A tag moiety or tag construct according to any one of items 1-34, a complex according to any one of items 35-45, or a composition according to any one of items 50-51 , for use as a diagnostic agent in vitro, as a diagnostic agent in vivo as a prognostic agent in vitro and / or as a prognostic agent in vitro.

[0634] 54. Tag moiety, tag construct, complex or composition for use according to item 52 in the treatment or prevention of cancer, an autoimmune disease or infection.

[0635] 55. Tag moiety, tag construct, complex or composition for use according to item 54 in the treatment or prevention of cancer.

[0636] 56. A method of treatment or prevention of cancer, an autoimmune disease or infection, comprising administering to a subject in need thereof of an effective amount of a tag moiety or tag construct according to any one of items 1-34, a complex according to any one of items 35-45, or a composition according to any one of items 50-51.

[0637] 57. Method according to item 56, which is for treatment or prevention of cancer.

[0638] 58. Use of a tag moiety or tag construct according to any one of items 1-34, a complex according to any one of items 35-45, or a composition according to any one of items 50-51 in the manufacture of a medicament.

[0639] 59. Use according to item 58, wherein said medicament is intended for treatment or prevention of cancer, an autoimmune disease or infection.

[0640] 60. Use according to item 59, wherein said medicament is intended for treatment or prevention of cancer.

[0641] 61. 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.

[0642] 62. 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. Diagnostic or prognostic method for determining the presence of a disease, disorder or infection in a subject comprising the steps of: a. contacting the subject, or a sample isolated from the subject, with a tag moiety or tag construct according to any one of items 1-34, a complex according to any one of items 35-45, or a composition according to any one of items 50-51, and b. obtaining a value corresponding to the amount of the tag moiety, tag construct, complex or composition that has bound in said subject or to said sample. 64. The diagnostic or prognostic method according to item 63, which is a method for diagnosis in vivo or prognosis in vivo, for example via medical imaging, in which said contacting in step a) consists of contacting said subject with said tag moiety, tag construct, complex or composition.

[0643] 65. The diagnostic or prognostic method according to item 63, which is an in vitro method and in which said contacting in step a) consists of contacting a sample previously isolated from said subject with said tag moiety, tag construct, complex or composition.

Claims

CLAIMS A tag moiety, consisting of the amino acid sequenceFIGITELXsX9(SEQ ID NO:1) whereinXs is selected from K, L, and H; andX9 is K or absent; said tag moiety comprising an epitope for binding by a binding molecule specific therefor. Tag moiety according to claim 1 , consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2-7. A tag construct comprising a tag moiety according to any preceding claim; and at least one cargo moiety. Tag construct according to claim 3, wherein said at least one cargo moiety is an antigen moiety. Tag construct according to claim 4, wherein said at least one antigen moiety is a peptidic antigen moiety comprising a target antigen amino acid sequence. Tag construct according to claim 5, wherein said target antigen amino acid sequence is selected from the group consisting of a cancer antigen, a selfantigen and an antigen derived from a pathogen. Tag construct according to claim 6, wherein said target antigen amino acid sequence is a cancer antigen, for example a cancer antigen selected from the group consisting of a neoantigen, a tumor-associated antigen and an antigen derived from an oncovirus. Tag construct according to claim 3, wherein said at least one cargo moiety is a nucleic acid moiety, such as an siRNA molecule.

9. Tag moiety or tag construct according to any preceding claim, which is capable of binding to said binding molecule such that the KD value of the interaction is at most 1 x 10’9M, for example at most 1 x 10'1° M, for example at most 1 x 10'11M.

10. Tag moiety or construct according to any preceding claim, wherein said binding molecule specific for said epitope in the tag moiety is an antibody construct, for example an antibody or antigen binding fragment thereof, or for example an scFv.11 . A complex comprising: a tag construct comprising a tag moiety according to any one of claims 1-2 and 9-10; a binding molecule specific for said tag moiety.

12. Complex according to claim 11 , wherein said tag construct is a tag construct according to any one of claims 3-10.

13. Complex according to any one of claims 11-12, wherein said binding molecule is as defined in claim 10.

14. Complex according to any one of claims 11-13, in which said binding molecule is covalently linked to a second moiety which is an antibody or antigen binding fragment thereof, for example 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, for example an anti-CD40 antibody or antigen binding fragment thereof.

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

16. A composition comprising either a tag moiety or tag construct according to any one of claims 1-10 or a complex according to any one of claims 11-14, and at least one pharmaceutically acceptable excipient or carrier.

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

18. Tag moiety, tag construct, complex or composition for use according to claim 17 in the treatment or prevention of cancer, an autoimmune disease or an infection.

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

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